Guiding bone formation using semi-onlay calcium phosphate implants in an ovine calvarial model.

The restoration of cranio-maxillofacial deformities often requires complex reconstructive surgery in a challenging anatomical region, with abnormal soft tissue structures and bony deficits. In this proof-of-concept, the possibility of vertical bone augmentation was explored by suspending hemispherically shaped titanium-reinforced porous calcium phosphate (CaP) implants (n = 12) over the frontal bone in a sheep model (n = 6). The animals were euthanized after week 13 and the specimens were subject to micro-computed tomography (µCT) and comprehensive histological analysis. Histology showed that the space between implant and the recipient bone was filled with a higher percentage of newly formed bone (NFB) versus soft tissue with a median of 53% and 47%, respectively. Similar results were obtained from the µ-CT analysis, with a median of 56% NFB and 44% soft tissue filling the void. Noteworthy, significantly higher bone-implant contact was found for the CaP (78%, range 14%-94%) versus the Titanium (29%, range 0%-75%) portion of the implant exposed to the surrounding bone. The histological analysis indicates that the CaP replacement by bone is driven by macrophages over time, emphasized by material-filled macrophages found in close vicinity to the CaP with only a small number of single osteoclasts found actively remodeling the NFB. This study shows that CaP based implants can be assembled with the help of additive manufacturing to guide vertical bone formation without decortification or administration of growth factors. Furthermore, it highlights the potential disadvantage of a seamless fit between the implant and the recipient's bone.

Targeted ToF-SIMS Analysis of Macrophage Content from a Human Cranial Triphasic Calcium Phosphate Implant.

Macrophages play a key role in determining the fate of implanted biomaterials, especially for biomaterials such as calcium phosphates (CaPs) where these cells play a vital role in material resorption and osteogenesis, as shown in different models, including clinical samples. Although substantial consideration is given to the design and validation of different CaPs, relatively little is known about their material-cell interaction. Specifically, the intracellular content of different CaP phases remains to be assessed, even though CaP-filled macrophages have been observed in several studies. In this study, 2D/3D ToF-SIMS imaging and multivariate analysis were directly applied on the histology samples of an explant to reveal the content of macrophages. The cellular content of the macrophages was analyzed to distinguish three CaP phases, monetite, beta-tricalcium phosphate, and pyrophosphate, which are all part of the monetite-based CaP implant composition under study. ToF-SIMS combined with histology revealed that the content of the identified macrophages was most similar to that of the pyrophosphate phase. This study is the first to uncover distinct CaP phases in macrophages from a human multiphasic CaP explant by targeted direct cell content analysis. The uncovering of pyrophosphate as the main phase found inside the macrophages is of great importance to understand the impact of the selected material in the process of biomaterial-instructed osteogenesis.

In vitro biological responses to nanofibrillated cellulose by human dermal, lung and immune cells: surface chemistry aspect.

BACKGROUND: Nanocellulose, and particularly nanofibrillated cellulose (NFC), has been proposed for a diversity of applications in industry and in the biomedical field. Its unique physicochemical and structural features distinguish nanocellulose from traditional materials and enable its use as an advance nanomaterial. However, its nanoscale features may induce unknown biological responses. Limited studies with NFC are available and the biological impacts of its use have not been thoroughly explored. This study assesses the in vitro biological responses elicited by wood-derived NFC gels, when human dermal fibroblasts, lung MRC-5 cells and THP-1 macrophage cells are exposed to the nanomaterial. Furthermore, whether the presence of surface charged groups (i.e. carboxymethyl and hydroxypropyltrimethylammonium groups) on NFC can induce distinct biological responses is investigated. RESULTS: The introduction of surface charged groups resulted in individual nanofibrils, while fibril aggregates predominated in the unmodified NFC gel suspensions as observed by transmission electron microscopy. In the presence of proteins, the surface modified NFCs formed compact agglomerates while the agglomeration pattern of the unmodified NFC was similar in the presence of proteins and in physiological buffer. Unmodified and modified NFC gels did not induce cytotoxicity in human dermal fibroblasts, lung and macrophage cells. No significant ROS production by THP-1 macrophages was found and no cellular uptake was observed. However, an inflammatory response was detected when THP-1 macrophages were treated with unmodified NFC as assessed by an increase in TNF-α and IL1-ß levels, an effect that was absent when surface charged groups were introduced into NFC. CONCLUSIONS: Taken together, the data presented here show the absence of cytotoxic effects associated with the exposure to unmodified, carboxymethylated and hydroxypropyltrimethylammonium-modified NFCs. Unmodified NFC presented a pro-inflammatory effect which can be further moderated by introducing surface modifications such as carboxymethyl and hydroxypropyltrimethylammonium groups into the nanofibrils. The present findings suggest that the inflammatory response to NFC might be driven by the material surface chemistry, and thus open up for the possibility of designing safe nanocellulose materials.

Dose-dependent autophagic effect of titanium dioxide nanoparticles in human HaCaT cells at non-cytotoxic levels.

BACKGROUND: Interactions between nanoparticles and cells are now the focus of a fast-growing area of research. Though many nanoparticles interact with cells without any acute toxic responses, metal oxide nanoparticles including those composed of titanium dioxide (TiO2-NPs) may disrupt the intracellular process of macroautophagy. Autophagy plays a key role in human health and disease, particularly in cancer and neurodegenerative diseases. We herein investigated the in vitro biological effects of TiO2-NPs (18 nm) on autophagy in human keratinocytes (HaCaT) cells at non-cytotoxic levels. RESULTS: TiO2-NPs were characterized by transmission electron microscopy (TEM) and dynamic light scattering techniques. Cellular uptake, as evaluated by TEM and NanoSIMS revealed that NPs internalization led to the formation of autophagosomes. TiO2-NPs treatment did not reduce cell viability of HaCaT cells nor increased oxidative stress. Cellular autophagy was additionally evaluated by confocal microscopy using eGFP-LC3 keratinocytes, western blotting of autophagy marker LC3I/II, immunodetection of p62 and NBR1 proteins, and gene expression of LC3II, p62, NBR1, beclin1 and ATG5 by RT-qPCR. We also confirmed the formation and accumulation of autophagosomes in NPs treated cells with LC3-II upregulation. Based on the lack of degradation of p62 and NBR1 proteins, autophagosomes accumulation at a high dose (25.0 µg/ml) is due to blockage while a low dose (0.16 µg/ml) promoted autophagy. Cellular viability was not affected in either case. CONCLUSIONS: The uptake of TiO2-NPs led to a dose-dependent increase in autophagic effect under non-cytotoxic conditions. Our results suggest dose-dependent autophagic effect over time as a cellular response to TiO2-NPs. Most importantly, these findings suggest that simple toxicity data are not enough to understand the full impact of TiO2-NPs and their effects on cellular pathways or function.

Morphological, toxicological and molecular characterization of a benthic Nodularia isolated from Atlantic estuarine environments.

A polyphasic study of a benthic Nodularia isolate (LEGE06071) from an Atlantic environment, specifically salt pans, was performed. LEGE06071 resembled both type strains of Nodularia sphaerocarpa and Nodularia harveyana, while ACOI00729 (purchased isolate) was identified as N. sphaerocarpa. The length and width of vegetative cells varied from 3.10 to 3.15 microm and from 3.71 to 4.25 microm, respectively, while heterocyts were 3.91-4.89 microm long and 4.20-4.74 microm wide. None of the isolates had aerotopes. The sequencing of the 16S rRNA gene from the two Nodularia isolates indicated that they belonged neither to Nodularia spumigena nor N. harveyana. Nodularin and other cyanotoxin synthesis-associated genes could not be detected, nor could nodularin production be detected by ELISA. However, MALDI-TOF analysis of LEGE06071 revealed the presence of other compounds, namely, glycolipids. Hence, toxicological screenings against Artemia nauplii, Escherichia coli and Salmonella typhimurium were performed. Toxic effects could only be observed against Artemia, with 48 h-LC(50) values for the aqueous and crude extract of methanol of 53.21 mg ml(-1) and 17.81 mg ml(-1), respectively. This study presents the first evidence of a non-nodularin-producing Nodularia isolate in Atlantic salt pan ecosystems and its potential ecotoxicity against Artemia sp.