Manufacturing 3D Biomimetic Tissue: A Strategy Involving the Integration of Electrospun Nanofibers with a 3D-Printed Framework for Enhanced Tissue Regeneration.

3D printing and electrospinning are versatile techniques employed to produce 3D structures, such as scaffolds and ultrathin fibers, facilitating the creation of a cellular microenvironment in vitro. These two approaches operate on distinct working principles and utilize different polymeric materials to generate the desired structure. This review provides an extensive overview of these techniques and their potential roles in biomedical applications. Despite their potential role in fabricating complex structures, each technique has its own limitations. Electrospun fibers may have ambiguous geometry, while 3D-printed constructs may exhibit poor resolution with limited mechanical complexity. Consequently, the integration of electrospinning and 3D-printing methods may be explored to maximize the benefits and overcome the individual limitations of these techniques. This review highlights recent advancements in combined techniques for generating structures with controlled porosities on the micro-nano scale, leading to improved mechanical structural integrity. Collectively, these techniques also allow the fabrication of nature-inspired structures, contributing to a paradigm shift in research and technology. Finally, the review concludes by examining the advantages, disadvantages, and future outlooks of existing technologies in addressing challenges and exploring potential opportunities.

PAI-1 transfected-conditioned media promotes osteogenic differentiation of hBMSCs.

Reconstruction of injured bone remains challenging in the clinic owing to the lack of suitable bone grafts. The utilization of PAI-1 transfected-conditioned media (P-CM) has demonstrated its ability to facilitate the differentiation process of mesenchymal stem cells (MSCs), potentially serving as a crucial mediator in tissue regeneration. This research endeavored to explore the therapeutic potential of P-CM concerning the differentiation of human bone marrow mesenchymal stem cells (hBMSCs). To assess new bone formation, a rat calvaria critical defect model was employed, while in vitro experiments involved the use of the alizarin Red-S mineral induction test. In the rat calvaria critical defect model, P-CM treatment resulted in significan new bone formation. In vitro, P-CM treated hBMSCs displayed robust osteogenesis compared to the control group, as demonstrated by the mineral induction test using alizarin Red-S. P-CM with hydroxyapatite/ß-tricalcium phosphate/fibrin gel treatment significantly exhibited new bone formation, and the expression of osteogenic associated markers was enhanced in the P-CM-treated group. In conclusion, results demonstrate that P-CM treatment significantly enhanced the osteogenic differantiation efficiency and new bone formation, thus could be used as an ideal therapeutic biomolecule for constructing bone-specific implants, especially for orthopedic and dental applications.

Plant-Actuated Micro-Nanorobotics Platforms: Structural Designs, Functional Prospects, and Biomedical Applications.

Plants are anatomically and physiologically different from humans and animals; however, there are several possibilities to utilize the unique structures and physiological systems of plants and adapt them to new emerging technologies through a strategic biomimetic approach. Moreover, plants provide safe and sustainable results that can potentially solve the problem of mass-producing practical materials with hazardous and toxic side effects, particularly in the biomedical field, which requires high biocompatibility. In this review, it is investigated how micro-nanostructures available in plants (e.g., nanoparticles, nanofibers and their composites, nanoporous materials, and natural micromotors) are adapted and utilized in the design of suitable materials for a micro-nanorobot platform. How plants' work on micro- and nanoscale systems (e.g., surface roughness, osmotically induced movements such as nastic and tropic, and energy conversion and harvesting) that are unique to plants, can provide functionality on the platform and become further prospective resources are examined. Furthermore, implementation across organisms and fields, which is promising for future practical applications of the plant-actuated micro-nanorobot platform, especially on biomedical applications, is discussed. Finally, the challenges following its implementation in the micro-nanorobot platform are also presented to provide advanced adaptation in the future.

Development and characterization of waste equine bone-derived calcium phosphate cements with human alveolar bone-derived mesenchymal stem cells.

Calcium phosphate cements (CPCs) are regarded as promising graft substitutes for bone tissue engineering. However, their wide use is limited by the high cost associated with the complex synthetic processes involved in their fabrication. Cheaper xenogeneic calcium phosphate (CaP) materials derived from waste animal bone may solve this problem. Moreover, the surface topography, mechanical strength, and cellular function of CPCs are influenced by the ratio of micro- to nano-sized CaP (M/NCaP) particles. In this study, we developed waste equine bone (EB)-derived CPCs with various M/NCaP particle ratios to examine the potential capacity of EB-CPCs for bone grafting materials. Our study showed that increasing the number of NCaP particles resulted in reductions in roughness and porosity while promoting smoother surfaces of EB-CPCs. Changes in the chemical properties of EB-CPCs by NCaP particles were observed using X-ray diffractometry. The mechanical properties and cohesiveness of the EB-CPCs improved as the NCaP particle content increased. In an in vitro study, EB-CPCs with a greater proportion of MCaP particles showed higher cell adhesion. Alkaline phosphatase activity indicated that osteogenic differentiation by EB-CPCs was promoted with increased NCaP particle content. These results could provide a design criterion for bone substitutes for orthopedic disease, including periodontal bone defects.

Graphene Oxide-Based Stimuli-Responsive Platforms for Biomedical Applications.

Graphene is a two-dimensional sp2 hybridized carbon material that has attracted tremendous attention for its stimuli-responsive applications, owing to its high surface area and excellent electrical, optical, thermal, and mechanical properties. The physicochemical properties of graphene can be tuned by surface functionalization. The biomedical field pays special attention to stimuli-responsive materials due to their responsive abilities under different conditions. Stimuli-responsive materials exhibit great potential in changing their behavior upon exposure to external or internal factors, such as pH, light, electric field, magnetic field, and temperature. Graphene-based materials, particularly graphene oxide (GO), have been widely used in stimuli-responsive applications due to their superior biocompatibility compared to other forms of graphene. GO has been commonly utilized in tissue engineering, bioimaging, biosensing, cancer therapy, and drug delivery. GO-based stimuli-responsive platforms for wound healing applications have not yet been fully explored. This review describes the effects of different stimuli-responsive factors, such as pH, light, temperature, and magnetic and electric fields on GO-based materials and their applications. The wound healing applications of GO-based materials is extensively discussed with cancer therapy and drug delivery.

Mushroom-Derived Bioactive Molecules as Immunotherapeutic Agents: A Review.

Mushrooms with enhanced medicinal properties focus on finding such compounds that could modulate the human body's immune systems. Mushrooms have antimicrobial, antidiabetic, antiviral, hepatoprotective, antitumor, and immunomodulatory properties due to the presence of various bioactive components. ß-glucans are the major constituent of the mushroom cell wall and play a significant role in their biological activity. This review described the techniques used in the extraction of the active ingredients from the mushroom. We highlighted the structure of the bioactive polysaccharides present in the mushrooms. Therapeutic applications of different mushrooms were also described. It is interesting to note that mushrooms have the potential sources of many bioactive products that can regulate immunity. Thus, the development of functional medicinal food based on the mushroom is vital for human welfare.

Protaetia brevitarsis seulensis Derived Protein Isolate with Enhanced Osteomodulatory and Antioxidative Property.

The osteogenic differentiation of stem cells is profoundly affected by their microenvironmental conditions. The differentiation behavior of stem cells can be tuned by changing the niche environments. The proteins or peptides that are derived by living organisms facilitate the osteogenic differentiation of stem cells. Here, we have evaluated the osteoinductive and antioxidative potential of the Protaetia brevitarsis seulensis insect-derived protein for human bone marrow-derived mesenchymal stem cells (hBMSCs). The amino acid contents in the isolated protein were determined by an amino acid analyzer. Fourier transform infrared (FTIR) spectroscopy and scanning electron microscopy (SEM) were used to analyze the extract's functional groups and surface morphology. The extracted protein exhibited 51.08% ß-sheet conformation. No adverse effects were observed in extract-treated cells, indicating their biocompatibility. The protein isolate showed an excellent antioxidative property. Besides this, an enhancement in the hBMSCs' mineralization has been observed in the presence of treated protein isolates. Notably, osteogenic marker genes and proteins were effectively expressed in the treated cells. These results indicated that the P. brevitarsis-derived protein isolate can be used as a potential antioxidative biomaterial for bone tissue engineering applications.

Stimuli-Mediated Macrophage Switching, Unraveling the Dynamics at the Nanoplatforms-Macrophage Interface.

Macrophages play an essential role in immunotherapy and tissue regeneration owing to their remarkable plasticity and diverse functions. Recent bioengineering developments have focused on using external physical stimuli such as electric and magnetic fields, temperature, and compressive stress, among others, on micro/nanostructures to induce macrophage polarization, thereby increasing their therapeutic potential. However, it is difficult to find a concise review of the interaction between physical stimuli, advanced micro/nanostructures, and macrophage polarization. This review examines the present research on physical stimuli-induced macrophage polarization on micro/nanoplatforms, emphasizing the synergistic role of fabricated structure and stimulation for advanced immunotherapy and tissue regeneration. A concise overview of the research advancements investigating the impact of physical stimuli, including electric fields, magnetic fields, compressive forces, fluid shear stress, photothermal stimuli, and multiple stimulations on the polarization of macrophages within complex engineered structures, is provided. The prospective implications of these strategies in regenerative medicine and immunotherapeutic approaches are highlighted. This review will aid in creating stimuli-responsive platforms for immunomodulation and tissue regeneration.

Sunflower Pollen-Morphology Mimicked Spiky Zinc Nanomotors as a Photosensitizer for Killing Bacteria and Cancer Cells.

Photosensitizing agents have received increased attention from the medical community, owing to their higher photothermal efficiency, induction of hyperthermia, and sustained delivery of bioactive molecules to their targets. Micro/nanorobots can be used as ideal photosensitizing agents by utilizing various physical stimuli for the targeted killing of pathogens (e.g., bacteria) and cancer cells. Herein, we report sunflower-pollen-inspired spiky zinc oxide (s-ZnO)-based nanorobots that effectively kill bacteria and cancer cells under near-infrared (NIR) light irradiation. The as-fabricated s-ZnO was modified with a catechol-containing photothermal agent, polydopamine (PDA), to improve its NIR-responsive properties, followed by the addition of antimicrobial (e.g., tetracycline/TCN) and anticancer (e.g., doxorubicin/DOX) drugs. The fabricated s-ZnO/PDA@Drug nanobots exhibited unique locomotory behavior with an average speed ranging from 13 to 14 µm/s under 2.0 W/cm2 NIR light irradiation. Moreover, the s-ZnO/PDA@TCN nanobots exhibited superior antibacterial activity against E. coli and S. epidermidis under NIR irradiation. The s-ZnO/PDA@DOX nanobots also displayed sufficient reactive oxygen species (ROS) amplification in B16F10 melanoma cells and induced apoptosis under NIR light, indicating their therapeutic efficacy. We hope the sunflower pollen-inspired s-ZnO nanorobots have tremendous potential in biomedical engineering from the phototherapy perspective, with the hope to reduce pathogen infections.

Stimuli-Responsive 3D Printable Conductive Hydrogel: A Step toward Regulating Macrophage Polarization and Wound Healing.

Conductive hydrogels (CHs) are promising alternatives for electrical stimulation of cells and tissues in biomedical engineering. Wound healing and immunomodulation are complex processes that involve multiple cell types and signaling pathways. 3D printable conductive hydrogels have emerged as an innovative approach to promote wound healing and modulate immune responses. CHs can facilitate electrical and mechanical stimuli, which can be beneficial for altering cellular metabolism and enhancing the efficiency of the delivery of therapeutic molecules. This review summarizes the recent advances in 3D printable conductive hydrogels for wound healing and their effect on macrophage polarization. This report also discusses the properties of various conductive materials that can be used to fabricate hydrogels to stimulate immune responses. Furthermore, this review highlights the challenges and limitations of using 3D printable CHs for future material discovery. Overall, 3D printable conductive hydrogels hold excellent potential for accelerating wound healing and immune responses, which can lead to the development of new therapeutic strategies for skin and immune-related diseases.

Zn@TA assisted dual cross-linked 3D printable glycol grafted chitosan hydrogels for robust antibiofilm and wound healing.

Rapid regeneration of the injured tissue or organs is necessary to achieve the usual functionalities of the damaged parts. However, bacterial infections delay the regeneration process, a severe challenge in the personalized healthcare sector. To overcome these challenges, 3D-printable multifunctional hydrogels of Zn/tannic acid-reinforced glycol functionalized chitosan for rapid wound healing were developed. Polyphenol strengthened intermolecular connections, while glutaraldehyde stabilized 3D-printed structures. The hydrogel exhibited enhanced viscoelasticity (G'; 1.96 × 104 Pa) and adhesiveness (210 kPa). The dual-crosslinked scaffolds showed remarkable antibacterial activity against Bacillus subtilis (∼81 %) and Escherichia coli (92.75 %). The hydrogels showed no adverse effects on human dermal fibroblasts (HDFs) and macrophages (RAW 264.7), indicating their superior biocompatibility. The Zn/TA-reinforced hydrogels accelerate M2 polarization of macrophages through the activation of anti-inflammatory transcription factors (Arg-1, VEGF, CD163, and IL-10), suggesting better immunomodulatory effects, which is favorable for rapid wound regeneration. Higher collagen deposition and rapid re-epithelialization occurred in scaffold-treated rat groups vis-à-vis controls, demonstrating superior wound healing. Taken together, the developed multifunctional hydrogels have great potential for rapidly regenerating bacteria-infected wounds in the personalized healthcare sector.

Extracellular Matrix-Bioinspired Anisotropic Topographical Cues of Electrospun Nanofibers: A Strategy of Wound Healing through Macrophage Polarization.

The skin serves as the body's outermost barrier and is the largest organ, providing protection not only to the body but also to various internal organs. Owing to continuous exposure to various external factors, it is susceptible to damage that can range from simple to severe, including serious types of wounds such as burns or chronic wounds. Macrophages play a crucial role in the entire wound-healing process and contribute significantly to skin regeneration. Initially, M1 macrophages infiltrate to phagocytose bacteria, debris, and dead cells in fresh wounds. As tissue repair is activated, M2 macrophages are promoted, reducing inflammation and facilitating restoration of the dermis and epidermis to regenerate the tissue. This suggests that extracellular matrix (ECM) promotes cell adhesion, proliferation, migrationand macrophage polarization. Among the numerous strategies, electrospinning is a versatile technique for obtaining ECM-mimicking structures with anisotropic and isotropic topologies of micro/nanofibers. Various electrospun biomaterials influence macrophage polarization based on their isotropic or anisotropic topologies. Moreover, these fibers possess a high surface-area-to-volume ratio, promoting the effective exchange of vital nutrients and oxygen, which are crucial for cell viability and tissue regeneration. Micro/nanofibers with diverse physical and chemical properties can be tailored to polarize macrophages toward skin regeneration and wound healing, depending on specific requirements. This review describes the significance of micro/nanostructures for activating macrophages and promoting wound healing.

Unzipped carbon nanotubes assisted 3D printable functionalized chitosan hydrogels for strain sensing applications.

Developing multifunctional hydrogels for wearable strain sensors has received significant attention due to their diverse applications, including human motion detection, personalized healthcare, soft robotics, and human-machine interfaces. However, integrating the required characteristics into one component remains challenging. To overcome these limitations, we synthesized multifunctional hydrogels using carboxymethyl chitosan (CMCS) and unzipped carbon nanotubes (f-CNTs) as strain sensor via a one-pot strategy. The polar groups in CMCS and f-CNTs enhance the properties of the hydrogels through different interactions. The hydrogels show superior printability with a uniformity factor (U) of 0.996 ± 0.049, close to 1. The f-CNTs-assisted hydrogels showed improved storage modulus (8.8 × 105 Pa) than the pure polymer hydrogel. The hydrogels adequately adhered to different surfaces, including human skin, plastic, plastic/glass interfaces, and printed polymers. The hydrogels demonstrated rapid self-healing and good conductivity. The biocompatibility of the hydrogels was assessed using human fibroblast cells. No adverse effects were observed with hydrogels, showing their biocompatibility. Furthermore, hydrogels exhibited antibacterial potential against Escherichia coli. The developed hydrogel exhibited unidirectional motion and complex letter recognition potential with a strain sensitivity of 2.4 at 210 % strain. The developed hydrogels could explore developing wearable electronic devices for detecting human motion.

Recent advances and biomedical application of 3D printed nanocellulose-based adhesive hydrogels: A review.

Nanocellulose-based tissue adhesives show promise for achieving rapid hemostasis and effective wound healing. Conventional methods, such as sutures and staples, have limitations, prompting the exploration of bioadhesives for direct wound adhesion and minimal tissue damage. Nanocellulose, a hydrolysis product of cellulose, exhibits superior biocompatibility and multifunctional properties, gaining interest as a base material for bioadhesive development. This study explores the potential of nanocellulose-based adhesives for hemostasis and wound healing using 3D printing techniques. Nanocellulose enables the creation of biodegradable adhesives with minimal adverse effects and opens avenues for advanced wound healing and complex tissue regeneration, such as skin, blood vessels, lungs, cartilage, and muscle. This study reviews recent trends in various nanocellulose-based 3D printed hydrogel patches for tissue engineering applications. The review also introduces various types of nanocellulose and their synthesis, surface modification, and bioadhesive fabrication techniques via 3D printing for smart wound healing.

A Review on Electroactive Polymer-Metal Composites: Development and Applications for Tissue Regeneration.

Electroactive polymer-metal composites (EAPMCs) have gained significant attention in tissue engineering owing to their exceptional mechanical and electrical properties. EAPMCs develop by combining an electroactive polymer matrix and a conductive metal. The design considerations include choosing an appropriate metal that provides mechanical strength and electrical conductivity and selecting an electroactive polymer that displays biocompatibility and electrical responsiveness. Interface engineering and surface modification techniques are also crucial for enhancing the adhesion and biocompatibility of composites. The potential of EAPMC-based tissue engineering revolves around its ability to promote cellular responses, such as cell adhesion, proliferation, and differentiation, through electrical stimulation. The electrical properties of these composites can be used to mimic natural electrical signals within tissues and organs, thereby aiding tissue regeneration. Furthermore, the mechanical characteristics of the metallic components provide structural reinforcement and can be modified to align with the distinct demands of various tissues. EAPMCs have extraordinary potential as regenerative biomaterials owing to their ability to promote beneficial effects in numerous electrically responsive cells. This study emphasizes the characteristics and applications of EAPMCs in tissue engineering.

Nanocellulose-assisted 3D-printable, transparent, bio-adhesive, conductive, and biocompatible hydrogels as sensors and moist electric generators.

Transparent hydrogels have found increasing applications in wearable electronics, printable devices, and tissue engineering. Integrating desired properties, such as conductivity, mechanical strength, biocompatibility, and sensitivity, in one hydrogel remains challenging. To address these challenges, multifunctional hydrogels of methacrylate chitosan, spherical nanocellulose, and ß-glucan with distinct physicochemical characteristics were combined to develop multifunctional composite hydrogels. The nanocellulose facilitated the self-assembly of the hydrogel. The hydrogels exhibited good printability and adhesiveness. Compared with the pure methacrylated chitosan hydrogel, the composite hydrogels exhibited improved viscoelasticity, shape memory, and conductivity. The biocompatibility of the composite hydrogels was monitored using human bone marrow-derived stem cells. Their motion-sensing potential was analyzed on different parts of the human body. The composite hydrogels also possessed temperature-responsiveness and moisture-sensing abilities. These results suggest that the developed composite hydrogels demonstrate excellent potential to fabricate 3D-printable devices for sensing and moist electric generator applications.

Electrically stimulated 3D bioprinting of gelatin-polypyrrole hydrogel with dynamic semi-IPN network induces osteogenesis via collective signaling and immunopolarization.

In recent years, three-dimensional (3D) bioprinting of conductive hydrogels has made significant progress in the fabrication of high-resolution biomimetic structures with gradual complexity. However, the lack of an effective cross-linking strategy, ideal shear-thinning, appropriate yield strength, and higher print fidelity with excellent biofunctionality remains a challenge for developing cell-laden constructs, hindering the progress of extrusion-based 3D printing of conductive polymers. In this study, a highly stable and conductive bioink was developed based on polypyrrole-grafted gelatin methacryloyl (GelMA-PPy) with a triple cross-linking (thermo-photo-ionically) strategy for direct ink writing-based 3D printing applications. The triple-cross-linked hydrogel with dynamic semi-inner penetrating polymer network (semi-IPN) displayed excellent shear-thinning properties, with improved shape fidelity and structural stability during 3D printing. The as-fabricated hydrogel ink also exhibited "plug-like non-Newtonian" flow behavior with minimal disturbance. The bioprinted GelMA-PPy-Fe hydrogel showed higher cytocompatibility (93%) of human bone mesenchymal stem cells (hBMSCs) under microcurrent stimulation (250 mV/20 min/day). Moreover, the self-supporting and tunable mechanical properties of the GelMA-PPy bioink allowed 3D printing of high-resolution biological architectures. As a proof of concept, we printed a full-thickness rat bone model to demonstrate the structural stability. Transcriptomic analysis revealed that the 3D bioprinted hBMSCs highly expressed gene hallmarks for NOTCH/mitogen-activated protein kinase (MAPK)/SMAD signaling while down-regulating the Wnt/ß-Catenin and epigenetic signaling pathways during osteogenic differentiation for up to 7 days. These results suggest that the developed GelMA-PPy bioink is highly stable and non-toxic to hBMSCs and can serve as a promising platform for bone tissue engineering applications.

Recent Advances in 3D Printing of Photocurable Polymers: Types, Mechanism, and Tissue Engineering Application.

The conversion of liquid resin into solid structures upon exposure to light of a specific wavelength is known as photopolymerization. In recent years, photopolymerization-based 3D printing has gained enormous attention for constructing complex tissue-specific constructs. Due to the economic and environmental benefits of the biopolymers employed, photo-curable 3D printing is considered an alternative method for replacing damaged tissues. However, the lack of suitable bio-based photopolymers, their characterization, effective crosslinking strategies, and optimal printing conditions are hindering the extensive application of 3D printed materials in the global market. This review highlights the present status of various photopolymers, their synthesis, and their optimization parameters for biomedical applications. Moreover, a glimpse of various photopolymerization techniques currently employed for 3D printing is also discussed. Furthermore, various naturally derived nanomaterials reinforced polymerization and their influence on printability and shape fidelity are also reviewed. Finally, the ultimate use of those photopolymerized hydrogel scaffolds in tissue engineering is also discussed. Taken together, it is believed that photopolymerized 3D printing has a great future, whereas conventional 3D printing requires considerable sophistication, and this review can provide readers with a comprehensive approach to developing light-mediated 3D printing for tissue-engineering applications.

Transcriptomic Changes toward Osteogenic Differentiation of Mesenchymal Stem Cells on 3D-Printed GelMA/CNC Hydrogel under Pulsatile Pressure Environment.

Biomimetic soft hydrogels used in bone tissue engineering frequently produce unsatisfactory outcomes. Here, it is investigated how human bone-marrow-derived mesenchymal stem cells (hBMSCs) differentiated into early osteoblasts on remarkably soft 3D hydrogel (70 ± 0.00049 Pa). Specifically, hBMSCs seeded onto cellulose nanocrystals incorporated methacrylate gelatin hydrogels are subjected to pulsatile pressure stimulation (PPS) of 5-20 kPa for 7 days. The PPS stimulates cellular processes such as mechanotransduction, cytoskeletal distribution, prohibition of oxidative stress, calcium homeostasis, osteogenic marker gene expression, and osteo-specific cytokine secretions in hBMSCs on soft substrates. The involvement of Piezo 1 is the main ion channel involved in mechanotransduction. Additionally, RNA-sequencing results reveal differential gene expression concerning osteogenic differentiation, bone mineralization, ion channel activity, and focal adhesion. These findings suggest a practical and highly scalable method for promoting stem cell commitment to osteogenesis on soft matrices for clinical reconstruction.

Cellulose nanocrystals vs. cellulose nanospheres: A comparative study of cytotoxicity and macrophage polarization potential.

Nanocellulose application has been increasing owing to its appealing physicochemical properties. Monitoring of the crystallinity, surface topography, and reactivity of this high-aspect-ratio nanomaterial is crucial for efficient tissue engineering. Controlling macrophage polarization phenotype remains a challenge in regenerative medicine and tissue engineering. Herein, we monitored the effects of shape-regulated (rod and spherical) nanocellulose on the macrophage modulatory potential of RAW 246.7 cells in vitro. Spherical nanocellulose (s-NC) exhibited higher thermal stability and biocompatibility than rod nanocellulose. Macrophage polarization was profoundly affected by nanocellulose topography and incubation period. M2 polarization was observed in vitro after 1 day of treatment with s-NC, followed by M1 polarization after treatment for longer periods. Transcriptome analysis similarly revealed that M1 polarization was dominant after 1 day h of incubation with both nanocellulose types. These findings demonstrate that macrophage polarization can be controlled by selecting suitable nanocellulose shape and incubation time for desired applications.