Aided Porous Medium Emulsification for Functional Hydrogel Microparticles Synthesis.

Despite the substantial advancement in developing various hydrogel microparticle (HMP) synthesis methods, emulsification through porous medium to synthesize functional hybrid protein-polymer HMPs has yet to be addressed. Here, the aided porous medium emulsification for hydrogel microparticle synthesis (APME-HMS) system, an innovative approach drawing inspiration from porous medium emulsification is introduced. This method capitalizes on emulsifying immiscible phases within a 3D porous structure for optimal HMP production. Using the APME-HMS system, synthesized responsive bovine serum albumin (BSA) and polyethylene glycol diacrylate (PEGDA) HMPs of various sizes are successfully synthesized. Preserving protein structural integrity and functionality enable the formation of cytochrome c (cyt c) - PEGDA HMPs for hydrogen peroxide (H2O2) detection at various concentrations. The flexibility of the APME-HMS system is demonstrated by its ability to efficiently synthesize HMPs using low volumes (≈50 µL) and concentrations (100 µm) of proteins within minutes while preserving proteins' structural and functional properties. Additionally, the capability of the APME-HMS method to produce a diverse array of HMP types enriches the palette of HMP fabrication techniques, presenting it as a cost-effective, biocompatible, and scalable alternative for various biomedical applications, such as controlled drug delivery, 3D printing bio-inks, biosensing devices, with potential implications even in culinary applications.

Recent advances of bone tissue engineering: carbohydrate and ceramic materials, fundamental properties and advanced biofabrication strategies ‒ a comprehensive review.

Bone is a dynamic tissue that can always regenerate itself through remodeling to maintain biofunctionality. This tissue performs several vital physiological functions. However, bone scaffolds are required for critical-size damages and fractures, and these can be addressed by bone tissue engineering. Bone tissue engineering (BTE) has the potential to develop scaffolds for repairing critical-size damaged bone. BTE is a multidisciplinary engineered scaffold with the desired properties for repairing damaged bone tissue. Herein, we have provided an overview of the common carbohydrate polymers, fundamental structural, physicochemical, and biological properties, and fabrication techniques for bone tissue engineering. We also discussed advanced biofabrication strategies and provided the limitations and prospects by highlighting significant issues in bone tissue engineering. There are several review articles available on bone tissue engineering. However, we have provided a state-of-the-art review article that discussed recent progress and trends within the last 3-5 years by emphasizing challenges and future perspectives.

Droplet-based microfluidics for engineering shape-controlled hydrogels with stiffness gradient.

Current biofabrication strategies are limited in their ability to replicate native shape-to-function relationships, that are dependent on adequate biomimicry of macroscale shape as well as size and microscale spatial heterogeneity, within cell-laden hydrogels. In this study, a novel diffusion-based microfluidics platform is presented that meets these needs in a two-step process. In the first step, a hydrogel-precursor solution is dispersed into a continuous oil phase within the microfluidics tubing. By adjusting the dispersed and oil phase flow rates, the physical architecture of hydrogel-precursor phases can be adjusted to generate spherical and plug-like structures, as well as continuous meter-long hydrogel-precursor phases (up to 1.75 m). The second step involves the controlled introduction a small molecule-containing aqueous phase through a T-shaped tube connector to enable controlled small molecule diffusion across the interface of the aqueous phase and hydrogel-precursor. Application of this system is demonstrated by diffusing co-initiator sodium persulfate (SPS) into hydrogel-precursor solutions, where the controlled SPS diffusion into the hydrogel-precursor and subsequent photo-polymerization allows for the formation of unique radial stiffness patterns across the shape- and size-controlled hydrogels, as well as allowing the formation of hollow hydrogels with controllable internal architectures. Mesenchymal stromal cells are successfully encapsulated within hollow hydrogels and hydrogels containing radial stiffness gradient and found to respond to the heterogeneity in stiffness through the yes-associated protein mechano-regulator. Finally, breast cancer cells are found to phenotypically switch in response to stiffness gradients, causing a shift in their ability to aggregate, which may have implications for metastasis. The diffusion-based microfluidics thus finds application mimicking native shape-to-function relationship in the context of tissue engineering and provides a platform to further study the roles of micro- and macroscale architectural features that exist within native tissues.

Manufacture Dependent Differential Biodegradation of 3D Printed Shape Memory Polymers.

In the field of tissue engineering, 3D printed shape memory polymers (SMPs) are drawing increased interest. Understanding how these 3D printed SMPs degrade is critical for their use in the clinic, as small changes in material properties can significantly change how they behave after in vivo implantation. Degradation of 3D printed acrylated poly(glycerol-dodecanedioate) (APGD) was examined via in vitro hydrolytic, enzymatic, and in vivo subcutaneous implantation assays. Three APGD manufacturing modalities were assessed to determine differences in degradation. Material extrusion samples showed significantly larger mass and volume loss at 2 months, compared to lasercut and vat photopolymerization samples, under both enzymatic and in vivo degradation. Critically, melt transition temperatures of degraded PGD increased over time in vitro, but not in vivo. Histology of tissue surrounding APGD implants showed no significant signs of inflammation compared to controls, providing a promising outlook for use of 3D printed APGD devices in the clinic.

Sustainable biofabrication: from bioprinting to AI-driven predictive methods.

Biofabrication is potentially an inherently sustainable manufacturing process of bio-hybrid systems based on biomaterials embedded with cell communities. These bio-hybrids promise to augment the sustainability of various human activities, ranging from tissue engineering and robotics to civil engineering and ecology. However, as routine biofabrication practices are laborious and energetically disadvantageous, our society must refine production and validation processes in biomanufacturing. This opinion highlights the research trends in sustainable material selection and biofabrication techniques. By modeling complex biosystems, the computational prediction will allow biofabrication to shift from an error-trial method to an efficient, target-optimized approach with minimized resource and energy consumption. We envision that implementing bionomic rationality in biofabrication will render bio-hybrid products fruitful for greening human activities.

Mass Spectrometry of Collagen-Containing Allogeneic Human Bone Tissue Material.

The current paper highlights the active development of tissue engineering in the field of the biofabrication of living tissue analogues through 3D-bioprinting technology. The implementation of the latter is impossible without important products such as bioinks and their basic components, namely, hydrogels. In this regard, tissue engineers are searching for biomaterials to produce hydrogels with specified properties both in terms of their physical, mechanical and chemical properties and in terms of local biological effects following implantation into an organism. One of such effects is the provision of the optimal conditions for physiological reparative regeneration by the structural components that form the basis of the biomaterial. Therefore, qualitative assessment of the composition of the protein component of a biomaterial is a significant task in tissue engineering and bioprinting. It is important for predicting the behaviour of printed constructs in terms of their gradual resorption followed by tissue regeneration due to the formation of a new extracellular matrix. One of the most promising natural biomaterials with significant potential in the production of hydrogels and the bioinks based on them is the polymer collagen of allogeneic origin, which plays an important role in maintaining the structural and biological integrity of the extracellular matrix, as well as in the morphogenesis and cellular metabolism of tissues, giving them the required mechanical and biochemical properties. In tissue engineering, collagen is widely used as a basic biomaterial because of its availability, biocompatibility and facile combination with other materials. This manuscript presents the main results of a mass spectrometry analysis (proteomic assay) of the lyophilized hydrogel produced from the registered Lyoplast® bioimplant (allogeneic human bone tissue), which is promising in the field of biotechnology. Proteomic assays of the investigated lyophilized hydrogel sample showed the presence of structural proteins (six major collagen fibers of types I, II, IV, IX, XXVII, XXVIII were identified), extracellular matrix proteins, and mRNA-stabilizing proteins, which participate in the regulation of transcription, as well as inducer proteins that mediate the activation of regeneration, including the level of circadian rhythm. The research results offer a new perspective and indicate the significant potential of the lyophilized hydrogels as an effective alternative to synthetic and xenogeneic materials in regenerative medicine, particularly in the field of biotechnology, acting as a matrix and cell-containing component of bioinks for 3D bioprinting.

Personalized compression therapeutic textiles: digital design, development, and biomechanical evaluation.

Physical-based external compression medical modalities could provide sustainable interfacial pressure dosages for daily healthcare prophylaxis and clinic treatment of chronic venous disease (CVD). However, conventional ready-made compression therapeutic textiles (CTs) with improper morphologies and ill-fitting of pressure exertions frequently limit patient compliance in practical application. Therefore, the present study fabricated the personalized CTs for various subjects through the proposed comprehensive manufacturing system. The individual geometric dimensions and morphologic profiles of lower extremities were characterized according to three-dimensional (3D) body scanning and reverse engineering technologies. Through body anthropometric analysis and pressure optimization, the knitting yarn and machinery variables were determined as the digital design strategies for 3D seamless fabrication of CTs. Next, to visually simulate the generated pressure mappings of developed CTs, the subject-specific 3D finite element (FE) CT-leg modelings with high accuracy and acceptability (pressure prediction error ratio: 11.00% ± 7.78%) were established based on the constructed lower limb models and determined tissue stiffness. Moreover, through the actual in vivo trials, the prepared customized CTs efficiently (Sig. <0.05; ρ = 0.97) distributed the expected pressure requirements referring to the prescribed compression magnitudes (pressure error ratio: 10.08% ± 7.75%). Furthermore, the movement abilities and comfortable perceptions were evaluated subjectively for the ergonomic wearing comfort (EWC) assessments. Thus, this study promotes the precise pressure management and clinical efficacy for targeted users and leads an operable development approach for related medical biomaterials in compression therapy.

Coumarin-Based Photodegradable Hydrogels Enable Two-Photon Subtractive Biofabrication at 300†mm s-1.

Spatiotemporally controlled two-photon photodegradation of hydrogels has gained increasing attention for high-precision subtractive tissue engineering. However, conventional photolabile hydrogels often have poor efficiency upon two-photon excitation in the near-infrared (NIR) region and thus require high laser dosage that may compromise cell activity. As a result, high-speed two-photon hydrogel erosion in the presence of cells remains challenging. Here we introduce the design and synthesis of efficient coumarin-based photodegradable hydrogels to overcome these limitations. A set of photolabile coumarin-functionalized polyethylene glycol linkers are synthesized through a Passerini multicomponent reaction. After mixing these linkers with thiolated hyaluronic acid, semi-synthetic photodegradable hydrogels are formed in situ via Michael addition crosslinking. The efficiency of photodegradation in these hydrogels is significantly higher than that in nitrobenzyl counterparts upon two-photon irradiation at 780 nm. A complex microfluidic network mimicking the bone microarchitecture is successfully fabricated in preformed coumarin hydrogels at high speeds of up to 300 mm s-1 and low laser dosage down to 10 mW. Further, we demonstrate fast two-photon printing of hollow microchannels inside a hydrogel to spatiotemporally direct cell migration in 3D. Collectively, these hydrogels may open new avenues for fast laser-guided tissue fabrication at high spatial resolution.

Various Antibacterial Strategies Utilizing Titanium Dioxide Nanotubes Prepared via Electrochemical Anodization Biofabrication Method.

Currently, titanium and its alloys have emerged as the predominant metallic biomaterials for orthopedic implants. Nonetheless, the relatively high post-operative infection rate (2-5%) exacerbates patient discomfort and imposes significant economic costs on society. Hence, urgent measures are needed to enhance the antibacterial properties of titanium and titanium alloy implants. The titanium dioxide nanotube array (TNTA) is gaining increasing attention due to its topographical and photocatalytic antibacterial properties. Moreover, the pores within TNTA serve as excellent carriers for chemical ion doping and drug loading. The fabrication of TNTA on the surface of titanium and its alloys can be achieved through various methods. Studies have demonstrated that the electrochemical anodization method offers numerous significant advantages, such as simplicity, cost-effectiveness, and controllability. This review presents the development process of the electrochemical anodization method and its applications in synthesizing TNTA. Additionally, this article systematically discusses topographical, chemical, drug delivery, and combined antibacterial strategies. It is widely acknowledged that implants should possess a range of favorable biological characteristics. Clearly, addressing multiple needs with a single antibacterial strategy is challenging. Hence, this review proposes systematic research into combined antibacterial strategies to further mitigate post-operative infection risks and enhance implant success rates in the future.

Design and evaluation of a bilayered dermal/hypodermal 3D model using a biomimetic hydrogel formulation.

Due to the limitations of the current skin wound treatments, it is highly valuable to have a wound healing formulation that mimics the extracellular matrix (ECM) and mechanical properties of natural skin tissue. Here, a novel biomimetic hydrogel formulation has been developed based on a mixture of Agarose-Collagen Type I (AC) combined with skin ECM-related components: Dermatan sulfate (DS), Hyaluronic acid (HA), and Elastin (EL) for its application in skin tissue engineering (TE). Different formulations were designed by combining AC hydrogels with DS, HA, and EL. Cell viability, hemocompatibility, physicochemical, mechanical, and wound healing properties were investigated. Finally, a bilayered hydrogel loaded with fibroblasts and mesenchymal stromal cells was developed using the Ag-Col I-DS-HA-EL (ACDHE) formulation. The ACDHE hydrogel displayed the best in vitro results and acceptable physicochemical properties. Also, it behaved mechanically close to human native skin and exhibited good cytocompatibility. Environmental scanning electron microscopy (ESEM) analysis revealed a porous microstructure that allows the maintenance of cell growth and ECM-like structure production. These findings demonstrate the potential of the ACDHE hydrogel formulation for applications such as an injectable hydrogel or a bioink to create cell-laden structures for skin TE.

Biofabrication of Living Actuators.

The impact of tissue engineering has extended beyond a traditional focus in medicine to the rapidly growing realm of biohybrid robotics. Leveraging living actuators as functional components in machines has been a central focus of this field, generating a range of compelling demonstrations of robots capable of muscle-powered swimming, walking, pumping, gripping, and even computation. In this review, we highlight key advances in fabricating tissue-scale cardiac and skeletal muscle actuators for a range of functional applications. We discuss areas for future growth including scalable manufacturing, integrated feedback control, and predictive modeling and also propose methods for ensuring inclusive and bioethics-focused pedagogy in this emerging discipline. We hope this review motivates the next generation of biomedical engineers to advance rational design and practical use of living machines for applications ranging from telesurgery to manufacturing to on- and off-world exploration.

Apoptotic Induction by Biosynthesized Gold Nanoparticles Using Phormidesmis communis Strain AB_11_10 against Osteosarcoma Cancer.

Phormidesmis communis strain AB_11_10 was isolated and identified using microscopy and 16s rRNA sequencing, and its phytochemical constituents were determined using liquid chromatography-quadrupole time-of-flight mass spectrometry. The isolate had a segmented filamentous shape with a blue-green color. Many biomolecules, including organic compounds, amino acids, and fatty acids, were detected. P. communis strain AB_11_10 was used to synthesize gold nanoparticles (Ph-AuNPs) by adjusting the optimum reaction conditions. The concentration, algal/precursor ratio, temperature, reaction time, and pH significantly influenced the synthesis of the Ph-AuNPs. Mixing 1 mL of 0.5 mM of HAuCl4 with 1 mL of algal extract and exposing the mixture to 100 °C for 30 min at pH 5.6 were the optimum conditions for the biosynthesis of Ph-AuNPs at a wavelength of 524.5 nm. The Ph-AuNPs were characterized using TEM, SEM, EDX, and mapping Zeta sizer and FTIR. The Ph-AuNPs had quasi-spherical to triangular shapes with an average diameter of 9.6 ± 4.3 nm. Ph-AuNPs composed of 76.10 ± 3.14% of Au and trace amounts of carbon and oxygen were detected, indicating that the P. communis strain AB_11_10 successfully synthesized Ph-AuNPs. The hydrodynamic diameter of the Ph-AuNPs was 28.5 nm, and their potential charge was -17.7 mV. O-H, N-H, C=C, N-O, C-H, and C-O were coated onto the surfaces of the Ph-AuNPs. These groups correspond to algal phytochemicals, which may have been the main reducing and stabilizing substances during the Ph-AuNP synthesis. The therapeutic activity of the Ph-AuNPs against osteosarcoma cancers was examined in MG-63 and SAOS-2 cell lines, while their biocompatibility was tested against Vero cell lines using a sulforhodamine B assay. The Ph-AuNPs had potent antitumor activity against the MG-63 and SAOS-2 cells, with a low toxicity toward Vero cells. Flow cytometry and cell cycle arrest analyses revealed that the Ph-AuNPs enhanced the apoptotic pathway and arrested the cell cycle in the MG-63 and SAOS-2 cells. P. communis strain AB_11_10 provides a new source to synthesize small, stable, and biocompatible AuNPs that act as apoptotic enhancers in osteosarcoma.

Advancing Synthetic Hydrogels through Nature-Inspired Materials Chemistry.

Synthetic extracellular matrix (ECM) mimics that can recapitulate the complex biochemical and mechanical nature of native tissues are needed for advanced models of development and disease. Biomedical research has heavily relied on the use of animal-derived biomaterials, which is now impeding their translational potential and convoluting the biological insights gleaned from in vitro tissue models. Natural hydrogels have long served as a convenient and effective cell culture tool, but advances in materials chemistry and fabrication techniques now present promising new avenues for creating xenogenic-free ECM substitutes appropriate for organotypic models and microphysiological systems. However, significant challenges remain in creating synthetic matrices that can approximate the structural sophistication, biochemical complexity, and dynamic functionality of native tissues. This review summarizes key properties of the native ECM, and discusses recent approaches used to systematically decouple and tune these properties in synthetic matrices. The importance of dynamic ECM mechanics, such as viscoelasticity and matrix plasticity, is also discussed, particularly within the context of organoid and engineered tissue matrices. Emerging design strategies to mimic these dynamic mechanical properties are reviewed, such as multi-network hydrogels, supramolecular chemistry, and hydrogels assembled from biological monomers.

Advances in 3D bioprinting for regenerative medicine applications.

Biofabrication techniques allow for the construction of biocompatible and biofunctional structures composed from biomaterials, cells and biomolecules. Bioprinting is an emerging 3D printing method which utilizes biomaterial-based mixtures with cells and other biological constituents into printable suspensions known as bioinks. Coupled with automated design protocols and based on different modes for droplet deposition, 3D bioprinters are able to fabricate hydrogel-based objects with specific architecture and geometrical properties, providing the necessary environment that promotes cell growth and directs cell differentiation towards application-related lineages. For the preparation of such bioinks, various water-soluble biomaterials have been employed, including natural and synthetic biopolymers, and inorganic materials. Bioprinted constructs are considered to be one of the most promising avenues in regenerative medicine due to their native organ biomimicry. For a successful application, the bioprinted constructs should meet particular criteria such as optimal biological response, mechanical properties similar to the target tissue, high levels of reproducibility and printing fidelity, but also increased upscaling capability. In this review, we highlight the most recent advances in bioprinting, focusing on the regeneration of various tissues including bone, cartilage, cardiovascular, neural, skin and other organs such as liver, kidney, pancreas and lungs. We discuss the rapidly developing co-culture bioprinting systems used to resemble the complexity of tissues and organs and the crosstalk between various cell populations towards regeneration. Moreover, we report on the basic physical principles governing 3D bioprinting, and the ideal bioink properties based on the biomaterials' regenerative potential. We examine and critically discuss the present status of 3D bioprinting regarding its applicability and current limitations that need to be overcome to establish it at the forefront of artificial organ production and transplantation.

[n]Cycloparaphenylenes as Compatible Fluorophores for Melt Electrowriting.

Fluorescent probes are an indispensable tool in the realm of bioimaging technologies, providing valuable insights into the assessment of biomaterial integrity and structural properties. However, incorporating fluorophores into scaffolds made from melt electrowriting (MEW) poses a challenge due to the sustained, elevated temperatures that this processing technique requires. In this context, [n]cycloparaphenylenes ([n]CPPs) serve as excellent fluorophores for MEW processing with the additional benefit of customizable emissions profiles with the same excitation wavelength. Three fluorescent blends are used with distinct [n]CPPs with emission wavelengths of either 466, 494, or 533 nm, identifying 0.01 wt% as the preferred concentration. It is discovered that [n]CPPs disperse well within poly(ε-caprolactone) (PCL) and maintain their fluorescence even after a week of continuous heating at 80 °C. The [n]CPP-PCL blends show no cytotoxicity and support counterstaining with commonly used DAPI (Ex/Em: 359 nm/457 nm), rhodamine- (Ex/Em: 542/565 nm), and fluorescein-tagged (Ex/Em: 490/515 nm) phalloidin stains. Using different color [n]CPP-PCL blends, different MEW fibers are sequentially deposited into a semi-woven scaffold and onto a solution electrospun membrane composed of [8]CPP-PCL as a contrasting substrate for the [10]CPP-PCL MEW fibers. In general, [n]CPPs are potent fluorophores for MEW, providing new imaging options for this technology.

Diverse Shape Design and Physical Property Evaluation of In-Body Tissue Architecture-Induced Tissues.

Autologous-engineered artificial tissues constitute an ideal alternative for radical surgery in terms of natural anticoagulation, self-repair, tissue regeneration, and the possibility of growth. Previously, we focused on the development and practical application of artificial tissues using "in-body tissue architecture (iBTA)", a technique that uses living bodies as bioreactors. This study aimed to further develop iBTA by fabricating tissues with diverse shapes and evaluating their physical properties. Although the breaking strength increased with tissue thickness, the nominal breaking stress increased with thinner tissues. By carving narrow grooves on the outer periphery of an inner core with narrow grooves, we fabricated approximately 2.2 m long cord-shaped tissues and net-shaped tissues with various designs. By assembling the two inner cores inside the branched stainless-steel pipes, a large graft with branching was successfully fabricated, and its aortic arch replacement was conducted in a donor goat without causing damage. In conclusion, by applying iBTA technology, we have made it possible, for the first time, to create tissues of various shapes and designs that are difficult using existing tissue-engineering techniques. Thicker iBTA-induced tissues exhibited higher rupture strength; however, rupture stress was inversely proportional to thickness. These findings broaden the range of iBTA-induced tissue applications.

Advanced optical assessment and modeling of extrusion bioprinting.

In the context of tissue engineering, biofabrication techniques are employed to process cells in hydrogel-based matrices, known as bioinks, into complex 3D structures. The aim is the production of functional tissue models or even entire organs. The regenerative production of biological tissues adheres to a multitude of criteria that ultimately determine the maturation of a functional tissue. These criteria are of biological nature, such as the biomimetic spatial positioning of different cell types within a physiologically and mechanically suitable matrix, which enables tissue maturation. Furthermore, the processing, a combination of technical procedures and biological materials, has proven highly challenging since cells are sensitive to stress, for example from shear and tensile forces, which may affect their vitality. On the other hand, high resolutions are pursued to create optimal conditions for subsequent tissue maturation. From an analytical perspective, it is prudent to first investigate the printing behavior of bioinks before undertaking complex biological tests. According to our findings, conventional shear rheological tests are insufficient to fully characterize the printing behavior of a bioink. For this reason, we have developed optical methods that, complementarily to the already developed tests, allow for quantification of printing quality and further viscoelastic modeling of bioinks.

Biofabrication of Tissue-Engineered Cartilage Constructs Through Faraday Wave Bioassembly of Cell-Laden Gelatin Microcarriers.

Acoustic biofabrication is an emerging strategy in tissue engineering due to its mild and fast manufacturing process. Herein, tissue-engineered cartilage constructs with high cell viability are fabricated from cell-laden gelatin microcarriers (GMs) through Faraday wave bioassembly, a typical acoustic "bottom-up" manufacturing process. Assembly modules are first prepared by incorporating cartilage precursor cells, the chondrogenic cell line ATDC5, or bone marrow-derived mesenchymal stem cells (BMSCs), into GMs. Patterned structures are formed by Faraday wave bioassembly of the cell-laden GMs. Due to the gentle and efficient assembly process and the protective effects of microcarriers, cells in the patterned structures maintain high activity. Subsequently, tissue-engineered cartilage constructs are obtained by inducing cell differentiation of the patterned structures. Comprehensive evaluations are conducted to verify chondrocyte differentiation and the formation of cartilage tissue constructs in terms of cell viability, morphological analysis, gene expression, and matrix production. Finally, implantation studies with a rat cartilage defect model demonstrate that these tissue-engineered cartilage constructs are beneficial for the repair of articular cartilage damage in vivo. This study provides the first biofabrication of cartilage tissue constructs using Faraday wave bioassembly, extending its application to engineering tissues with a low cell density.

A sustainable green-approach for biofabrication of chitosan nanoparticles, optimization, characterization, its antifungal activity against phytopathogenic Fusarium culmorum and antitumor activity.

Chitosan is a natural non-toxic, biocompatible, biodegradable, and mucoadhesive polymer. It also has a broad spectrum of applications such as agriculture, medical fields, cosmetics and food industries. In this investigation, chitosan nanoparticles were produced by an aqueous extract of Cympopogon citratus leaves as a reducing agent. According to the SEM and TEM micrographs, CNPs had a spherical shape, and size ranging from 8.08 to 12.01 nm. CNPs have a positively charged surface with a Zeta potential of + 26 mV. The crystalline feature of CNPs is determined by X-ray diffraction. There are many functional groups, including C꞊C, CH2-OH, C-O, C-S, N-H, CN, CH and OH were detected by FTIR analysis. As shown by the thermogravimetric study, CNPs have a high thermal stability. For the optimization of the green synthesis of CNPs, a Face centered central composite design (FCCCD) with 30 trials was used. The maximum yield of CNPs (13.99 mg CNPs/mL) was produced with chitosan concentration 1.5%, pH 4.5 at 40 °C, and incubation period of 30 min. The antifungal activity of CNPs was evaluated against phytopathogenic fungus; Fusarium culmorum. A 100% rate of mycelial growth inhibition was gained by the application of 20 mg CNPs/mL. The antitumor activity of the green synthesized CNPs was examined using 6 different cell lines, the viability of the cells reduced when the concentration of green synthesized CNPs increased, the IC50 dose of the green synthesized CNPs on the examined cell lines HePG-2, MCF-7, HCT-116, PC-3, Hela and WI-38 was 36.25 ± 2.3, 31.21 ± 2.2, 67.45 ± 3.5, 56.30 ± 3.3, 44.62 ± 2.6 and 74.90 ± 3.8; respectively.