Background:Most data related to high-grade glioma and glioblastoma (GBM) 3D models refer to ultra-low/suspension cultivation systems, for several reasons: easy to initiate, high penetrability of treatments, ease of harvesting for downstream analysis. However, the major limitation of these models is the absence of the extracellular matrix, which enables the specific, local invasiveness of these aggressive brain tumors.Aim:This study provides a comparison between ultra-low and several extracellular matrix (ECM) and hydrogel culture conditions for various grades of patient-derived glioma organoids, in terms of cell growth, invasiveness, and release of cytokines/chemokines, as a model for downstream analysis of soluble factors.Materials and Methods:Patient-derived glioma organoids were initiated in three different conditions: ultra-low attachment(UL), 3D Matrigel, and dextran-based hydrogels. Human endothelial cells were used for tube assay co-cultures and human normal monocyte cell line SC to assess monocyte invasion and interaction with tumor organoids.Results:UL conditions supported organoid formation with better growth rate and easy passaging, and reliable detection of secreted cytokines and chemokines, closely mirroring qualitative secretory profiles observed in tumor explants. In contrast, ECM-based systems enabled invasive growth patterns and co-culture assays, but with a restricted immune cell infiltration and with a reduced detectable level of soluble factors. While qualitative cytokine signatures were preserved across culture platforms, substantial quantitative differences were observed, highlighting the influence of matrix composition on measurable outputs. Preliminary data showed that organoid-forming capacity indicated no clear association with Isocitrate Dehydrogenase 1 (IDH1) mutational status in the tested samples.Conclusion:Our findings indicate that UL and ECM-based models provide complementary information and recommend a dual-platform cell culture initiation strategy to maximize robustness and translational relevance in patient-derived glioma research.

CD36 is a transmembrane protein that plays a role in various biological processes, including oxidized low-density lipoprotein and fatty acid uptake as well as regulatory control for inflammation signaling. Its robust expression in monocytes and macrophages associated with its ability to translocate fatty acids linked this scavenger receptor to foam cell formation and atherosclerosis. In the context of ischemic stroke, CD36 has been shown to contribute to brain injury and inflammation. Preclinical studies have demonstrated that CD36 expression increases in the brain after stroke and that inhibiting CD36 can reduce infarction size and improve neurological outcomes in animal models. These findings suggest that CD36 may be a potential therapeutic target for ischemic stroke. However, no clinical trials addressing CD36 and acute ischemic stroke are registered in the American or European databases. This review will discuss the relationship between CD36 and ischemic stroke and present some clinical findings in patients with single nucleotide polymorphisms of the CD36 gene.

Background: Phytocarriers are advanced drug delivery systems that use biocompatible and biodegradable materials to enhance the efficacy, stability, and bioavailability of natural products. The sea buckthorn (Hippophae rhamnoides L.) berry extract is rich in essential fatty acids and antioxidants, including vitamin C, vitamin E, and anthocyanins, which contribute to its wide-ranging health benefits. In this study, we assessed the morphology, intracellular delivery, and anti-inflammatory effect of sodium cholate (NaC) and sodium deoxycholate (NaDC)-based phytocarriers loaded with ethanolic extract from sea buckthorn berries (sea buckthorn carrier nanostructures, further defined as phytocarriers). Methods: Negative and electron cryo-microscopy were used to analyze hollow and loaded nanocarriers. The cyto-compatibility of nanocarriers was assessed by endpoint (LDH and MTS) and real-time cell assays, on both human fibroblasts (HS27) and human normal monocytes (SC). The anti-inflammatory effect of hollow and loaded nanocarriers was tested by multiplexing. Results: The negative and electron cryo-microscopy analyses showed that NaC-based phytocarriers were spherical, whilst NaDC-based phytocarriers were predominantly polymorphic. Moreover, the NaDC-based phytocarriers frequently formed large lipid networks or “plaques”. Although 24 h cytotoxicity testing showed both types of nanocarriers are biocompatible with human fibroblasts and monocytes, based on a long-term real-time assay, NaDC delayed fibroblast proliferation. NaC sea buckthorn phytocarriers did not impair fibroblast proliferation in the long term and they were uptaken by cells, as shown by hyperspectral microscopy. NaC nanocarriers and NaC sea buckthorn phytocarriers induced an anti-inflammatory effect, lowering IL-8 cytokine production in normal human monocytes as soon as 4 h of treatment lapsed. Conclusions: NaC-derived phytocarriers loaded with sea buckthorn alcoholic extract are a cell-compatible delivery system with anti-inflammatory properties.

Background: To fill the knowledge gap regarding the antiviral immunity in oncologic patients, we performed a comparative study on natural/vaccine-induced SARS-CoV-2 immunity in head and neck cancer (HNC) in Romania. Methods: Blood was collected from HNC (n=49) and controls (n=14), stratified as vaccinated (RNA/adenovirus-based vaccines), convalescent, and hybrid immunity. Plasma IgG/IgA antibodies (Abs) against Spike (S1/S2), receptor binding domain (RBD), and nucleocapsid (NC), and cytokines were quantified using the MILLIPLEX; technology. The frequency/phenotype/isotype of RBD-specific B-cells were studied by flow cytometry using tetramers (Tet++). Cell proliferation in response to Spike/NC peptides was monitored by carboxyfluorescein succinimidyl ester (CFSE) assay. A longitudinal follow-up was performed on n=25 HNC. Findings: Levels of S1/S2/RBD-specific IgG/IgA Abs were similarly high in HNC and controls, but significantly increased in convalescent/hybrid versus vaccinated HNC. NC-specific IgG/IgA Abs were only detected in convalescent/hybrid immunity groups. The frequency of Tet++ B-cells in HNC was similar to controls, irrespective of the immunization status, and correlated positively with RBD IgG/IgA Abs and negatively with the time since immunization (TSI). Compared to total B-cells, Tet++ were enriched in CD27+ memory phenotype and IgG/IgA isotype. A linear regression model identified Spike S2 IgG and NC IgA Abs as strong positive predictors of Tet++ frequencies, while IL-6 was a marginally significant negative predictor. Tet++ frequency remained stable at median TSI of 341 versus 117 days, despite a decline in memory phenotype. Interpretation: HNC participants mount efficient and durable SARS-CoV-2 humoral immunity, with RBD-specific IgG/IgA Abs and Tet++ B-cells representing the major immunization outcomes. ### Competing Interest Statement The authors have declared no competing interest.

Background: Despite all the available treatments, psoriasis remains incurable; therefore, finding personalized therapies is a continuous challenge. Psoriasis is linked to a gut microbiota imbalance, highlighting the importance of the gut–skin axis and its inflammatory mediators. Restoring this imbalance can open new perspectives in psoriasis therapy. We investigated the effect of purified IgY raised against pathological human bacteria antibiotic-resistant in induced murine psoriatic dermatitis (PSO). Methods: To evaluate the immune portrayal in an imiquimod experimental model, before and after IgY treatment, xMAP array and flow cytometry were used. Results: There were significant changes in IL-1α,β, IL-5, IL-6, IL-9, IL-10, IL-12 (p70), IL-13, IL-15, IL-17a, IFN-γ, TNF-α, IP-10/CXCL10, MCP-1/CCL2, MIP-1α/CCL3, MIP-1β/CCL4, MIG/CXCL9, and KC/CXCL1 serum levels. T (CD3ε+), B (CD19+) and NK (NK1.1+) cells were also quantified. In our model, TNF-α, IL-6, and IL-1β cytokines and CXCL1 chemokine have extremely high circulatory levels in the PSO group. Upon experimental therapy, the cytokine serum values were not different between IgY-treated groups and spontaneously remitted PSO. Conclusions: Using the murine model of psoriatic dermatitis, we show that the orally purified IgY treatment can lead to an improvement in skin lesion healing along with the normalization of cellular and humoral immune parameters.

Healthcare professionals face an ongoing challenge in managing both acute and chronic wounds, given the potential impact on patients' quality of life and the limited availability of expensive treatment options. Hydrogel wound dressings offer a promising solution for effective wound care due to their affordability, ease of use, and ability to incorporate bioactive substances that enhance the wound healing process. Our study aimed to develop and evaluate hybrid hydrogel membranes enriched with bioactive components such as collagen and hyaluronic acid. We utilized both natural and synthetic polymers and employed a scalable, non-toxic, and environmentally friendly production process. We conducted extensive testing, including an in vitro assessment of moisture content, moisture uptake, swelling rate, gel fraction, biodegradation, water vapor transmission rate, protein denaturation, and protein adsorption. We evaluated the biocompatibility of the hydrogel membranes through cellular assays and performed instrumental tests using scanning electron microscopy and rheological analysis. Our findings demonstrate that the biohybrid hydrogel membranes exhibit cumulative properties with a favorable swelling ratio, optimal permeation properties, and good biocompatibility, all achieved with minimal concentrations of bioactive agents.

Complicated wounds often require specialized medical treatments, and hydrogels have emerged as a popular choice for wound dressings in such cases due to their unique properties and the ability to incorporate and release therapeutic agents. Our focus was to develop and characterize a new optimized formula for biohybrid hydrogel membranes, which combine natural and synthetic polymers, bioactive natural compounds, like collagen and hyaluronic acid, and pharmacologically active substances (doxycycline or npAg). Dynamic (oscillatory) rheometry confirmed the strong gel-like properties of the obtained hydrogel membranes. Samples containing low-dose DOXY showed a swelling index of 285.68 ± 6.99%, a degradation rate of 71.6 ± 0.91% at 20 h, and achieved a cumulative drug release of approximately 90% at pH 7.4 and 80% at pH 8.3 within 12 h. The addition of npAg influenced the physical properties of the hydrogel membranes. Furthermore, the samples containing DOXY demonstrated exceptional antimicrobial efficacy against seven selected bacterial strains commonly associated with wound infections and complications. Biocompatibility assessments revealed that the samples exhibited over 80% cell viability. However, the addition of smaller-sized nanoparticles led to decreased cellular viability. The obtained biohybrid hydrogel membranes show favorable properties that render them suitable for application as wound dressings.

Background: The oral squamous cell carcinoma (OSCC) tumor microenvironment (TME) is a complex interweb of cells and mediators balancing carcinogenesis, inflammation, and the immune response. However, cytokines are not only secreted within the TME but also released by a variety of other cells that do not comprise the TME; therefore, a thorough assessment of humoral changes in OSCC should include the measurement of serum cytokines. Methods: We assessed the role of various serum cytokines in the evolution of OSCC, before and after treatment, versus a control group. We measured the serum concentrations of MIP-1α, IL-1β, IL-4, IL-6, IL-8, IL-10, and TNF-α. Results: Significantly higher values (p < 0.01) were noted for IL-1β, IL-6, IL-8, IL-10, and TNF-α in the OSCC group before treatment (n = 13) compared with the control group (n = 14), and the increased concentrations persisted after treatment (n = 11). Furthermore, the variations in the values of MIP-1α, IL-1β, IL-10, and TNF-α are correlated both before and after treatment (p < 0.01). In the pretherapeutic group, IL-6 and IL-8 concentrations also correlate with IL-1β and IL-10 serum levels (p < 0.01), while in the posttherapeutic group, IL-4 varies with MIP-1α and TNF-α (p < 0.01). Conclusion: In OSCC patients, serum cytokine levels are significantly higher compared with control, but they are not significantly altered by treatment, therefore implying that they are also influenced by systemic factors. The interactions between all involved cytokines and the various pathways they regulate warrant further studies to clarify their definitive roles.

Glioblastoma (GBM) is one of the most aggressive tumors of the central nervous system, characterized by a wide range of inter- and intratumor heterogeneity. Accumulation of fatty acids (FA) metabolites was associated with a low survival rate in high-grade glioma patients. The diversity of brain lipids, especially polyunsaturated fatty acids (PUFAs), is greater than in all other organs and several classes of proteins, such as FA transport proteins (FATPs), and FA translocases are considered principal candidates for PUFAs transport through BBB and delivery of PUFAs to brain cells. Among these, the CD36 FA translocase promotes long-chain FA uptake as well as oxidated lipoproteins. Moreover, CD36 binds and recognizes thrombospondin-1 (TSP-1), an extracellular matrix protein that was shown to play a multifaceted role in cancer as part of the tumor microenvironment. Effects on tumor cells are mediated by TSP-1 through the interaction with CD36 as well as CD47, a member of the immunoglobulin superfamily. TSP-1/CD47 interactions have an important role in the modulation of glioma cell invasion and angiogenesis in GBM. Separately, FA, the two membrane receptors CD36, CD47, and their joint ligand TSP-1 all play a part in GBM pathogenesis. The last research has put in light their interconnection/interrelationship in order to exert a cumulative effect in the modulation of the GBM molecular network.

Fatty acids (FAs) have been shown to exhibit a pro-inflammatory response in various cell types, but astrocytes have been mostly overlooked. FAs, both saturated and unsaturated, have previously been shown to induce pro-inflammatory responses in astrocytes at high concentrations of hundreds of µg/mL. SSO (Sulfo-N-succinimidyl Oleate sodium), an inhibitor of FA translocase CD36, has been shown to prevent inflammation in the mouse brain by acting on local microglia and infiltrating monocytes. Our hypothesis was that SSO treatment would also impact astrocyte pro-inflammatory response to FA. In order to verify our assumption, we evaluated the expression of pro- and anti-inflammatory cytokines in normal human astrocyte cell culture pre-treated (or not) with SSO, and then exposed to low concentrations of both saturated (palmitic acid) and unsaturated (oleic acid) FAs. As a positive control for astrocyte inflammation, we used fibrillary amyloid. Neither Aβ 1–42 nor FAs induced CD36 protein expression in human astrocytes in cell culture At low concentrations, both types of FAs induced IL-8 protein secretion, and this effect was specifically inhibited by SSO pre-treatment. In conclusion, low concentrations of oleic acid are able to induce an early increase in IL-8 expression in normal human astrocytes, which is specifically downregulated by SSO.

Background: Human monocyte lines are widely used in basic research as model of inflammation, mostly following adherence with phorbol 12-myristate 13-acetate (PMA). However, the SC line, of normal human monocytes is not well documented, unlike tumour-derived cell lines, such as THP-1. Aim: The purpose of this study was to determine the phenotype of adherent macrophages, induced after the treatment with PMA in three different concentrations, starting from the most widely reported concentration in the literature. Methods: Normal human monocytes SC (ATCC CRL-9855) were routinely maintained according to manufacturer’s instructions. Cells were treated with Phorbol 12-myristate 13-acetate (PMA Sigma Aldrich P1585), in concentrations of 200 ng/mL, 100 ng/mL, 25 ng/mL and adhesion was documented using an Evos phase-contrast inverted microscope. Cell behaviour was validated by real-time impedance readings. The adhered cells were treated with bacterial lipopolysaccharide (LPS) in concentrations of 50 ng/mL (mimicking chronic inflammation) and 1 μg/mL (mimicking acute inflammation). The supernatant was collected twice, after 4 hours, respectively after 18 hours of treatment with LPS. A screening of pro- and anti-inflammatory cytokines was performed using the multiplexing platform Luminex 200. ELISA tests were performed to validate the cytokines secretion: IL-6, IL-8, IL-10, IL-23 and TNF-ɑ, using a LEGEND MAX Human ELISA kit specific to each cytokine. Results: Cell adhesion was studied by time-lapse microscopy for 48 hrs. The lowest concentration of PMA which induced cell adherence was 25 ng/mL. Multiplex screening of cytokines showed a pro-inflammatory phenotype of macrophages stimulated with LPS. This finding was validated by ELISA tests for IL-6, IL-8, IL-23 and TNF-ɑ (as pro-inflammatory cytokine) and IL-10 (an anti-inflammatory molecule). For the first category, we noticed a time-dependent response, present in adherent macrophages, but not in circulating monocytes. Regarding the second category of cytokines, the secretion is present only for the adhered and LPS treated cells. It is also present in a time-dependent manner (a higher concentration can be noticed in the collected supernatant after 18 hours of treatment compared with the one collected after 4 hours of treatment). Conclusion: The macrophages obtained from normal human monocytes with PMA are M1 type, regardless of the concentration used for differentiation.

Glioma is the most common primary intracranial tumor and has the greatest prevalence of all brain tumors. Treatment resistance and tumor recurrence in GBM are mostly explained by considerable alterations within the tumor microenvironment, as well as extraordinary cellular and molecular heterogeneity. Soluble factors, extracellular matrix components, tissue-resident cell types, resident or newly recruited immune cells together make up the GBM microenvironment. Regardless of many immune cells, a profound state of tumor immunosuppression is supported and developed, posing a considerable hurdle to cancer cells' immune-mediated destruction. Several studies have suggested that various GBM subtypes present different modifications in their microenvironment, although the importance of the microenvironment in treatment response has yet to be determined. Understanding the microenvironment and how it changes after therapies is critical because it can influence the remaining invasive GSCs and lead to recurrence. This review article sheds light on the various components of the GBM microenvironment and their roles in tumoral development, as well as immune-related biological processes that support the interconnection/interrelationship between different cell types. Also, we summarize the current understanding of the modulation of soluble factors and highlight the dysregulated inflammatory chemokine/specific receptors cascades/networks and their significance in tumorigenesis, cancer-related inflammation, and metastasis.

Studies on the immunopharmacological activities of various plant species have provided evidence for the high therapeutic potential of different extracts. These represent a promising alternative to reduce the inflammatory processes and, thus, diseases related to inflammation. Numerous scientific studies strongly suggest that diet plays an essential role in inflammation, and that certain dietary factors can act as preventive or treatment methods to lower inflammation. In the present study, a novel lingonberry-based dietary supplement was investigated for the ability to suppress the inflammatory response in activated monocytes/macrophages. Based on cell viability/proliferation and cytotoxicity tests, concentrations between 40 and 130 µg/ml of the extracts showed a high viability/proliferation effect and no cytotoxic activity in monocyte/macrophage cells. To further investigate the anti-inflammatory potential of our novel lingonberry-based dietary supplement, we studied the effect of the extract on the inflammatory response in lipopolysaccharide (LPS)-stimulated macrophages. We found that the extract exhibited a strong anti-inflammatory potential by inhibiting the expression of major inflammatory cytokines [interleukin (IL)-6, IL-8 and tumor necrosis factor (TNF)α] in activated monocyte/macrophage cells. The expression of IL-6 and IL-8 was subsequently validated by enzyme-linked immunosorbent assay (ELISA). In conclusion, we demonstrated that our product exhibits no cytotoxicity and suppresses inflammation, and thus can be considered a natural important tool for inflammation control.

Past decades demonstrate an increasing interest in herbal remedies in the public eye, with as many as 80% of people worldwide using these remedies as healthcare products, including those for skin health. Sea buckthorn and its derived products (oil; alcoholic extracts), rich in flavonoids and essential fatty acids, are among these healthcare products. Specifically, sea buckthorn and its derivatives are reported to have antioxidant and antitumor activity in dysplastic skin cells. On the other hand, evidence suggests that the alteration of lipid metabolism is related to increased malignant behavior. Given the paradoxical involvement of lipids in health and disease, we investigated how sea-buckthorn seed oil, rich in long-chain fatty acids, modifies the proliferation of normal and dysplastic skin cells in basal conditions, as well as under ultraviolet A (UVA) radiation. Using real-time analysis of normal and dysplastic human keratinocytes, we showed that sea-buckthorn seed oil stimulated the proliferation of dysplastic cells, while it also impaired the ability of both normal and dysplastic cells to migrate over a denuded area. Furthermore, UVA exposure increased the expression of CD36/SR-B2, a long-chain fatty acid translocator that is related to the metastatic behavior of tumor cells.

In recent years, natural product's research gained momentum, fueled by technological advancement and open availability of research data. To date, sea buckthorn (Hippophae rhamnoides L. [Elaeagnaceae]) plant parts, especially berries, are well characterized and repeatedly tested for antioxidant activity and regenerative properties, in various cell types and tissues. However, fatty acids (FA) have been less investigated in term of biological effects, although, they are important bioactive components of the sea buckthorn fruit and oil. The aim of our work was to determine whether sea buckthorn seed oil is a suitable source of FA with regenerative properties on normal skin cells. Using high-performance liquid chromatography (HPLC) and liquid chromatography - mass spectrometry (LC-MS), we purified and characterized four fractions enriched in saturated (palmitic) and non-saturated (linoleic, alfa-linolenic, oleic) FA, which were tested for cytotoxicity, cytokine and growth factor production, and regenerative effect on normal keratinocytes and skin fibroblasts. Evidence is presented that the palmitic acid enriched fraction was a suitable sea buckthorn seed oil derived product with cell proliferation properties on both skin cell types.

Purpose Chronic low-grade inflammation and oxidative stress are present in most of the pathologic mechanisms underlying non-communicable diseases. Inflammation and redox biomarkers might therefore have a value in disease prognosis and therapy response. In this context, we performed a case–control study for assessing in whole blood the expression profile of inflammation and redox-related genes in elderly subjects with various comorbidities. Patients and Methods In the blood of 130 elderly subjects with various pathologies (cardiovascular disease, hypertension, dyslipidemia including hypercholesterolemia, type 2 diabetes mellitus), kept under control by polyvalent disease-specific medication, we investigated by pathway-focused qRT-PCR a panel comprising 84 inflammation-related and 84 redox-related genes. Results The study highlights a distinctive expression profile of genes critically involved in NF-κB-mediated inflammation and redox signaling in the blood of patients with cardiovascular disease, characterized by significant down-regulation of the genes NFKB2, NFKBIA, RELA, RELB, AKT1, IRF1, STAT1, CD40, LTA, TRAF2, PTGS1, ALOX12, DUOX1, DUOX2, MPO, GSR, TXNRD2, HSPA1A, MSRA, and PDLIM1. This gene expression profile defines the transcriptional status of blood leukocytes in stable disease under medication control, without discriminating between disease- and therapy-related changes. Conclusion The study brings preliminary proof on a minimally invasive strategy for monitoring disease in patients with cardiovascular pathology, from the point of view of inflammation or redox dysregulation in whole blood.

Caveolae are membrane microdomains described in many cell types involved in endocytocis, transcytosis, cell signaling, mechanotransduction, and aging. They are found at the interface with the extracellular environment and are structured by caveolin and cavin proteins. Caveolae and caveolins mediate transduction of chemical messages via signaling pathways, as well as non-chemical messages, such as stretching or shear stress. Various pathogens or signals can hijack these gates, leading to infectious, oncogenic and even caveolin-related diseases named caveolinopathies. By contrast, preclinical and clinical research have fallen behind in their attempts to hijack caveolae and caveolins for therapeutic purposes. Caveolae involvement in human disease is not yet fully explored or understood and, of all their scaffold proteins, only caveolin-1 is being considered in clinical trials as a possible biomarker of disease. This review briefly summarizes current knowledge about caveolae cell signaling and raises the hypothesis whether these microdomains could serve as hijackable "gatekeepers" or "gateways" in cell communication. Furthermore, because cell signaling is one of the most dynamic domains in translating data from basic to clinical research, we pay special attention to translation of caveolae, caveolin, and cavin research into clinical practice.

Starting from the recent identification of CD36 and CD97 as a novel marker combination of fibroblast quiescence in lung during fibrosis, we aimed to survey the literature in search for facts about the separate (or concomitant) expression of clusters of differentiation CD36 and CD97 in either tumor- or pancreatic-cancer-associated cells. Here, we provide an account of the current knowledge on the diversity of the cellular functions of CD36 and CD97 and explore their potential (common) contributions to key cellular events in oncogenesis or metastasis development. Emphasis is placed on quiescence as an underexplored mechanism and/or potential target in therapy. Furthermore, we discuss intricate signaling mechanisms and networks involving CD36 and CD97 that may regulate different subpopulations of tumor-associated cells, such as cancer-associated fibroblasts, adipocyte-associated fibroblasts, tumor-associated macrophages, or neutrophils, during aggressive pancreatic cancer. The coexistence of quiescence and activated states in cancer-associated cell subtypes during pancreatic cancer should be better documented, in different histological forms. Remodeling of the local microenvironment may also change the balance between growth and dormant state. Taking advantage of the reported data in different other tissue types, we explore the possibility to induce quiescence (similar to that observed in normal cells), as a therapeutic option to delay the currently observed clinical outcome.

Lead, a high-risk environmental pollutant and extensively used by industry, is one of the most widely outspread toxic metal today. Lead toxicity is a public health problem both for the children and for the adults. Lead does not have any useful functions in the body, instead it produces only harmful effects once it gets into the body. In this study, we investigated the toxicity of lead in an animal model of non-acute exposure. Experimental groups received treatment of aqueous solution lead acetate at different doses and time of administration. Toxic effects of lead were investigated on haematological and behaviour of treated rats. We noticed the disturbances of both haematological parameters and behaviour. Our results indicated that non-acute exposure to lead induced toxic effects in the blood, and central nervous system of adult Wistar rats.

Recently, a large spectrum of biomaterials emerged, with emphasis on various pure, blended, or doped calcium phosphates (CaPs). Although basic cytocompatibility testing protocols are referred by International Organization for Standardization (ISO) 10993 (parts 1-22), rigorous in vitro testing using cutting-edge technologies should be carried out in order to fully understand the behavior of various biomaterials (whether in bulk or low-dimensional object form) and to better gauge their outcome when implanted. In this review, current molecular techniques are assessed for the in-depth characterization of angiogenic potential, osteogenic capability, and the modulation of oxidative stress and inflammation properties of CaPs and their cation- and/or anion-substituted derivatives. Using such techniques, mechanisms of action of these compounds can be deciphered, highlighting the signaling pathway activation, cross-talk, and modulation by microRNA expression, which in turn can safely pave the road toward a better filtering of the truly functional, application-ready innovative therapeutic bioceramic-based solutions.