Effect of DMPEI coating against biofilm formation on PVC catheter surface.

Urinary tract infections (UTIs) are a significant cause of morbidity in healthcare systems and are prominently associated with applying urethral catheters, particularly in surgeries. Polyvinyl chloride (PVC) is extensively utilized in the fabrication of catheters. Biofilms, complex polymeric constructions, provide a protective milieu for cell multiplication and the enhancement of antibiotic resistance. Strategies to counteract biofilm development on medical apparatuses' surfaces incorporate antimicrobial agents such as N,N-dodecyl, and methyl polyethylenimine (DMPEI). This research endeavored to characterize the morphology of PVC and PVC-DMPEI surfaces utilizing Scanning Electron Microscopy (SEM) and Atomic Force Microscopy (AFM) and to gauge hydrophobicity through contact angle measurements. Employing Escherichia coli, Staphylococcus aureus, and Candida albicans in adhesion assays enabled the assessment of DMPEI's efficacy in preventing microbial adherence to PVC. Butanol successfully solubilized 2 mg.mL-1 DMPEI without altering the PVC structure. SEM results substantiated the formation of a DMPEI layer on the PVC surface, which led to decreased surface roughness, as validated by AFM, and increased hydrophilicity, as demonstrated by contact angle evaluations. E. coli, S. aureus, and C. albicans exhibited significant adhesion reduction, 89.3%, 94.3%, and 86.6% on PVC-DMPEI surfaces. SEM visualizations confirmed reduced cellular colonization on PVC-DMPEI and highlighted considerable morphological modifications in E. coli. Consequently, DMPEI films effectively minimize the adhesion of E. coli, S. aureus, and C. albicans on PVC surfaces. DMPEI, with its potential as a protective coating for innovative medical devices, promises to inhibit biofilm adherence effectively.

Titanium dioxide nanoparticles affect osteoblast-derived exosome cargos and impair osteogenic differentiation of human mesenchymal stem cells.

Titanium (Ti) and its alloys are the most widely used metallic biomaterials in total joint replacement; however, increasing evidence supports the degradation of its surface due to corrosion and wear processes releasing debris (ions, and micro and nanoparticles) and contribute to particle-induced osteolysis and implant loosening. Cell-to-cell communication involving several cell types is one of the major biological processes occurring during bone healing and regeneration at the implant-bone interface. In addition to the internal response of cells to the uptake and intracellular localization of wear debris, a red flag is the ability of titanium dioxide nanoparticles (mimicking wear debris) to alter cellular communication with the tissue background, disturbing the balance between osseous tissue integrity and bone regenerative processes. This study aims to understand whether titanium dioxide nanoparticles (TiO2 NPs) alter osteoblast-derived exosome (Exo) biogenesis and whether exosomal protein cargos affect the communication of osteoblasts with human mesenchymal stem/stromal cells (HMSCs). Osteoblasts are derived from mesenchymal stem cells coexisting in the bone microenvironment during development and remodelling. We observed that TiO2 NPs stimulate immature osteoblast- and mature osteoblast-derived Exo secretion that present a distinct proteomic cargo. Functional tests confirmed that Exos derived from both osteoblasts decrease the osteogenic differentiation of HMSCs. These findings are clinically relevant since wear debris alter extracellular communication in the bone periprosthetic niche, contributing to particle-induced osteolysis and consequent prosthetic joint failure.

Large-Scale, Automated Production of Adipose-Derived Stem Cell Spheroids for 3D Bioprinting.

Adipose-derived stromal/stem cells (ASCs) are a subpopulation of cells found in the stromal vascular fraction of human subcutaneous adipose tissue recognized as a classical source of mesenchymal stromal/stem cells. Many studies have been published with ASCs for scaffold-based tissue engineering approaches, which mainly explored the behavior of these cells after their seeding on bioactive scaffolds. However, scaffold-free approaches are emerging to engineer tissues in vitro and in vivo, mainly by using spheroids, to overcome the limitations of scaffold-based approaches. Spheroids are 3D microtissues formed by the self-assembly process. They can better mimic the architecture and microenvironment of native tissues, mainly due to the magnification of cell-to-cell and cell-to-extracellular matrix interactions. Recently, spheroids are mainly being explored as disease models, drug screening studies, and building blocks for 3D bioprinting. However, for 3D bioprinting approaches, numerous spheroids, homogeneous in size and shape, are necessary to biofabricate complex tissue and organ models. In addition, when spheroids are produced automatically, there is little chance for microbiological contamination, increasing the reproducibility of the method. The large-scale production of spheroids is considered the first mandatory step for developing a biofabrication line, which continues in the 3D bioprinting process and finishes in the full maturation of the tissue construct in bioreactors. However, the number of studies that explored the large-scale ASC spheroid production are still scarce, together with the number of studies that used ASC spheroids as building blocks for 3D bioprinting. Therefore, this article aims to show the large-scale production of ASC spheroids using a non-adhesive micromolded hydrogel technique spreading ASC spheroids as building blocks for 3D bioprinting approaches.

Nanomaterial Exposure, Extracellular Vesicle Biogenesis and Adverse Cellular Outcomes: A Scoping Review.

The progressively increasing use of nanomaterials (NMs) has awakened issues related to nanosafety and its potential toxic effects on human health. Emerging studies suggest that NMs alter cell communication by reshaping and altering the secretion of extracellular vesicles (EVs), leading to dysfunction in recipient cells. However, there is limited understanding of how the physicochemical characteristics of NMs alter the EV content and their consequent physiological functions. Therefore, this review explored the relevance of EVs in the nanotoxicology field. The current state of the art on how EVs are modulated by NM exposure and the possible regulation and modulation of signaling pathways and physiological responses were assessed in detail. This review followed the manual for reviewers produced by The Joanna Brigs Institute for Scoping Reviews and the PRISMA extension for Scoping Reviews (PRISMA-ScR): checklist and explanation. The research question, "Do NMs modulate cellular responses mediated by EVs?" was analyzed following the PECO model (P (Population) = EVs, E (Exposure) = NMs, C (Comparator) = EVs without exposure to NMs, O (Outcome) = Cellular responses/change in EVs) to help methodologically assess the association between exposure and outcome. For each theme in the PECO acronym, keywords were defined, organized, and researched in PubMed, Science Direct, Scopus, Web of Science, EMBASE, and Cochrane databases, up to 30 September 2021. In vitro, in vivo, ex vivo, and clinical studies that analyzed the effect of NMs on EV biogenesis, cargo, and cellular responses were included in the analysis. The methodological quality assessment was conducted using the ToxRTool, ARRIVE guideline, Newcastle Ottawa and the EV-TRACK platform. The search in the referred databases identified 2944 articles. After applying the eligibility criteria and two-step screening, 18 articles were included in the final review. We observed that depending on the concentration and physicochemical characteristics, specific NMs promote a significant increase in EV secretion as well as changes in their cargo, especially regarding the expression of proteins and miRNAs, which, in turn, were involved in biological processes that included cell communication, angiogenesis, and activation of the immune response, etc. Although further studies are necessary, this work suggests that molecular investigations on EVs induced by NM exposure may become a potential tool for toxicological studies since they are widely accessible biomarkers that may form a bridge between NM exposure and the cellular response and pathological outcome.

Impact of DMPEI on Biofilm Adhesion on Latex Urinary Catheter.

BACKGROUND: Microorganisms can migrate from the external environment to the patient's organism through the insertion of catheters. Despite being indispensable medical device, the catheter surface can be colonized by microorganisms and become a starting point for biofilm formation. Therefore, new technologies are being developed in order to modify surfaces to prevent the adhesion and survival of microorganisms. Patents with the use of DMPEI have been filed. OBJECTIVE: In the present work, we coated latex catheter surfaces with 2 mg mL-1 DMPEI in different solvents, evaluated the wettability of the surface and the anti- biofilm activity of the coated catheter against Escherichia coli, Staphylococcus aureus, and Candida albicans. METHODS: We coated the inner and outer catheter surfaces with 2 mg mL-1 of DMPEI solubilized in butanol, dimethylformamide, and cyclohexanone and the surfaces were analyzed visually. Contact angle measurement allowed the analysis of the wettability of the surfaces. The CFU mL-1 count evaluated E. coli, S. aureus, and C. albicans adhesion onto the control and treated surfaces. RESULTS: The contact angle decreased from 50.48º to 46.93º on the inner surface and from 55.83º to 50.91º on the outer surface of latex catheters coated with DMPEI. The catheter coated with DMPEI showed anti-biofilm activity of 83%, 88%, and 93% on the inner surface and 100%, 92%, and 86% on the outer surface for E. coli, S. aureus, and C. albicans, respectively. CONCLUSION: Latex catheter coated with DMPEI efficiently impaired the biofilm formation both on the outer and inner surfaces, showing a potential antimicrobial activity along with a high anti-biofilm activity for medical devices.

In Vitro and In Vivo Evaluation of Nanostructured Biphasic Calcium Phosphate in Granules and Putty Configurations.

Synthetic biphasic calcium phosphate (BCP) granules and powder are biocompatible biomaterials with a well-known capacity for osteoconduction, presenting very satisfactory clinical and histological results. It remains unanswered if the putty configuration impacts the biological response to the material. In this study, we aimed to compare the cytocompatibility and biocompatibility of nanostructured BCP in the putty configuration (moldable nanostructured calcium phosphate, MnCaP) on the healing of critical-sized bone defects (8 mm) in rat calvaria. Cytocompatibility was determined through the viability of fibroblast cells (V-79) to the extracts of different concentrations of MnCaP. Forty-five Wistar rats were randomly divided into three groups (n = 15)-clot, MnCaP, and commercial biphasic calcium phosphate in granules configurations (Nanosynt®)-and subdivided into three experimental periods (1, 3, and 6 months). Histological, histomorphometric, and microtomographic analyses allowed the evaluation of newly formed bone, residual biomaterial, and connective tissue. The in vitro evaluation showed that MnCaP was cytocompatible. The histomorphometric results showed that the Nanosynt® group granted the highest new-formed bone values at six months (p < 0.05), although the biomaterial volume did not differ between groups. The putty configuration was easier to handle, and both configurations were biocompatible and osteoconductive, presented similar biosorption rates, and preserved the calvaria architecture.

A Scaffold- and Serum-Free Method to Mimic Human Stable Cartilage Validated by Secretome.

A stabilized cartilage construct without signs of hypertrophy in chondrocytes is still a challenge. Suspensions of adipose stem/stromal cells (ASCs) and cartilage progenitor cells (CPCs) were seeded into micromolded nonadhesive hydrogel to produce spheroids (scaffold- and serum-free method) characterized by size, immunohistochemistry, fusion, and biomechanical properties. After cell dissociation, they were characterized for mesenchymal cell surface markers, cell viability, and quantitative real-time polymerase chain reaction. Both targeted and nontargeted (shotgun mass spectrometry) analyses were conducted on the culture supernatants. Induced ASC spheroids (ø = 350 µm) showed high cell viability and CD73 downregulation contrasting to CD90. The transforming growth factor (TGF)-ß3/TGF-ß1 ratio and SOX9 increased (p < 0.05), whereas interleukin (IL)-6, IL-8, RUNX2, and ALPL decreased. Induced ASC spheroids were able to completely fuse and showed a higher force required to compression at day 14 (p < 0.0001). Strong collagen type II in situ was associated with gradual decrease of collagen type X and a lower COLXA1 gene expression at day 14 compared with day 7 (p = 0.0352). The comparison of the secretome content of induced and non-induced ASCs and CPCs identified 138 proteins directly relevant to chondrogenesis of 704 proteins in total. Although collagen X was absent, thrombospondin-1 (TSP-1), described as antiangiogenic and antihypertrophic, and cartilage oligomeric matrix protein (COMP), a biomarker of chondrogenesis, were upregulated in induced ASC spheroids. Our scaffold- and serum-free method mimics stable cartilage acting as a tool for biomarker discovery and for regenerative medicine protocols. Impact Statement Promising adult stem cell sources for cartilage regeneration include adipose stem/stromal cells (ASCs) from subcutaneous adipose tissue. Our main objective was the development of a reproducible and easy-to-handle scaffold- and serum-free method to obtain stable cartilage from induced ASC spheroids. In addition to targeted protein profiling and biomechanical analysis, we provide the first characterization of the secretome composition for ASC spheroids, providing a useful tool to monitor in vitro chondrogenesis and a noninvasive quality control of tissue-engineered constructs. Furthermore, our secretome analysis revealed a potential novel biomarker-thrombospondin-1 (TSP-1), known by its antiangiogenic properties and recently described as an antihypertrophic protein.

The role of apoptosis associated speck-like protein containing a caspase-1 recruitment domain (ASC) in response to bone substitutes.

The apoptosis-associated Speck-like protein containing a caspase-1 recruitment domain (ASC), present in inflammasomes, regulates inflammation events and is involved in osteogenic phenotype. Nevertheless, its function in bone repair induced by bone substitute biomaterials is unclear. This study aimed to unveil the role of ASC on osteoprogenitor and tissue response to stoichiometric-hydroxyapatite (HA), nanostructured carbonated-hydroxyapatite (CHA), and CHA containing 5% Strontium (SrCHA), characterized previously by XRD, uXRF-SR, and FTIR spectroscopy implants. Thereafter, conditioned media by the biomaterials were used later to treat pre-osteoblasts and an osteogenic stimulus was shown in response to the materials, with higher expression of Runx2, Osterix, ALP, and Collagen 1a1 genes, with significant involvement of inflammatory-related genes. Thus, to better address the involvement of inflammasome, primary cells obtained from both genotypes [Wild-Type (WT) and ASC Knockout (ASC-KO) mice] were subjected to conditioned media up to 7 days, and our data reinforces both HA and CHA induces lower levels of alkaline phosphatase (ALP) than SrCHA, considering both genotypes (p < 0.01), and ASC seems contribute with osteogenic stimulus promoted by SrCHA. Complimentarily, the biomaterials were implanted into both subcutaneous and bone defects in tibia. Histological analysis on 28 days after implantation of biomaterials into mice's subcutaneous tissue revealed moderate inflammatory response to them. Both histomorphometry and µCT analysis of tibias indicated that the biomaterials did not reverse the delay in bone repair of ASC KO, reinforcing the involvement of ASC on bone regeneration and bone de novo deposition. Also, the bone density in CHA was >2-fold higher in WT than ASC-KO samples. HA was virtually not resorbed throughout the experimental periods, in opposition to CHA in the WT group. CHA reduced to half-area after 28 days, and the bone deposition was higher in CHA for WT mice than HA. Taken together, our results show that biomaterials did not interfere with the healing pattern of the ASC KO, but CHA promoted higher bone deposition in the WT group, probably due to its greater biodegradability. These results reinforce the importance of ASC during bone de novo deposition and healing.

In Vitro Evaluation of Neutral Aryloximes as Reactivators for Electrophorus eel Acetylcholinesterase Inhibited by Paraoxon.

Casualties caused by organophosphorus pesticides are a burden for health systems in developing and poor countries. Such compounds are potent acetylcholinesterase irreversible inhibitors, and share the toxic profile with nerve agents. Pyridinium oximes are the only clinically available antidotes against poisoning by these substances, but their poor penetration into the blood-brain barrier hampers the efficient enzyme reactivation at the central nervous system. In searching for structural factors that may be explored in future SAR studies, we evaluated neutral aryloximes as reactivators for paraoxon-inhibited Electrophorus eel acetylcholinesterase. Our findings may result into lead compounds, useful for development of more active compounds for emergencies and supportive care.

In vitro and in vivo evaluations of nanocrystalline Zn-doped carbonated hydroxyapatite/alginate microspheres: zinc and calcium bioavailability and bone regeneration.

Background: Zinc-doped hydroxyapatite has been proposed as a graft biomaterial for bone regeneration. However, the effect of zinc on osteoconductivity is still controversial, since the release and resorption of calcium, phosphorus, and zinc in graft-implanted defects have rarely been studied. Methods: Microspheres containing alginate and either non-doped carbonated hydroxyapatite (cHA) or nanocrystalline 3.2 wt% zinc-doped cHA (Zn-cHA) were implanted in critical-sized calvarial defects in Wistar rats for 1, 3, and 6 months. Histological and histomorphometric analyses were performed to evaluate the volume density of newly formed bone, residual biomaterial, and connective tissue formation. Biomaterial degradation was characterized by transmission electron microscopy (TEM) and synchrotron radiation-based X-ray microfluorescence (SR-µXRF), which enabled the elemental mapping of calcium, phosphorus, and zinc on the microsphere-implanted defects at 6 months post-implantation. Results: The bone repair was limited to regions close to the preexistent bone, whereas connective tissue occupied the major part of the defect. Moreover, no significant difference in the amount of new bone formed was found between the two microsphere groups. TEM analysis revealed the degradation of the outer microsphere surface with detachment of the nanoparticle aggregates. According to SR-µXRF, both types of microspheres released high amounts of calcium, phosphorus, and zinc, distributed throughout the defective region. The cHA microsphere surface strongly adsorbed the zinc from organic constituents of the biological fluid, and phosphorus was resorbed more quickly than calcium. In the Zn-cHA group, zinc and calcium had similar release profiles, indicating a stoichiometric dissolution of these elements and non-preferential zinc resorption. Conclusions: The nanometric size of cHA and Zn-cHA was a decisive factor in accelerating the in vivo availability of calcium and zinc. The high calcium and zinc accumulation in the defect, which was not cleared by the biological medium, played a critical role in inhibiting osteoconduction and thus impairing bone repair.

Impact of crystallinity and crystal size of nanostructured carbonated hydroxyapatite on pre-osteoblast in vitro biocompatibility.

Nanostructured carbonated hydroxyapatite (nCHA) is a promising biomaterial for bone tissue engineering due to its chemical properties, similar to those of the bone mineral phase and its enhanced in vivo bioresorption. However, the biological effects of nCHA nanoparticles on cells and tissues are not sufficiently known. This study assessed the impact of exposing pre-osteoblasts to suspensions with high doses of nCHA nanoparticles with high or low crystallinity. MC3T3-E1 pre-osteoblasts were cultured for 1 or 7 days in a culture medium previously exposed to CHA nanoparticles for 1 day. Control groups were produced by centrifugation for removal of bigger nCHA aggregates before exposure. Interaction of nanoparticles with the culture medium drastically changed medium composition, promoting Ca, P, and protein adsorption. Transmission Electron microscopy revealed that exposed cells were able to internalize both materials, which seemed concentrated inside endosomes. No cytotoxicity was observed for both materials, regardless of centrifugation, and the exposure did not induce alterations in the release of pro-and anti-inflammatory cytokines. Morphological analysis revealed strong interactions of nCHA aggregates with cell surfaces, however without marked alterations in morphological features and cytoskeleton ultrastructure. The overall in vitro biocompatibility of nCHA materials, regardless of physicochemical characteristics such as crystallinity, encourages further studies on their clinical applications.

Apoptosis-associated speck-like protein containing a caspase-1 recruitment domain (ASC) contributes to osteoblast differentiation and osteogenesis.

The role of apoptosis-associated speck-like protein containing a caspase-1 recruitment domain (ASC) in bone healing remains to be understood. To address this issue, we investigated the requirement of inflammasome-related genes in response to bone morphogenetic protein 7 (BMP7)-induced osteoblast differentiation in vitro. To validate the importance of ASC on osteogenesis, we subjected wild-type (WT) and ASC knockout C57BL/6 mice (ASC KO) to tibia defect to evaluate the bone healing process (up to 28 days). Our in vitro data showed that there is an involvement of ASC during BMP7-induced osteoblast differentiation, concomitant to osteogenic biomarker expression. Indeed, primary osteogenic cells from ASC KO presented a lower osteogenic profile than those obtained from WT mice. To validate this hypothesis, we evaluated the bone healing process of tibia defects on both WT and ASC KO mice genotypes and the ASC KO mice were not able to fully heal tibia defects up to 28 days, whereas WT tibia defects presented a higher bone de novo volume at this stage, evidencing ASC as an important molecule during osteogenic phenotype. In addition, we have shown a higher involvement of runt-related transcription factor 2 in WT sections during bone repair, as well as circulating bone alkaline phosphatase isoform when both were compared with ASC KO mice behavior. Altogether, our results showed for the first time the involvement of inflammasome during osteoblast differentiation and osteogenesis, which opens new avenues to understand the pathways involved in bone healing.

Molecular Modeling and In Vitro Studies of a Neutral Oxime as a Potential Reactivator for Acetylcholinesterase Inhibited by Paraoxon.

The present work aimed to compare the small, neutral and monoaromatic oxime, isatin-3-oxime (isatin-O), to the commercial ones, pralidoxime (2-PAM) and obidoxime, in a search for a new potential reactivator for acetylcholinesterase (AChE) inhibited by the pesticide paraoxon (AChE/POX) as well as a novel potential scaffold for further synthetic modifications. The multicriteria decision methods (MCDM) allowed the identification of the best docking poses of those molecules inside AChE/POX for further molecular dynamic (MD) studies, while Ellman's modified method enabled in vitro inhibition and reactivation assays. In corroboration with the theoretical studies, our experimental results showed that isatin-O have a reactivation potential capable of overcoming 2-PAM at the initial moments of the assay. Despite not achieving better results than obidoxime, this molecule is promising for being an active neutral oxime with capacity of crossing the blood⁻brain barrier (BBB), to reactivate AChE/POX inside the central and peripheral nervous systems. Moreover, the fact that isatin-O can also act as anticonvulsant makes this molecule a possible multipotent reactivator. Besides, the MCDM method showed to be an accurate method for the selection of the best docking poses generated in the docking studies.

PVR/CD155 Ala67Thr Mutation and Cleft Lip/Palate.

The 19q13 locus has been linked to cleft lip and palate by our group and independently by others. Here we fine mapped the region in an attempt to identify an etiological variant that can explain cleft lip and palate occurrence. A total of 2739 individuals born with cleft lip and palate, related to individuals born with cleft lip and palate, and unrelated were studied. We used linkage and association approaches to fine map the interval between D19S714 and D19S433 and genotypes were defined by the use of TaqMan chemistry. We confirmed our previous findings that markers in PVR/CD155 are associated with cleft lip and palate. We studied the mutation Ala67Thr further and calculated its penetrance. We also attempted to detect PVR/CD155 expression in human whole saliva. Our results showed that markers in PVR/CD155 are associated with cleft lip and palate and the penetrance of the Ala67Thr is very low (between 1% and 5%). We could not detect PVR/CD155 expression in adult human whole saliva and PVR/CD155 possibly interacts with maternal infection to predispose children to cleft lip only.

Matrix Metalloproteinases in Bone Resorption, Remodeling, and Repair.

Matrix metalloproteinases (MMPs) are the major protease family responsible for the cleavage of the matrisome (global composition of the extracellular matrix (ECM) proteome) and proteins unrelated to the ECM, generating bioactive molecules. These proteins drive ECM remodeling, in association with tissue-specific and cell-anchored inhibitors (TIMPs and RECK, respectively). In the bone, the ECM mediates cell adhesion, mechanotransduction, nucleation of mineralization, and the immobilization of growth factors to protect them from damage or degradation. Since the first description of an MMP in bone tissue, many other MMPs have been identified, as well as their inhibitors. Numerous functions have been assigned to these proteins, including osteoblast/osteocyte differentiation, bone formation, solubilization of the osteoid during bone resorption, osteoclast recruitment and migration, and as a coupling factor in bone remodeling under physiological conditions. In turn, a number of pathologies, associated with imbalanced bone remodeling, arise mainly from MMP overexpression and abnormalities of the ECM, leading to bone osteolysis or bone formation. In this review, we will discuss the functions of MMPs and their inhibitors in bone cells, during bone remodeling, pathological bone resorption (osteoporosis and bone metastasis), bone repair/regeneration, and emergent roles in bone bioengineering.

Bio-camouflage of anatase nanoparticles explored by in situ high-resolution electron microscopy.

While titanium is the metal of choice for most prosthetics and inner body devices due to its superior biocompatibility, the discovery of Ti-containing species in the adjacent tissue as a result of wear and corrosion has been associated with autoimmune diseases and premature implant failures. Here, we utilize the in situ liquid cell transmission electron microscopy (TEM) in a liquid flow holder and graphene liquid cells (GLCs) to investigate, for the first time, the in situ nano-bio interactions between titanium dioxide nanoparticles and biological medium. This imaging and spectroscopy methodology showed the process of formation of an ionic and proteic bio-camouflage surrounding Ti dioxide (anatase) nanoparticles that facilitates their internalization by bone cells. The in situ understanding of the mechanisms of the formation of the bio-camouflage of anatase nanoparticles may contribute to the definition of strategies aimed at the manipulation of these NPs for bone regenerative purposes.

Successful Low-Cost Scaffold-Free Cartilage Tissue Engineering Using Human Cartilage Progenitor Cell Spheroids Formed by Micromolded Nonadhesive Hydrogel.

The scaffold-free tissue engineering using spheroids is pointed out as an approach for optimizing the delivery system of cartilage construct. In this study, we aimed to evaluate the micromolded nonadhesive hydrogel (MicroTissues®) for spheroid compaction (2-day culture) and spontaneous chondrogenesis (21-day culture) using cartilage progenitors cells (CPCs) from human nasal septum without chondrogenic stimulus. CPC spheroids showed diameter stability (486 µm ± 65), high percentage of viable cells (88.1 ± 2.1), and low percentage of apoptotic cells (2.3%). After spheroid compaction, the synthesis of TGF-ß1, TGF-ß2, and TGF-ß3 was significantly higher compared to monolayer (p < 0.005). Biomechanical assay revealed that the maximum forces applied to spheroids after chondrogenesis were 2.6 times higher than for those cultured for 2 days. After spontaneous chondrogenesis, CPC spheroids were entirely positive for N-cadherin, collagen type II and type VI, and aggrecan and chondroitin sulfate. Comparing to monolayer, the expression of SOX5 and SOX6 genes analyzed by qPCR was significantly upregulated (p < 0.01). Finally, we observed the capacity of CPC spheroids starting to fuse. To the best of our knowledge, this is the first time in the scientific literature that human CPC spheroids were formed by micromolded nonadhesive hydrogel, achieving a successful scaffold-free cartilage engineering without chondrogenic stimulus (low cost).

Hydrophilic surface of Ti6Al4V-ELI alloy improves the early bone apposition of sheep tibia.

OBJECTIVE: The aim of this preclinical study was to compare histologically and histomorphometrically both sandblasted/acid-etched implant surfaces with or without maintained in an isotonic solution of 0.9% sodium chloride in early stages of osseointegration. MATERIAL AND METHODS: Both implant surfaces were composed of a titanium/aluminum/vanadium alloy (Ti6Al4V-ELI), but they had different surface chemistries: sandblasted/acid-etched titanium surface (FN) or sandblasted/acid-etched surface maintained in an isotonic solution of 0.9% sodium chloride (FA). The surface morphology, topography and chemistry were evaluated by scanning electron microscopy (SEM), confocal microscopy (CM) and X-ray photoelectron spectroscopy (XPS), respectively. Dynamic contact angle (DCA) was employed for wettability evaluation. One implant from each group was placed in the left tibia of twenty healthy, skeletally mature Santa Ines sheep (n = 5). Bone area (BA) and bone-to-implant contact (BIC) were performed on thin sections (30 µm) at 7, 14, 21 and 28 days after implant installation. RESULTS: Despite the roughness and morphology similarities between the groups, at the XPS evaluation, the FA group presented 2.3 times less carbon on the surface (FN: 27.3% and FA: 11.6%), sharply enhanced hydrophilicity and significantly enhanced BA and BIC at 14, 21 and 28 days of healing (P < 0.05) compared with the FN. CONCLUSION: The data suggest that the hydrophilic FA accelerates the BA apposition and BIC interface around the implants during early stages of bone formation, providing highest degree of osseointegration.

Delivery of Human Adipose Stem Cells Spheroids into Lockyballs.

Adipose stem cells (ASCs) spheroids show enhanced regenerative effects compared to single cells. Also, spheroids have been recently introduced as building blocks in directed self-assembly strategy. Recent efforts aim to improve long-term cell retention and integration by the use of microencapsulation delivery systems that can rapidly integrate in the implantation site. Interlockable solid synthetic microscaffolds, so called lockyballs, were recently designed with hooks and loops to enhance cell retention and integration at the implantation site as well as to support spheroids aggregation after transplantation. Here we present an efficient methodology for human ASCs spheroids biofabrication and lockyballs cellularization using micro-molded non-adhesive agarose hydrogel. Lockyballs were produced using two-photon polymerization with an estimated mechanical strength. The Young's modulus was calculated at level 0.1362 +/-0.009 MPa. Interlocking in vitro test demonstrates high level of loading induced interlockability of fabricated lockyballs. Diameter measurements and elongation coefficient calculation revealed that human ASCs spheroids biofabricated in resections of micro-molded non-adhesive hydrogel had a more regular size distribution and shape than spheroids biofabricated in hanging drops. Cellularization of lockyballs using human ASCs spheroids did not alter the level of cells viability (p > 0,999) and gene fold expression for SOX-9 and RUNX2 (p > 0,195). The biofabrication of ASCs spheroids into lockyballs represents an innovative strategy in regenerative medicine, which combines solid scaffold-based and directed self-assembly approaches, fostering opportunities for rapid in situ biofabrication of 3D building-blocks.

Osteogenic effect of tricalcium phosphate substituted by magnesium associated with Genderm® membrane in rat calvarial defect model.

Beta-tricalcium phosphate (ß-TCP) is one of the most widely employed bioresorbable materials for bone repair since it shows excellent biological compatibility, osteoconductivity and resorbability. The incorporation of divalent cations such as magnesium onto the ß-TCP structure (ß-TCMP) may improve the biological response to the material through the release of bioactive ions. The objective of this study was to evaluate, on a rat calvarial critical size grafting model, the bone regeneration process using ß-TCP and ß-TMCP granules by histomorphometric analysis. Results demonstrated that six months after bone grafting, the association of GBR (guided bone regeneration) using a membrane (GenDerm®) and granules of ß-TCP and ß-TCMP significantly improves bone repair in the treatment of critical-size defect in rat skulls, in comparison to untreated defects or GBR alone, leading to a bone level approximately four to five-fold greater than in the blood clot group. The ß-TCMP+GenDerm® membrane group presented 40.5% of the defect area filled by newly-formed bone, even at the central part of the defect, rather than only at the border, as seen in the other experimental groups.