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Research on biomaterials typically starts with cytocompatibility evaluation, using the ISO 10993-5 standard as a reference that relies on extract tests to determine whether the material is safe (cell metabolic activity should exceed 70 %). However, the generalized approach within the standard may not accurately reflect the material's behavior in direct contact with cells, raising concerns about its effectiveness. Calcium phosphates (CaPs) are a group of materials that, despite being highly biocompatible and promoting bone formation, still exhibit inconsistencies in basic cytotoxicity evaluations. Hence, in order to test the cytocompatibility dependence on different experimental setups and material-cell interactions, we used amorphous calcium phosphate, α-tricalcium phosphate, hydroxyapatite, and octacalcium phosphate (0.1 mg/mL to 5 mg/mL) with core cell lines of bone microenvironment: mesenchymal stem cells, osteoblast-like and endothelial cells. All materials have been characterized for their physicochemical properties before and after cellular contact and once in vitro assays were finalized, groups identified as 'cytotoxic' were further analyzed using a modified Annexin V apoptosis assay to accurately determine cell death. The obtained results showed that indirect contact following ISO standards had no sensitivity of tested cells to the materials, but direct contact tests at physiological concentrations revealed decreased metabolic activity and viability. In summary, our findings offer valuable guidelines for handling biomaterials, especially in powder form, to better evaluate their biological properties and avoid false negatives commonly associated with the traditional standard approach., Competing Interests: Declaration of competing interest Authors do not have any conflicts of interest to declare., (Copyright © 2024 The Authors. Published by Elsevier B.V. All rights reserved.)

Background: Nano-hydroxyapatite (nHA) is a potential ideal biomaterial for bone regeneration. However, studies have yet to characterize the behavior of human osteoblasts derived from alveolar bone on nHA. Thus, the aim of the present study was to evaluate the influence of nHA on the adhesion, proliferation and differentiation of these alveolar bone-derived cells., Methods: Primary human alveolar osteoblasts were collected from the alveolar ridge of a male periodontal patient during osseous resective surgery and grown on culture plates coated with either polylysine or polylysine with nano-hydroxyapatite (POL/nHA) composite. The cells were grown and observed for 14 days, and then assessed for potential modifications to osteoblasts homeostasis as evaluated by quantitative reverse transcriptase-polymerase chain reaction (real time RT-PCR), scanning electron microscopy and atomic force microscopy., Results: Real time PCR revealed a significant increase in the expression of the selected markers of osteoblast differentiation (bone morphogenetic protein (BMP)-2,-5,-7, ALP, COLL-1A2, OC, ON) in cells grown on the POL/nHA substrate. In addition, as compared with the POL surface, cells grown on the POL/nHA substrate demonstrated better osteoconductive properties, as demonstrated by the increase in adhesion and spreading, likely as a result of the increased surface roughness of the composite., Conclusions: The increased expression of BMPs and osteoinductive biomarkers suggest that nano-hydroxyapatite may stimulate the proliferation and differentiation of local alveolar osteoblasts and thus encourage bone regeneration at sites of alveolar bone regeneration.

Purpose: No clinical evidence-based data on a possible negative influence of osteopenia and harmful lifestyles on implant osseointegration have been published to date. However, chronic alcohol abuse has been related to a progressive osteoporotic state. The continuous effort on developing better performing surfaces might offer improvement in osseointegration rates in conditions of poor bone quality. The aim of the present study was to investigate, in vitro, the behavior of osteoblasts obtained from rats exposed to ethanol vapor for 7 weeks and cultured on an innovative titanium surface achieved by a double anodic spark deposition., Materials and Methods: Ethanol-exposed and unexposed (control) osteoblasts were cultured on an anodic spark-deposited titanium surface and compared to those cultured on an acid-etched one. Cell proliferation and synthesis of alkaline phosphatase, osteocalcin, collagen I, transforming growth factor-β1, interleukin-6, and tumor necrosis factor-α were measured at 3, 7, and 14 days of culture., Results: When cultured on the anodic spark-deposited titanium surface, ethanol-exposed osteoblasts recovered proliferation impairment. Anodic spark-deposited titanium surfaces significantly improved both ethanol-exposed and unexposed osteoblast bioactivity, particularly with regard to alkaline phosphatase and transforming growth factor-β1. Moreover, interleukin-6 release for ethanol-exposed osteoblasts was significantly reduced by the anodic spark-deposited titanium surface., Conclusion: Proliferative and synthetic cell processes altered by ethanol contact were positively and partially restored by contact with the tested innovative surface.

Titanium and its alloys are the current materials to manufacture oral implants because of their excellent mechanical properties and biocompatibility. However the increasing needs of the patients to receive fast and reliable rehabilitation have forced materials scientists to modified the surface of the materials in order to increase the rate of osseointegration and minimize the times for healing. The presence of a reconstituted extracellular matrix (ECM), constituted of proteins and polysaccharides is a key factor for healing and regeneration of the tissues. The nano-fibrous feature of ECM improves cells proliferation and addresses their phenotype. Electrospinning technique is an efficient processing method to manufacture micro- and nano-sized fibrous structures mimicking the ECM. In this work we describe a method to obtain collagen coating made of nano-fibers onto titanium for oral implant manufacturing, using electrospinning. The obtained collagen coatings showed morphology, structure and chemical composition similar to that of ECM. Moreover the stem cells cultured onto titanium samples coated with electrospun collagen showed faster osteoblastic differentiation and more efficient deposition of mineralized matrix in comparison with those onto uncoated substrates. This effect was amplified using osteogenetic media.

In bone tissue reconstruction, the use of engineered constructs created by mesenchymal stem cells (MSCs) that differentiate and proliferate into three-dimensional porous scaffolds is an appealing alternative to autologous and heterologous bone grafts. Scaffolds considered in this work are represented by polyurethane (PU) foams. Two PU foams (EC-1 and EC-2) were synthesized and characterized for morphology, mechanical properties and in vitro interaction with the osteoblast-like cell line MG63 and MSCs from human bone marrow. EC-1 and EC-2 showed similar densities (0.20 g cm(-3)) with different morphologies: EC-1 showed a more homogeneous pore size (average Phi = 691 microm) and distribution, with a 35% open porosity, whereas EC-2 evidenced a wide range of pore dimension, with an average pore size of 955 microm and a 74% open porosity. The compressive properties of the two foams were similar in the dry condition and both showed a strong decrease in the wet condition. In vitro tests showed good MG63 cell proliferation, as confirmed by the results of the MTT assay and scanning electron microscopy (SEM) observations, with a higher cell viability on EC-2 foam 7 days post-seeding. In the experiments with MSCs, SEM observations showed the presence of an inorganic phase deposition starting day 7 onto EC-1, day 14 onto EC-2. The inorganic particles (CaP) deposition was much more evident onto the pore surface of both foams at day 30, indicating good differentiation of MSCs into osteoblasts. Both PU foams therefore appeared to stimulate cell adhesion and proliferation in vitro, sustaining the MSCs' growth and differentiation into osteoblasts.

Chronic alcohol consumption is associated with pathological effects on bone, and it is correlated with the increasing risk of osteoporosis and fractures. The negative effects of alcohol intake also influence bone repair processes and the osseointegration of implants. The aim of the present in vitro study was to investigate the proliferation and synthetic activity of osteoblasts isolated from the trabecular bone of rats previously exposed to 7-week intermittent exposure to ethanol vapour (EE-OB), and sham-aged rats (SA-OB), when cultured on standard commercially pure Ti (cpTi). Osteoblast proliferation (WST-1), alkaline phosphatase (ALP), osteocalcin (OC), collagen type I (CICP), tumor necrosis factor-alpha (TNF-alpha), interleukin-6 (IL-6), and transforming growth factor-beta1 (TGF-beta1) were measured at 1, 7, and 14 days of culture. Our results showed a decrease in the cell viability and synthetic activity of osteoblasts exposed to ethanol when cultured on cpTi. Moreover, the release of local regulatory factors from osteoblasts was imbalanced: TGF-beta1 production was reduced and TNF-alpha and IL-6 were up-regulated. These in vitro data suggest that alcohol abuse affects bone repair and decreases the ability to form bone around standard cpTi. Innovative surfaces and adjuvant therapies could be useful when implants are required in alcoholics.

With rising rates of alcohol consumption acute and chronic damage from alcohol is expected to increase all over the world. Habitual excessive alcohol consumption is associated with pathological effects on bone. The aim of the present in vitro study was to investigate comparatively the proliferation and synthetic activity of osteoblasts (OB) isolated from the trabecular bone of rats previously exposed to 7-week intermittent exposure to ethanol vapor, sham-aged rats and long-term estrogen deficient rats. Cell proliferation (WST1) and synthesis of alkaline phosphatase (ALP), osteocalcin (OC), collagen I (CICP), transforming growth factor beta1 (TGF-beta1), interleukin-6 (IL-6), tumor necrosis factor alfa (TNFalpha) were measured at 3, 7 and 14 days of culture. Osteoblast proliferation rate and TGF-beta1, IL-6 and TNFalpha syntheses were significantly affected by alcohol exposure. Estrogen deficiency and alcohol consumption share many common pathophysiological mechanisms of damage to bone, but alcohol affects OB proliferation and TNFalpha synthesis significantly more than menopause does. Therefore, these in vitro data suggest that alcohol has even more deleterious effects on bone than estrogen deficiency does.

The aim of this study was to characterize and compare various titanium (Ti) and hydroxyapatite (HA) coatings on Ti6Al4V, in view of their application on noncemented orthopedic implants. Two innovative vacuum plasma sprayed (VPS) coatings, the first of ultrahigh rough and dense Ti (PG60, Ra=74 microm) and the second of ultrahigh rough and dense Ti coated with HA (HPG60, Ra=52 microm), have been developed, and the response of osteoblast-like cells (MG-63) seeded on these new coatings was evaluated in comparison to: a low roughness and sandblasted (Ti/SA, Ra=4 microm) Ti6Al4V surface; Ti medium (TI01, Ra=18 microm), and high (TI60, Ra=40 microm) roughness VPS coatings; and the relative Ti plus HA duplex coatings (HT01, Ra=12 microm and HT60, Ra=36 microm respectively), also obtained by VPS. PG60 coating presented no open porosity, making it dense and potentially intrinsically stronger. Cell adhesion and proliferation on PG60 was similar to those of the smoothest one (Ti/SA) and adhesion on ultrahigh roughness was lower than the medium- and high-roughness coatings, whereas cell proliferation on PG60 was lower than TI60. The HA coating determined significant increases in cell proliferation at medium and high roughness levels when compared to the relative Ti coating, but not compared to the ultrahigh one; all HA-coated surfaces showed a decrease in alkaline phosphatase activity and collagen I production. Surface morphology and the HA coating strongly affected cell behavior. However, ultrahigh values of roughness are not correctly seen by cells, and the presence of HA has no improving effects., (Copyright (c) 2005 Wiley Periodicals, Inc.)

Over the past decade extracorporeal shock-wave therapy (ESWT) has been increasingly applied to orthopaedic and musculoskeletal pathologies, the aim of this study was to assess how the energy density of the shock waves and the number of impulses affect viability, differentiation and synthetic activity of osteoblasts. Primary sheep osteoblasts cultures were treated with ESWT with an electro-hydraulic shock wave generator by selecting three different energy levels (14-21-28 kV corresponding at 0.15-0.31-0.40 mJ/mm2) and two different total numbers of impulses (500, 1000) for each level. At the end of treatment, cell counts and viability were recorded. Cells were then cultivated for 48 hours starting from a concentration of 1 x 10(4) cells/ml. The biological activity and viability were evaluated at 24 and 48 hours after treatment. No cytodestructive effects were observed in Group A, while a cytodestructive effect of ESWT was seen in cultures receiving the highest energy treatments. The different shock wave treatment induced differences in MTT assays after 24 and 48 hours, in particular the highest level showed a detrimental effect on cell respiration at both experimental times as compared to the Control Group and the protein metabolism was generally depressed by ESWT with impulses at the highest energy level. After 24 hours such effect further increased with the growing number of impulses. The lowest energy level appeared to significantly improve the metabolic parameter in primary cell cultures as compared to controls when 500 impulses were selected. The current study has demonstrated that one of the most important aspects to be considered is not the total number of impulses used but the energy level of the shock waves, thus confirming that ESWT has a dose-dependent effect on cells.

Metal-Organic Chemical Vapor Deposition (MOCVD) has recently been proposed to coat orthopedic and dental prostheses with metal nanostructured oxide films through the decomposition of oxygenated compounds (single-source precursors) or the reaction of oxygen-free metal compounds with oxygenating agents. The present study was carried out to assess the in vitro biocompatibility in terms of cell proliferation and activation, of commercially pure Ti (control material: TI/MA) coated with nanostructured TiO2 film by MOCVD (Ti/MOCVD) using osteoblast-like cell cultures (MG-63). Evaluations were performed at 3, 7 and 14 days. Cell proliferation showed a similar trend for Ti/MA and TilMOCVD compared to polystyrene; cell number increased with time from seeding to day 7 (p < 0.005), and then decreased progressively until day 14 (ranging from -14% to -47%). The ALP level and OC production showed no significant differences between Ti/MOCVD and Ti/MA at each experimental time. Significantly higher ALP levels were found in Ti/MA at 3 days and in Ti/MOCVD at 7 and 14 days when compared to the polystyrene group. OC production decreased over time and the highest values were observed at 3 days, when it was significantly higher in the Ti/MA than in the polystyrene group (50%, p < 0.05). CICP synthesis was positively affected by the presence of Ti/MOCVD and was higher in Ti/MOCVD than in the polystyrene group. No significant differences were found between Ti/MOCVD and Ti/MA in terms of IL-6 and TGF-beta1 synthesis at any experimental time. In conclusion, the current findings demonstrate that the nanostructured TiO2 coating positively affects the osteoblast-like cell behavior in terms of cell proliferation and activity, thus confirming its high level of in vitro biocompatibility in accordance with expectations.

The effects of two surfaces with different roughness (Low Roughness, LR: Ra: 5.6-5.9 microm; High Roughness, HR: Ra: 21.5-22.5 microm), uncoated and fluorohydroxyapatite(FHA)-coated, were investigated in MG-63 osteoblasts. At 72 hours, cells proliferated on biomaterials more slowly than in the control group (p < 0.0001), the proliferation rate was higher on FHA-coated LR than uncoated HR (p = 0.037). Collagen-I production was positively affected by the LR surface (p = 0.001) as compared to controls, while it was significantly lower (p = 0.0001) in the HR surfaces. Compared to controls, LR and HR surfaces led to enhanced production of TGF-beta1, further improved by FHA (FHA-coated LR: p = 0.007; FHA-coated HR p < 0.0001 respectively). ALP, OC, IL-6 and TNF-alpha levels were not significantly different from the controls. Results suggest that collagen-I production could be useful in predicting the in vivo osteointegration rate of biocompatible biomaterials observed in previous studies.