Search Results (233)

Mesenchymal stem cells have been widely investigated in the field of regenerative medicine and also used as a model to study the differentiation-induction properties of a variety of biomaterials. This study evaluates the osteoinductive potential of novel hydroxyapatite scaffolds functionalized with a phage-displayed peptide (SC1) selected via biopanning for its similarity to bone matrix proteins. The peptide, identified through sequence alignment as a mimotope of osteonectin (SPARC), was used to functionalize scaffolds. Results from SC1 were gathered at different time points (14, 28 and 46 days) and compared with those from nonfunctionalized hydroxyapatite (HA) scaffolds. In vitro experiments, by seeding human adipose-derived stem cells (hASCs), indicated satisfactory biocompatibility for both types of scaffolds. Histochemical observations showed that SC1, better than HA scaffolds, was able to improve hASC osteogenic differentiation, as evaluated through Alizarin Red staining (showing on average a darker staining of 100%). An increase was also observed, especially at early stages (14 days), for osterix (up to 60% increase) and osteonectin immunoexpression (up to 50% increase). In in vivo experiments, cell-free scaffolds of both types were subcutaneously implanted into the backs of mice and analyzed after 2, 4, 8 and 16 weeks. Also, in this case, SC1 more effectively promoted the osteogenic differentiation of infiltrated resident cells. In particular, increased immunoexpression of osterix and osteonectin (+30% and 35%, respectively) was found already at 2 weeks. It can be concluded that SC1 scaffolds may represent a valuable tool to address critical-sized bone defects. Full article

The attachment of the oral epithelium to the abutment surface is crucial for the long-term success of dental implants. This study aimed to evaluate the attachment of human epithelial cells to anodized titanium surfaces. Anodized titanium discs were used as the experimental group, while machined titanium discs served as the control. Surface roughness and wettability were first measured for each group. Next, human epithelial cells were seeded onto each disc at a density of 4.0 × 10⁴ cells/cm² and evaluated 3, 6, and 24 h later for cell proliferation, as well as mRNA expression and protein levels of laminin and integrin β₄. Surface roughness was comparable between the two groups; however, wettability was significantly higher in the experimental group. Cell proliferation increased over time in both groups and showed no significant difference. Notably, the expression levels of both laminin and integrin β₄ were significantly higher in the experimental group at 24 h. Furthermore, protein localization of laminin and integrin β₄ was observed along the cell margins on the anodized surface. These findings suggest that anodization enhances epithelial cell attachment by promoting the expression and peripheral organization of key adhesion molecules. Full article

Backdoor attacks in self-supervised learning pose an increasing threat. Recent studies have shown that knowledge distillation can mitigate these attacks by altering feature representations. In response, we propose BASK, a novel backdoor attack that remains effective after distillation. BASK uses feature weighting and representation alignment strategies to implant persistent backdoors into the encoder’s feature space. This enables transferability to student models. We evaluated BASK on the CIFAR-10 and STL-10 datasets and compared it with existing self-supervised backdoor attacks under four advanced defenses: SEED, MKD, Neural Cleanse, and MiMiC. Our experimental results demonstrate that BASK maintains high attack success rates while preserving downstream task performance. This highlights the robustness of BASK and the limitations of current defense mechanisms. Full article

Background: Subcutaneous pocket infection is a common morbidity associated with the integration of cardiac implantable electronic devices (CIEDs). A new antibiotic-eluting CIED bioenvelope has been developed as a prophylactic measure to mitigate infection and skin erosion caused by device migration. This study investigated the envelope’s regulatory properties in scar formation and vascularization. Methods: Fibroblasts were seeded on either plastic (n = 6) or small intestine submucosal extracellular matrix (SIS-ECM) (n = 6) for 24 h. The culture media were analyzed for proangiogenic and proinflammatory proteins with multiplex. Sham (n = 8) or SIS-ECM (n = 8) was randomly implanted into the dorsal subcutaneous pocket of mice. The implants were excised on day 7, cultured for 24 h, and the media analyzed. Rabbit models were implanted with either synthetic polymer HDPE (n = 12) or SIS-ECM (n = 11). The treatments were excised at weeks 2, 10, and 26 and then stained for analysis. Results: SIS-ECM significantly increased the fibroblasts’ paracrine release of proangiogenic and proinflammatory factors like VEGF-A (p < 0.05) and IL-6 (p < 0.05) compared with plastic. The murine tissue interacting with SIS-ECM released significantly more angiogenic proteins like VEGF-A (p < 0.05) than the sham. The histology analysis of rabbit subcutaneous tissue revealed a decreasing level of inflammation and fibrosis over time with SIS-ECM. Conclusions: The CIED bioenvelope elicited proangiogenic paracrine signaling and reduced fibrotic response in fibroblasts and animal models. Clinical translation of the CIED bioenvelope as an adjunct to regular prophylactic practice may be warranted in the future. Full article

A platform of two-component cross-linked hydrogel (cGEL) based on gelatinous peptides and anhydride-containing cross-linkers (oPNMA, oPDMA) is extended for use in peripheral nerve regeneration. Hybrid composites with bio-/chemical cues for enhanced biophysical and biochemical properties were fabricated by covalently grafting small molecular, heterobifunctional amines including the nerve growth factor mimetic LM11A-31 to the oligomeric cross-linkers prior to hydrogel formation. The cytocompatibility and growth-supportive conditions within the matrix are confirmed for pristine and modified hydrogels using L929 mouse fibroblasts and human adipose-derived stem cells (hASCs). For hASCs, cell behavior depends on the type of cross-linker and integrated amine. In a subsequent step, neonatal rat Schwann cells (SCs) are seeded on pristine and functionalized cGEL to investigate the materials’ capabilities to support SC growth and morphology. Within all formulations, cell viability, adherence, and cell extension are maintained though the cell elongation and orientation vary compared to the two-dimensional control. It is possible to merge adjustable two-component hydrogels with amines as biochemical signals, leading to improved nervous cell proliferation and activity. This indicates the potential of tunable bioactive cGEL as biomaterials in nerve implants, suggesting their use as a foundational component for nerve conduits. Full article

The rising incidence of colorectal cancer and ulcerative colitis underscores an urgent need for regenerative solutions to address functional deficits after colectomy. However, the creation of clinically applicable large intestine scaffolds remains underdeveloped. Here, we report the successful generation and thorough characterisation of transplantable-sized porcine large intestinal scaffolds via perfusion decellularisation. This method effectively preserved extracellular matrix (ECM) structural and biochemical integrity while minimising immunogenicity through cellular component removal. Crucially, native vasculature remained intact, confirmed by histology, DNA quantification, and high-resolution CT angiography. Despite efficient decellularisation, challenges including residual nucleic acids, ECM heterogeneity, and partial microvascular occlusion were noted, echoing ongoing limitations in engineered, perfusable, full-thickness scaffolds. In vivo implantation demonstrated favourable biocompatibility and host integration; however, thrombosis occurred due to the lack of pre-seeded cells, emphasising the necessity of recellularisation for functional perfusion prior to implantation. This study addresses significant field limitations, presenting the first reproducible approach for structurally intact, perfusable, full-thickness large intestinal scaffolds of transplantable dimensions. Our innovations offer a strong foundation for future integration of patient-derived cells, stem cells, and organoids, progressing toward clinically viable, scalable, tissue-engineered large intestine constructs, from xenogeneic sources, relevant for regenerative medicine, disease modelling, and pharmacological screening. Full article

Heat preconditioning has been shown to promote nutritive perfusion and tissue survival in autologous fat grafting as well as in flap and breast surgery. However, its impact on the vascularization properties of nanofat has not been investigated so far. Therefore, we exposed nanofat from donor mice to a temperature of 43 °C for 1 h and assessed the effects of this heat stress on cell viability and the expression of heat shock proteins (HSPs) and angiogenesis-related factors. Moreover, dermal substitutes seeded with heat-preconditioned and non-preconditioned control nanofat were implanted into dorsal skinfold chambers of recipient mice to study their vascularization and tissue integration in vivo by means of repeated intravital fluorescence microscopy, histology and immunohistochemistry. Heat preconditioning upregulated the expression of HSPs in nanofat without affecting cell viability. Moreover, it resulted in the downregulation of many pro-angiogenic factors and the increased expression of anti-angiogenic factors, indicating a shift towards an anti-angiogenic phenotype. Accordingly, implanted dermal substitutes seeded with heat-preconditioned nanofat exhibited a reduced vascularization and were not better integrated into the host tissue when compared to controls. These findings indicate that heat preconditioning cannot be recommended for enhancing the vascularization capacity of nanofat. Full article

(1) Autologous chondrocyte implantation (ACI) is a prominent method for treating cartilage damage, but periosteal patches can cause chondrocyte leakage. This study evaluates the potential of a decellularized membrane derived from the cell-produced extracellular matrix of 1-day-old porcine cartilage (pcECM-DM) to act as a substitute for periosteal patches. (2) The interaction between young rabbit chondrocyte cells and pcECM-DM was assessed through cytotoxicity, differentiation, cell viability, cell migration, and adhesive ability. Rabbit chondrocyte cells, cultivated until passage two, were seeded onto a 6 mm diameter membrane. Assessments included DAPI-PKH26 staining, histological staining, live/dead assay, WST-1 assay, and proteomics analysis. (3) Results: DAPI-PKH26 staining showed successful adhesion and the uniform distribution of cells on the membrane. Safranin-O and H&E staining confirmed that the membrane supports chondrocyte adhesion and extracellular matrix production with high cell density and typical chondrocyte morphology. The live/dead assay demonstrated a high proportion of viable cells at 24 and 48 h, with increased cell proliferation over time. The WST-1 assay showed a significant increase in OD450 values, confirming cell proliferation and biocompatibility. Proteomic analysis revealed the significant enrichment of genes associated with extracellular matrix organization, cell adhesion, and cartilage development. (4) Conclusions: This novel biomaterial holds the potential to enhance cartilage regeneration and offer a viable alternative to periosteal patches. Full article

This study presents a novel method to enhance the biocompatibility of decellularized porcine aortic segments while preserving their mechanical properties and histological structure. Detergent-decellularized aortic segments were treated with modified globular chitosan (Novochizol™) at varying concentrations (0.5%, 1%, 2%, and 3%) by sonication and subsequently subjected to mechanical testing. To further improve cell infiltration, blind-ended laser channels were created within the decellularized segments. The modified grafts were then seeded with porcine vascular interstitial cells in vitro for 7 days or implanted into the thoracic aorta of minipigs for 30 days. Histological analysis was performed at each stage of the study. Impregnation with Novochizol™ significantly increased the specific strength (from 0.97 ± 0.19 MPa to 4.99 ± 2.43 MPa) and Young’s modulus (from 0.73 ± 0.06 MPa to 14.66 ± 7.14 MPa) of the decellularized aortic segments. Histological examination confirmed the preservation of the connective tissue matrix’s morphological structure. Optimal modification conditions were identified as a 30 min sonication in a 1% Novochizol™ solution at 25 °C. A 35 ms continuous laser treatment was sufficient to create a 1 mm deep blind-ended channel, thereby promoting the seeding of vascular interstitial cells within the acellular graft, as confirmed by implantation in minipigs. Full article

(1) Background: For the reconstruction of a human vagina, various surgical procedures are available that are often associated with complications due to their failure to mimic the physiology of the human vagina. We recently developed a vascularized, organ-specific matrix from healthy human vaginal wall tissue with suitable biomechanical properties. A superior graft would require further extensive colonization with autologous vaginal cells to reduce complications upon implantation. However, reports on isolation of vaginal cells from biopsies are scarce, and published protocols rarely contain sufficient details. In this study, we aimed to examine protocols for inconsistencies and identify (where possible) the optimal protocol in terms of reproducibility and efficiency for isolation of human vaginal fibroblasts (FBs), epithelial cells (VECs), and smooth muscle cells (SMCs). Overall, this study aims to guide other researchers and aid future tissue engineering solutions that rely on autologous cells. (2) Methods: A total of 41 isolation protocols were tested: four protocols specific to FBs, 13 protocols for VECs, and 24 protocols for SMCs. Protocols were derived from published reports on cell isolation by enzymes, with exclusion criteria including the need for specialized equipment, surgical separation of tissue layers, or missing protocol details. Enzymatic digestion with collagenase-I, collagenase-IV, and dispase-II was used for isolation of VECs, collagenase-IV for isolation of SMCs, and collagenase-IA for isolation of FBs. Fluorescent immunostaining was applied to identify VECs with cytokeratin, SMCs with desmin, endothelial cells with UEA-1, and FBs with vimentin. Protocols were assessed based on (>95%) homogeneity, duplicate consistency, cell viability, and time to first passage. (3) Results: A total of 9 out of the 41 protocols resulted in isolation and expansion of vaginal FBs. This involved 1 out of 13 VEC protocols, 6 out of 24 SMC protocols, and 2 out of 2 FB protocols. Isolation of vaginal SMCs or VECs was not achieved. The best results were obtained after digestion with 0.1% collagenase-IV, where pure FB colonies formed with high cell viability. (4) Conclusions: Today, vaginoplasty is considered the gold standard for surgically creating a neovagina, despite its considerable drawbacks and limitations. Tissue-engineered solutions carry great potential as an alternative, but cell seeding is desired to prevent complications upon implantation of grafts. In this study, we examined isolation of human vaginal FBs, SMCs, and VECs, and identified the most efficient and reliable protocol for FBs. We further identified inconsistencies and irreproducible methods for isolation of VECs and SMCs. These findings aid the clinical translation of cell-based tissue engineering for the reconstruction and support of vaginas, fulfilling unmet medic needs. Full article

Ex vivo regional gene therapy is a promising tissue-engineering strategy for bone regeneration: osteogenic mesenchymal stem cells (MSCs) can be genetically modified to express an osteoinductive stimulus (e.g., bone morphogenetic protein-2), seeded onto an osteoconductive scaffold, and then implanted into a bone defect to exert a therapeutic effect. Compared to recombinant human BMP-2 (rhBMP-2), which is approved for clinical use, regional gene therapy may have unique benefits related to the addition of MSCs and the sustained release of BMP-2. However, the cellular and transcriptional mechanisms regulating the response to these two strategies for BMP-2 mediated bone regeneration are largely unknown. Here, for the first time, we performed single-cell RNA sequencing (10x Genomics) of hematoma tissue in six rats with critical-sized femoral defects that were treated with either regional gene therapy or rhBMP-2. Our unbiased bioinformatic analysis of 2393 filtered cells in each group revealed treatment-specific differences in their cellular composition, transcriptional profiles, and cellular communication patterns. Gene therapy treatment induced a more robust chondrogenic response, as well as a decrease in the proportion of fibroblasts and the expression of profibrotic pathways. Additionally, gene therapy was associated with an anti-inflammatory microenvironment; macrophages expressing canonical anti-inflammatory markers were more common in the gene therapy group. In contrast, pro-inflammatory markers were more highly expressed in the rhBMP-2 group. Collectively, the results of our study may offer insights into the unique pathways through which ex vivo regional gene therapy can augment bone regeneration compared to rhBMP-2. Furthermore, an improved understanding of the cellular pathways involved in segmental bone defect healing may allow for the further optimization of regional gene therapy or other bone repair strategies. Full article

Background and Objectives: Current craniofacial reconstruction surgical methods have limitations because they involve facial deformation. The craniofacial region includes many areas where the mucosa, exposed to air, is closely adjacent to bone, with the maxilla being a prominent example of this structure. Therefore, this study explored whether human neural-crest-derived stem cells (hNTSCs) aid bone and airway mucosal regeneration during craniofacial reconstruction using a rabbit model. Materials and Methods: hNTSCs were induced to differentiate into either mucosal epithelial or osteogenic cells in vitro. hNTSCs were seeded into polycaprolactone scaffold (three-dimensionally printed) that were implanted into rabbits with maxillary defects. Four weeks later, tissue regeneration was analyzed via histological evaluation and immunofluorescence staining. Results: In vitro, hNTSCs differentiated into both mucosal epithelial and osteogenic cells. hNTSC differentiation into respiratory epithelial cells was confirmed by Alcian Blue staining, cilia in SEM, and increased expression levels of FOXJ1 and E-cadherin through quantitative RT-PCR. hNTSC differentiation into bone was confirmed by Alizarin Red staining, increased mRNA expression levels of BMP2 (6.1-fold) and RUNX2 (2.3-fold) in the hNTSC group compared to the control. Four weeks post-transplantation, the rabbit maxilla was harvested, and H&E, SEM, and immunohistofluorescence staining were performed. H&E staining and SEM showed that new tissue and cilia around the maxillary defect were more prominent in the hNTSC group. Also, the hNTSCs group showed positive immunohistofluorescence staining for acetylated α-tubulin and cytokerin-5 compared to the control group. Conclusions: hNTSCs combined with PCL scaffold enhanced the regeneration of mucosal tissue and bone in vitro and promoted mucosal tissue regeneration in the in vivo rabbit model. Full article

This study aimed to evaluate zirconia dental implant surfaces patterned using Nd:YAG laser or conventional milling techniques against Streptococcus oralis adhesion and biofilm formation. Zirconia dental implant discs were subjected to surface patterning treatments and categorized into four groups: groove texturing by conventional milling (GM), pore texturing by conventional milling (PM), groove texturing by Nd:YAG laser (GL), and pore texturing by Nd: YAG laser (PL). Streptococcus oralis CECT 907T was cultivated on enriched blood agar plates and then transferred to a brain–heart infusion modified medium and incubated at 37 °C under anaerobic conditions until reaching the exponential growth phase. The bacterial suspension was then seeded on 24-well plates containing the treated discs. The viability of bacteria within the biofilm was determined based on colony-forming unit (CFU) counts, while the total biofilm was quantified by measuring its biomass. A qualitative analysis was conducted using scanning electron microscopy (SEM) images to evaluate the bacterial morphology. The statistical analysis of multigroup comparisons was performed using Kruskal–Wallis test with post hoc pairwise comparison, as well as Mann Whiney U test, with significance set at p < 0.05. After both 1 h and 24 h of incubation of Streptococcus oralis on the discs, all groups showed similar results, with no statistically significant differences (p > 0.05). A comparison of the Nd: YAG laser-treated surfaces with conventionally milled surfaces, as well as grooves versus pores for CFU counts, also revealed no statistically significant differences (p > 0.05) for both 1 h and 24 h of culture. Biomass quantification at both the 1 h and 24-h time points showed similar results across the groups, without statistical differences. When comparing the conventionally machined surfaces to Nd: YAG laser-treated surfaces in terms of biomass, no significant differences were observed (p > 0.05). Similarly, the comparison between groove-patterned surfaces and pore-patterned surfaces showed no statistically significant difference. The groove and pore patterns on zirconia surfaces with Nd: YAG laser or conventional milling did not change the Streptococcus oralis adhesion and biofilm formation behavior. Additional studies are recommended to expand our knowledge in this area. Full article

This study assessed the effects of titanium (Ti) surface modification with sodium hydroxide (NaOH) associated or not with Naringenin (NA) citrus flavonoid-coating on osteoblastic-like cells (Ob) metabolism. Ti discs were submitted to alkalinization by NaOH solution (5 M, 60 °C) for 24 h; then, the discs were impregnated or not with 100 µg/mL of NA and dried for 1 h at room temperature. The chemical composition, surface topography, and NA release were evaluated. For the biological assays, the discs were placed on 24-well cell culture plates and Ob (Saos-2; ATCC HTB-85) was seeded onto the discs. After different periods, cell adhesion and viability, alkaline phosphatase activity (ALP), and mineralized nodules deposition (MND) were assessed. In addition, cells stimulated with tumor necrosis factor-alpha (TNF-α) were submitted to matrix metalloproteinase (MMP)-2 synthesis and ALP gene expression assessment. Since data presented normal distribution and homogeneity (Shapiro-Wilk; Levene), Student’s t-test or one-way ANOVA/post-hoc tests were selected for data analysis (α = 0.05). Higher roughness was observed on Ti discs submitted to NaOH treatment, while the chemical and NA release evaluations indicated the successful adsorption of NA to alkali-treated Ti surface. Higher cell adhesion, cell viability (after 7 days of culture), ALP activity, and MND were observed on Ti NaOH coated with NA compared to the control group (Ti NaOH) (p < 0.05). Moreover, NA coating also promoted decreased MMP-2 synthesis and increased ALP gene expression in the presence of the inflammatory stimulus TNF-α (p < 0.05). The modification of Ti disks with NaOH associated with NA-coating enhanced bone cell metabolism, suggesting that this type of surface modification has a promising potential to accelerate bone repair and formation around dental implants. Full article

This study evaluates the potential of erbium, chromium-doped:yttrium, scandium, gallium, and garnet (Er,Cr:YSGG) laser irradiation to modify the titanium surface for optimal seeding of fibroblasts and osteoblasts in the treatment of peri-implantitis. Titanium discs were treated using the Er,Cr:YSGG laser, an ultrasonic device with a stainless tip, or titanium scalers. Changes in surface properties were analyzed by profilometer and scanning electron microscopy (SEM). Murine fibroblast and osteoblast adhesion and proliferation were evaluated qualitatively and quantitatively at 24 and 72 h. Profilometric surface topography and SEM showed that titanium scalers and ultrasonic debridement techniques significantly changed the structure of the machined and rough titanium surfaces. The Er,Cr:YSGG laser irradiation, on the other hand, did not alter titanium microstructures. The Er,Cr:YSGG laser irradiation with the 40 Hz group showed a significantly higher attached fibroblast cell numbers than the titanium scaler group at 72 h after treatment (p = 0.023). Additionally, the number of the attached osteoblasts in the Er,Cr:YSGG laser irradiation with the 40 Hz group was significantly higher than that of the no-treatment groups 24 h after treatment (p = 0.045). The Er,Cr:YSGG laser effectively promoted adherence of fibroblasts and osteoblasts to the titanium surface without significantly altering the titanium surface, suggesting its superiority for treating peri-implantitis. Full article