Double-Cross-Linked Hydrogel with Long-Lasting Underwater Adhesion: Enhancement of Maxillofacial In Situ and Onlay Bone Retention.

Bone retention is a usual clinical problem existing in a lot of maxillofacial surgeries involving bone reconstruction and bone transplantation, which puts forward the requirements for bone adhesives that are stable, durable, biosafe, and biodegradable in wet environment. To relieve the suffering of patients during maxillofacial surgery with one-step operation and satisfying repair, herein, we developed a double-cross-linked A-O hydrogel named by its two components: [(3-Aminopropyl) methacrylamide]-co-{[Tris(hydroxymethyl) methyl] acrylamide} and oxidated methylcellulose. With excellent bone adhesion ability, it can maintain long-lasting stable underwater bone adhesion for over 14 days, holding a maximum adhesion strength of 2.32 MPa. Schiff-base reaction and high-density hydrogen bonds endow the hydrogel with strong cohesion and adhesion performance as well as maneuverable properties such as easy formation and injectability. A-O hydrogel not only presents rarely reported long-lasting underwater adhesion of hard tissue but also owns inherent biocompatibility and biodegradation properties with a porous structure that facilitates the survival of bone graft. Compared to the commercial cyanoacrylate adhesive (3 M Vetbond Tissue Adhesive), the A-O hydrogel is confirmed to be safer, more stable, and more effective in calvarial in situ bone retention model and onlay bone retention model of rat, providing a practical solution for the everyday scenario of clinical bone retention.

Blue-ringed octopus-inspired microneedle patch for robust tissue surface adhesion and active injection drug delivery.

Intratissue topical medication is important for the treatment of cutaneous, mucosal or splanchnic diseases. However, penetrating surface barriers to providing adequate and controllable drug delivery while guaranteeing adhesion in bodily fluids remains challenging. Here, the predatory behavior of the blue-ringed octopus inspired us with a strategy to improve topical medication. For effective intratissue drug delivery, the active injection microneedles were prepared in a manner inspired by the teeth and venom secretion of blue-ringed octopus. With on demand release function guided by temperature-sensitive hydrophobic and shrinkage variations, these microneedles can supply adequate drug delivery at an early stage and then achieve the long-term release stage. Meanwhile, the bionic suction cups were developed to facilitate microneedles to stay firmly in place (>10 kilopascal) when wet. With wet bonding ability and multiple delivery mode, this microneedle patch achieved satisfactory efficacy, such as accelerating the ulcers' healing speed or halting early tumor progression.

Gold nanoparticles: promising biomaterials for osteogenic/adipogenic regulation in bone repair.

Bone defects are a common bone disease, which are usually caused by accidents, trauma and tumors. However, the treatment of bone defects is still a great clinical challenge. In recent years, research on bone repair materials has continued with great success, but there are few reports on the repair of bone defects at a high lipid level. Hyperlipidemia is a risk factor in the process of bone defect repair, which has a negative impact on the process of osteogenesis, increasing the difficulty of bone defect repair. Therefore, it is necessary to find materials that can promote bone defect repair under the condition of hyperlipidemia. Gold nanoparticles (AuNPs) have been applied in the fields of biology and clinical medicine for many years and developed to modulate osteogenic differentiation and adipogenic differentiation. In vitro and vivo studies displayed that they promoted bone formation and inhibited fat accumulation. Further, the metabolism and mechanisms of AuNPs acting on osteogenesis/adipogenesis were partially revealed by researchers. This review further clarifies the role of AuNPs in osteogenic/adipogenic regulation during the process of osteogenesis and bone regeneration by summarizing the related in vitro and in vivo research, discussing the advantages and challenges of AuNPs and highlighting several possible directions for future research, with the aim to provide a new strategy for dealing with bone defects in hyperlipidemic patients.

Alginate-based biomaterial-mediated regulation of macrophages in bone tissue engineering.

Many studies in the bone tissue engineering field have focused on the interactions between materials and bone marrow stem cells. With the development of osteoimmunology, the immune cells' essential role in biomaterial-mediated osteogenesis has increasingly been recognized. As a promising therapeutic candidate for bone defects due to their prominent biocompatibility, tuneability, and versatility, it is necessary to develop alginate-based biomaterials that can regulate immune cells, especially macrophages. Moreover, modified alginate-based biomaterials may facilitate better regulation of macrophage phenotypes by the newly endowed physicochemical properties, including stiffness, porosity, hydrophilicity, and electrical properties. This review summarizes the role of macrophages in bone regeneration and the recent research progress related to the effects of alginate-based biomaterials on macrophages applied in bone tissue engineering. This review also emphasizes the strategies adopted by material design to regulate macrophage phenotypes, the corresponding macrophage responses, and their contribution to osteogenesis.

Effects of photobiomodulation therapy on implant stability and postoperative recovery: a systematic review and meta-analysis.

The purpose of this study was to systemically analyse the effects of photobiomodulation therapy (PBMT) on implant stability and postoperative recovery. Electronic searches on MEDLINE (PubMed), Cochrane Library, EMBASE, and Web of Science were completed independently by two researchers in February 2021, and a manual search was performed for the references of the included articles. The primary outcome was implant stability. The secondary outcome was postoperative recovery, including postoperative pain, recovery of peri-implant hard tissue (marginal bone loss and bone mineral density), facial swelling, and peri-implant clinical parameters. Twenty studies were finally obtained (17 randomised controlled, and 3 controlled clinical studies). Meta-analysis revealed that PBMT increased implant stability at 10 days after insertion (MD 2.27, 95% CI: 0.40 to 4.13, P = 0.020), and reduced marginal bone loss at 6 months after insertion (MD -0.16, 95% CI: -0.23 to -0.08, P < 0.001). However, no significant improvements were noted in implant stability two weeks (P = 0.070), three weeks (P = 0.090), six weeks (P = 0.050), and 12 weeks (P = 0.080) after insertion. Qualitative analysis suggested that PBMT could not alleviate postoperative pain, increase bone mineral density, or improve peri-implant clinical parameters. It was effective only in reducing facial swelling. This study suggests that the effects of PBMT on implant stability and postoperative recovery may be limited.

Interimplant papilla reconstruction at second-stage surgery: A technique.

Reconstructing an esthetic interimplant papilla remains challenging with implant-supported restorations, especially for patients with a thin gingival biotype. This technique report describes a modified approach to rebuilding an interimplant papilla by joining 2 elevated connective tissue flaps at the second-stage surgery.

Effects of platelet concentrates on implant stability and marginal bone loss: a systematic review and meta-analysis.

BACKGROUND: Osseointegration is essential for the success and stability of implants. Platelet concentrates were reported to enhance osseointegration and improve implant stability. The purpose of this review is to systematically analyze the effects of platelet concentrates on implant stability and marginal bone loss. METHODS: Two researchers independently performed searches in the following databases (last searched on 21 July 2021): MEDLINE (PubMed), Cochrane Library, EMBASE, and Web of Science. In addition, a manual search was carried out on references of relevant reviews and initially included studies. All randomized controlled trials (RCTs) and controlled clinical trials (CCTs) on the application of platelet concentrates in the implant surgery procedure were included. The risk of bias of RCTs and CCTs were assessed with a revised Cochrane risk of bias tool for randomized trials (RoB 2.0) and the risk of bias in non-randomized studies-of interventions (ROBINS-I) tool, respectively. Meta-analyses on implant stability and marginal bone loss were conducted. Researchers used mean difference or standardized mean difference as the effect size and calculated the 95% confidence interval. In addition, subgroup analysis was performed based on the following factors: type of platelet concentrates, method of application, and study design. RESULTS: Fourteen studies with 284 participants and 588 implants were included in the final analysis. 11 studies reported implant stability and 5 studies reported marginal bone level or marginal bone loss. 3 studies had high risk of bias. The meta-analysis results showed that platelet concentrates can significantly increase implant stability at 1 week (6 studies, 302 implants, MD 4.26, 95% CI 2.03-6.49, P < 0.001) and 4 weeks (8 studies, 373 implants, MD 0.67, 95% CI 0.46-0.88, P < 0.001) after insertion, significantly reduced marginal bone loss at 3 months after insertion (4 studies, 95 implants, mesial: MD - 0.33, 95% CI - 0.46 to - 0.20, P < 0.001; distal: MD - 0.38, 95% CI - 0.54 to - 0.22, P < 0.001). However, the improvement of implant stability at 12 weeks after insertion was limited (P = 0.10). Subgroup analysis showed that PRP did not significantly improve implant stability at 1 week and 4 weeks after insertion (P = 0.38, P = 0.17). Platelet concentrates only placed in the implant sites did not significantly improve implant stability at 1 week after insertion (P = 0.20). CONCLUSIONS: Platelet concentrates can significantly improve implant stability and reduce marginal bone loss in the short term. Large-scale studies with long follow-up periods are required to explore their long-term effects and compare effects of different types. TRIAL REGISTRATION: This study was registered on PROSPERO, with the Registration Number being CRD42021270214.

A mussel-inspired film for adhesion to wet buccal tissue and efficient buccal drug delivery.

Administration of drugs via the buccal route has attracted much attention in recent years. However, developing systems with satisfactory adhesion under wet conditions and adequate drug bioavailability still remains a challenge. Here, we propose a mussel-inspired mucoadhesive film. Ex vivo models show that this film can achieve strong adhesion to wet buccal tissues (up to 38.72 ± 10.94 kPa). We also demonstrate that the adhesion mechanism of this film relies on both physical association and covalent bonding between the film and mucus. Additionally, the film with incorporated polydopamine nanoparticles shows superior advantages for transport across the mucosal barrier, with improved drug bioavailability (~3.5-fold greater than observed with oral delivery) and therapeutic efficacy in oral mucositis models (~6.0-fold improvement in wound closure at day 5 compared with that observed with no treatment). We anticipate that this platform might aid the development of tissue adhesives and inspire the design of nanoparticle-based buccal delivery systems.

Accelerated Bone Regeneration by MOF Modified Multifunctional Membranes through Enhancement of Osteogenic and Angiogenic Performance.

Owing to the insufficient guidance of new bone formation in orthopedic and craniomaxillofacial surgery, construction of a guided bone regeneration membrane to support vascularized bone regeneration remains a challenge. Herein, an electrospun asymmetric double-layer polycaprolactone/collagen (PCL/Col) membrane modified by metal-organic framework (MOF) crystals is developed. The optimization of the PCL/Col weight ratio (1:1 and 1:1.5) enables the composite membrane with a balanced tensile strength (only fell by 49.9% in wet conditions) and a controlled degradation rate (completely degraded at 12 weeks). The MOF crystals can provide a pH-responsive release of Zn2+ ions. In vitro experiments indicate that the barrier layer functions to prevent the infiltration of fibrous connective tissue. The MOF crystal layer functions to enhance osteogenesis and angiogenesis in vitro. Using a rat calvarial defect model, the MOF crystals exhibit a sign of osteoinductivity along with blood vessel formation after 8 weeks post-surgery. Strikingly, when assessed in a chick chorioallantoic membrane model, the MOF modified membrane demonstrates a significant angiogenic response, which can be envisaged as its outstanding merits over the commercially Col membrane. Therefore, the MOF crystals represent an exciting biomaterial option, with neovascularization capacity for bone tissue engineering and regenerative medicine.

Nanoscale Zeolitic Imidazolate Framework-8 Activator of Canonical MAPK Signaling for Bone Repair.

Zeolitic imidazolate framework-8 (ZIF-8) is an important type of metal organic framework and has found numerous applications in the biomedical field. Our previous studies have demonstrated that nano ZIF-8-based titanium implants could promote osseointegration; however, its osteogenic capacity and the related mechanisms in bone regeneration have not been fully clarified. Presented here is a nanoscale ZIF-8 that could drive rat bone mesenchymal stem cell (rBMSC) differentiation into osteoblasts both in vitro and in vivo, and interestingly, nano ZIF-8 exhibited a better osteogenic effect compared with ionic conditions of Zn at the same concentration of Zn2+. Moreover, the cellular uptake mechanisms of the nanoparticles were thoroughly clarified. Specifically, nano ZIF-8 could enter the rBMSC cytoplasm probably via caveolae-mediated endocytosis and macropinocytosis. The intracellular and extracellular Zn2+ released from nano ZIF-8 and the receptors involved in the endocytosis may play a role in inducing activation of key osteogenic pathways. Furthermore, through transcriptome sequencing, multiple osteogenic pathways were found to be upregulated, among which nano ZIF-8 primarily phosphorylated ERK, thus activating the canonical mitogen-activated protein kinase pathway and promoting the osteogenesis of rBMSCs. Taken together, this study helps to elucidate the mechanism by which nano ZIF-8 regulates osteogenesis and suggests it to be a potential biomaterial for constructing multifunctional composites in bone tissue engineering.

ZIF-8-Modified Multifunctional Bone-Adhesive Hydrogels Promoting Angiogenesis and Osteogenesis for Bone Regeneration.

Designing bone adhesives with adhesiveness, antideformation, biocompatibility, and biofunctional effects has great practical significance for bone defect reconstructive treatment, especially for bone graft repair surgery. Here, we designed zeolitic imidazolate framework-8 nanoparticle (ZIF-8 NP)-modified catechol-chitosan (CA-CS) multifunctional hydrogels (CA-CS/Z) to stabilize the bone graft environment, ensure blood supply, promote osteogenic differentiation, and accelerate bone reconstruction. Characterizations confirmed the successful synthesis of CA-CS/Z hydrogels. Hydrogels exhibited advanced rheological properties, reliable mechanical strength, and excellent adhesion for clinical applications. Based on excellent biocompatibility, it could enhance paracrine of the vascular endothelial growth factor (VEGF) in rat bone marrow mesenchymal stem cells (rBMSCs) to ensure blood supply reconstruction in bone defect areas. Furthermore, the ZIF-8 NPs released from the hydrogels could also up-regulate the production and secretion of alkaline phosphatase, collagen 1, and osteocalcin, promoting the osteogenic differentiation of rBMSCs. In addition, the antibacterial properties of CA-CS/Z could also be observed. In vivo experiments further provided a powerful proof that CA-CS/Z promoted vascularized osteogenesis in wound areas by stabilizing bone graft materials and greatly accelerated the speed and healing of bone reconstruction. These results indicate the promising potential of CA-CS/Z hydrogels with promoting implantation stability, angiogenesis, and osteogenesis for bone regeneration applications.

Development and validation of prognostic index based on autophagy-related genes in patient with head and neck squamous cell carcinoma.

Head and neck squamous cell carcinoma (HNSCC) is one of the most common cancers worldwide, accounting for almost 50% of all malignancies in developing nations. Autophagy plays a vital role in cancer initiation, malignant progression, and resistance to treatment. However, autophagy-related gene sets have rarely been analyzed in HNSCC. Hence, it is necessary to assess its clinical and pathological significance in a larger cohort of patients with HNSCC. The purpose of this study was to establish a novel autophagy-related prognostic marker for HNSCC. We screened 232 autophagy-related genes (ARGs) and identified 38 differentially expressed ARGs in The Cancer Genome Atlas (TCGA) cohorts. The prognosis-related ARGs signature, established using the univariate and multivariate Cox proportional regression models, consists of 10 ARGs that could divide patients into high-risk and low-risk groups. Survival analysis indicated that patients in the high-risk group had dramatically shorter overall survival compared with their low-risk counterparts. Cox regression analysis further confirmed the independent prognostic value of the autophagy-related signature, and the area under the receiver operating characteristic curve of the combined prognostic model was 0.722. Finally, the efficacy of autophagy-related signature was also validated by an independent cohort from the Gene Expression Omnibus (GEO) database. Collectively, we successfully constructed a novel autophagy-related signature for the prediction of prognosis in patients with HNSCC.

PEGylated nano-graphene oxide as a nanocarrier for delivering mixed anticancer drugs to improve anticancer activity.

Due to their high specific surface area, graphene oxide and graphene oxide-base nanoparticles have great potential both in dual-drug delivery and combination chemotherapy. Herein, we developed cisplatin (Pt) and doxorubicin (DOX) dual-drug-loaded PEGylated nano-graphene oxide (pGO) to facilitate combined chemotherapy in one system. In this study, nano-sized pGO-Pt/DOX ranged around 161.50 nm was fabricated and characterized using zeta-potential, AFM, TEM, Raman, UV-Vis, and FTIR analyses. The drug delivery efficacy of Pt was enhanced through the introduction of pGO, and the final weight ratio of DOX: Pt: pGO was optimized to 0.376: 0.376: 1. In vitro studies revealed that pGO-Pt/DOX nanoparticles could be effectively delivered into tumor cells, in which they induced prominent cell apoptosis and necrosis and exhibited higher growth inhibition than the single drug delivery system or free drugs. The pGO-Pt/DOX induced the most prominent cancer cell apoptosis and necrosis rate with 18.6%, which was observed almost 2 times higher than that of pGO-Pt or pGO-DOX groups. in the apoptosis and necrotic quadrants In vivo data confirmed that the pGO-Pt/DOX dual-drug delivery system attenuated the toxicity of Pt and DOX to normal organs compared to free drugs. The tumor inhibition data, histopathology observations, and immunohistochemical staining confirmed that the dual-drug delivery system presented a better anticancer effect than free drugs. These results clearly indicated that the pGO-Pt/DOX dual-drug delivery system provided the means for combination drug delivery in cancer treatment.

Guided residual root preparation for socket-shield procedures: A clinical report.

Tooth extraction in the esthetic area is usually accompanied by hard- and soft-tissue changes. The socket-shield technique is an effective method for preventing these undesirable changes. However, difficulty in preparing the socket shield and controlling the implant position might increase the complication rate along with the time needed for the surgery, limiting its widespread use. This clinical report demonstrates a digital protocol for accurate and rapid socket-shield preparation and implant placement with surgical guides.

The enhancement of osseointegration using a graphene oxide/chitosan/hydroxyapatite composite coating on titanium fabricated by electrophoretic deposition.

Titanium (Ti) has been commonly used as an implant material in dentistry and bone surgery for several decades. Meanwhile, surface modification of titanium can enhance the osseointegration of implants. In this study, a graphene oxide/chitosan/hydroxyapatite (GO/CS/HA) composite coating was fabricated by electrophoretic deposition on Ti substrates. Subsequently, the surface morphology, phase composition, wettability, and bonding strength of this composite coating were researched. Additionally, in vitro cytological examination was performed, including evaluations of cell adhesion, cell viability, cell differentiation, cell mineralization, and osteogenetic factor expression. Finally, the in vivo osteogenetic properties were evaluated through an animal study, including a histological analysis, a microcomputed tomography, and biomechanical tests. The results showed that a homogeneous and crack-free GO/CS/HA composite coating was coated on Ti, and the wettability and bonding strength of the GO/CS/HA composite coating were enhanced compared with HA, GO/HA, and CS/HA coatings. Furthermore, the GO/CS/HA coating greatly heightened the cell-material interactions in vitro. Additionally, this GO/CS/HA-Ti implant could enhance osseointegration in vivo. Consequently, GO/CS/HA-Ti may have potential applications in the field of dental implants. © 2018 Wiley Periodicals, Inc. J Biomed Mater Res Part B: Appl Biomater, 2018. © 2018 Wiley Periodicals, Inc. J Biomed Mater Res Part B: Appl Biomater 107B: 635-645, 2019.

Osteogenic activity and antibacterial effect of porous titanium modified with metal-organic framework films.

As a new class of crystalline nanoporous materials, metal-organic frameworks (MOFs) have recently been used for biomedical applications due to their large surface area, high porosity, and theoretically infinite structures. To improve the biological performance of titanium, MOF films were applied to surface modification of titanium. Zn-based MOF films composed of zeolitic imidazolate framework-8 (ZIF-8) crystals with nanoscale and microscale sizes (nanoZIF-8 and microZIF-8) were prepared on porous titanium surfaces by hydrothermal and solvothermal methods, respectively. The ZIF-8 films were characterized by powder X-ray diffraction (PXRD), Fourier transform infrared spectroscopy (FTIR), and scanning electron microscopy (SEM). The nanoZIF-8 film exhibited good biocompatibility, whereas the microZIF-8 film showed obvious cytotoxicity to MG63 cells. Compared to pure titanium and alkali- and heat-treated porous titanium, the nanoZIF-8 film not only enhanced alkaline phosphatase (ALP) activity, extracellular matrix mineralization, and expression of osteogenic genes (ALP, Runx2) in MG63 cells but also inhibited the growth of Streptococcus mutans. These results indicate that MOF films or coatings may be promising candidates for bone tissue engineering. © 2016 Wiley Periodicals, Inc. J Biomed Mater Res Part A: 105A: 834-846, 2017.

Synergistic Enhancement of Antitumor Efficacy by PEGylated Multi-walled Carbon Nanotubes Modified with Cell-Penetrating Peptide TAT.

In the present study, a cell-penetrating peptide, the transactivating transcriptional factor (TAT) domain from HIV, was linked to PEGylated multi-walled carbon nanotubes (MWCNTs) to develop a highly effective antitumor drug delivery system. FITC was conjugated on MWCNTs-polyethylene glycol (PEG) and MWCNTs-PEG-TAT to provide fluorescence signal for tracing the cellular uptake of the nanocarrier. After loaded with an anticancer agent, doxorubicin (DOX) via π - π stacking interaction, the physicochemical characteristics, release profile and biological evaluation of the obtained nano-sized drug carrier were investigated. The DOX loaded MWCNTs-PEG and MWCNTs-PEG-TAT drug carriers both displayed appropriate particle size, excellent stability, high drug loading, and pH-dependent drug release profile. Nevertheless, compared with DOX-MWCNTs-PEG, DOX-MWCNTs-PEG-TAT showed improved cell internalization, intracellular distribution and potentiated anticancer efficacy due to the TAT-mediated membrane translocation, endosomal escape and nuclear targeting. Furthermore, the therapeutic efficacy of DOX was not compromised after being conjugated with MWCNTs-PEG-TAT and the proposed nanocarrier was also confirmed to have a good biocompatibility. In conclusion, our results suggested that the unique combination of TAT and MWCNTs as a multifunctional drug delivery system might be a powerful tool for improved anticancer drug development.

Osteogenic activity and antibacterial effect of zinc oxide/carboxylated graphene oxide nanocomposites: Preparation and in vitro evaluation.

The aim of this study was to prepare nanocomposites of carboxylated graphene oxide (GO-COOH) sheets decorated with zinc oxide (ZnO) nanoparticles (NPs) and investigate their advantages in the field of bone tissue engineering. First, ZnO/GO-COOH nanocomposites were synthesized by facile reactions, including the carboxylation of graphene oxide (GO) and the nucleation of ZnO on GO-COOH sheets. The synthesized ZnO/GO-COOH nanocomposites were then characterized by X-ray diffraction (XRD), X-ray photoelectron spectroscopy (XPS), Raman spectra, and transmission electron microscopy (TEM). The biocompatibility, osteogenic activity and antibacterial effect of ZnO/GO-COOH nanocomposites were further investigated. In the nanocomposites, ZnO nanoparticles with a size of approximately 12nm were uniformly decorated on GO-COOH sheets. Compared with GO-COOH and the control group, ZnO/GO-COOH nanocomposites significantly enhanced ALP activity, osteocalcin production and extracellular matrix mineralization as well as up-regulated osteogenic-related genes (ALP, OCN, and Runx2) in MG63 osteoblast-like cells. Moreover, ZnO/GO-COOH nanocomposites had an antibacterial effect against Streptococcus mutans. These results indicated that ZnO/GO-COOH nanocomposites exhibited both osteogenic activity and antibacterial effect and had great potential for designing new biomaterials in the field of bone tissue engineering.

Multiwall carbon nanotubes/polycaprolactone composites for bone tissue engineering application.

In this study, the multiwall carbon nanotubes (MWNTs)/polycaprolactone composite scaffolds were fabricated by the solution evaporation technique. The morphology, phase composition and the mechanical properties of the composite scaffolds were characterized and the cellular bioactivity of the scaffolds was assessed by using rat bone-marrow-derived stroma cells (BMSCs). The attachment, proliferation and differentiation of the BMSCs on the composite scaffolds were analyzed by scanning electron microscopy (SEM), 4',6-diamidino-2-phenylindole dihydrochloride (DAPI) nuclear staining and fluorescein diacetate (FDA) and propidium iodide (PI) live/dead staining, methylthiazol tetrazolium (MTT) assay and alkaline phosphatase (ALP) activity assay, respectively. Results showed that mechanical properties of the composite scaffolds were improved with the addition of MWNTs (0.25-2 wt%). BMSCs on the composite scaffolds differentiated down the osteogenic lineage and expressed high levels of bone marker ALP. The scaffolds with low concentration (0.5 wt%) of MWNTs can enhance the proliferation and differentiation of the BMSCs more than that with higher concentration of MWNTs. It is concluded that MWNTs/PCL composite scaffolds have the potential for bone tissue engineering and the relatively low concentration of MWNTs (0.5 wt%) is preferred.