Mulberry Leaf Lipid Nanoparticles: a Naturally Targeted CRISPR/Cas9 Oral Delivery Platform for Alleviation of Colon Diseases.

Oral treatment of colon diseases with the CRISPR/Cas9 system has been hampered by the lack of a safe and efficient delivery platform. Overexpressed CD98 plays a crucial role in the progression of ulcerative colitis (UC) and colitis-associated colorectal cancer (CAC). In this study, lipid nanoparticles (LNPs) derived from mulberry leaves are functionalized with Pluronic copolymers and optimized to deliver the CRISPR/Cas gene editing machinery for CD98 knockdown. The obtained LNPs possessed a hydrodynamic diameter of 267.2 nm, a narrow size distribution, and a negative surface charge (-25.6 mV). Incorporating Pluronic F127 into LNPs improved their stability in the gastrointestinal tract and facilitated their penetration through the colonic mucus barrier. The galactose end groups promoted endocytosis of the LNPs by macrophages via asialoglycoprotein receptor-mediated endocytosis, with a transfection efficiency of 2.2-fold higher than Lipofectamine 6000. The LNPs significantly decreased CD98 expression, down-regulated pro-inflammatory cytokines (TNF-α and IL-6), up-regulated anti-inflammatory factors (IL-10), and polarized macrophages to M2 phenotype. Oral administration of LNPs mitigated UC and CAC by alleviating inflammation, restoring the colonic barrier, and modulating intestinal microbiota. As the first oral CRISPR/Cas9 delivery LNP, this system offers a precise and efficient platform for the oral treatment of colon diseases.

Natural lipid nanoparticles extracted from Morus nigra L. leaves for targeted treatment of hepatocellular carcinoma via the oral route.

The clinical application of conventional medications for hepatocellular carcinoma treatment has been severely restricted by their adverse effects and unsatisfactory therapeutic effectiveness. Inspired by the concept of 'medicine food homology', we extracted and purified natural exosome-like lipid nanoparticles (LNPs) from black mulberry (Morus nigra L.) leaves. The obtained MLNPs possessed a desirable hydrodynamic particle size (162.1 nm), a uniform size distribution (polydispersity index = 0.025), and a negative surface charge (-26.6 mv). These natural LNPs were rich in glycolipids, functional proteins, and active small molecules (e.g., rutin and quercetin 3-O-glucoside). In vitro experiments revealed that MLNPs were preferentially internalized by liver tumor cell lines via galactose receptor-mediated endocytosis, increased intracellular oxidative stress, and triggered mitochondrial damage, resulting in suppressing the viability, migration, and invasion of these cells. Importantly, in vivo investigations suggested that oral MLNPs entered into the circulatory system mainly through the jejunum and colon, and they exhibited negligible adverse effects and superior anti-liver tumor outcomes through direct tumor killing and intestinal microbiota modulation. These findings collectively demonstrate the potential of MLNPs as a natural, safe, and robust nanomedicine for oral treatment of hepatocellular carcinoma.

Coculture of adipose-derived mesenchymal stem cells/macrophages on decellularized placental sponge promotes differentiation into the osteogenic lineage.

BACKGROUND: Several factors like three-dimensional microstructure, growth factors, cytokines, cell-cell communication, and coculture with functional cells can affect the stem cells behavior and differentiation. The purpose of this study was to investigate the potential of decellularized placental sponge as adipose-derived mesenchymal stem cells (AD-MSCs) and macrophage coculture systems, and guiding the osteogenic differentiation of stem cells. METHODS: The decellularized placental sponge (DPS) was fabricated, and its mechanical characteristics were evaluated using degradation assay, swelling rate, and pore size determination. Its structure was also investigated using hematoxylin and eosin staining and scanning electron microscopy. Mouse peritoneal macrophages and AD-MSCs were isolated and characterized. The differentiation potential of AD-MSCs co-cultured with macrophages was evaluated by RT-qPCR of osteogenic genes on the surface of DPS. The in vivo biocompatibility of DPS was determined by subcutaneous implantation of scaffold and histological evaluations of the implanted site. RESULTS: The DPS had 67% porosity with an average pore size of 238 µm. The in vitro degradation assay showed around 25% weight loss during 30 days in PBS. The swelling rate was around 50% during 72 h. The coculture of AD-MSCs/macrophages on the DPS showed a significant upregulation of four differentiation osteogenic lineage genes in AD-MSCs on days 14 and 21 and a significantly higher mineralization rate than the groups without DPS. Subcutaneous implantation of DPS showed in vivo biocompatibility of scaffold during 28 days follow-up. CONCLUSIONS: Our findings suggest the decellularized placental sponge as an excellent bone substitute providing a naturally derived matrix substrate with biostructure close to the natural bone that guided differentiation of stem cells toward bone cells and a promising coculture substrate for crosstalk of macrophage and mesenchymal stem cells in vitro.

Smart Responsive Microneedles for Controlled Drug Delivery.

As an emerging technology, microneedles offer advantages such as painless administration, good biocompatibility, and ease of self-administration, so as to effectively treat various diseases, such as diabetes, wound repair, tumor treatment and so on. How to regulate the release behavior of loaded drugs in polymer microneedles is the core element of transdermal drug delivery. As an emerging on-demand drug-delivery technology, intelligent responsive microneedles can achieve local accurate release of drugs according to external stimuli or internal physiological environment changes. This review focuses on the research efforts in smart responsive polymer microneedles at home and abroad in recent years. It summarizes the response mechanisms based on various stimuli and their respective application scenarios. Utilizing innovative, responsive microneedle systems offers a convenient and precise targeted drug delivery method, holding significant research implications in transdermal drug administration. Safety and efficacy will remain the key areas of continuous efforts for research scholars in the future.

Bioengineering of fibroblast-conditioned polycaprolactone/gelatin electrospun scaffold for skin tissue engineering.

BACKGROUND: Synthetic tissue engineering scaffolds has poor biocompatiblity with very low angiogenic properties. Conditioning the scaffolds with functional groups, coating with biological components, especially extracellular matrix (ECM), is an excellent strategy for improving their biomechanical and biological properties. METHODS: In the current study, a composite of polycaprolactone and gelatin (PCL/Gel) was electrospun in the ratio of 70/30 and surface modified with 1% gelatin-coating (G-PCL/Gel) or plasma treatment (P-PCL/Gel). The surface modification was determined by SEM and ATR-FTIR spectroscopy, respectively. The scaffolds were cultured with fibroblast 3T3, then decellularized during freeze-thawing process to fabricate a fibroblast ECM-conditioned PCL/Gel scaffold (FC-PCL/Gel). The swelling and degaradtion as well as in vitro and in vivo biocompatibility and angiogenic properties of the scaffolds were evaluated. RESULTS: The structure of the surface-modified G-PCL/Gel and P-PCL/Gel were unique and not changed compared with the PCL/Gel scaffolds. ATR-FTIR analysis admitted the formation of oxygen-containing groups, hydroxyl and carboxyl, on the surface of the P-PCL/Gel scaffold. The SEM micrographs and DAPI staining confirmed the cell attachment and the ECM deposition on the platform and successful removal of the cells after decellularization. P-PCL/Gel showed better cell attachment, ECM secretion and deposition after decellularization compared with G-PCL/Gel. The FC-PCL/Gel was considered as an optimized scaffold for further assays in this study. The FC-PCL/Gel showed increased hydrophilic behavior and cytobiocompatibility compared with P-PCL/Gel. The ECM on the FC-PCL/Gel scaffold showed a gradual degradation during 30 days of degradation time, as a small amount of ECM remained over the FC-PCL/Gel scaffold at day 30. The FC-PCL/Gel showed significant biocompatibility and improved angiogenic property compared with P-PCL/Gel when subcutaneously implanted in a mouse animal model for 7 and 28 days. CONCLUSIONS: Our findings suggest FC-PCL/Gel as an excellent biomimetic construct with high angiogenic properties. This bioengineered construct can serve as a possible application in our future pre-clinical and clinical studies for skin regeneration.

Current Trends in Microfluidics and Biosensors for Cancer Research Applications.

Despite the significant amount of resources invested, cancer remains a considerable burden in our modern society and a leading cause of death. There is still a lack of knowledge about the mechanistic determinants of the disease, the mechanism of action of drugs, and the process of tumor relapse. Current methodologies to study all these events fail to provide accurate information, threatening the prognosis of cancer patients. This failure is due to the inadequate procedure in how tumorigenesis is studied and how drug discovery and screening are currently made. Traditionally, they both rely on seeding cells on static flat cultures and on the immunolabelling of cellular structures, which are usually limited in their ability to reproduce the complexity of the native cellular habitat and provide quantitative data. Similarly, more complex animal models are employed for-unsuccessfully-mimicking the human physiology and evaluating the etiology of the disease or the efficacy/toxicity of pharmacological compounds. Despite some breakthroughs and success obtained in understanding the disease and developing novel therapeutic approaches, cancer still kills millions of people worldwide, remaining a global healthcare problem with a high social and economic impact. There is a need for novel integrative methodologies and technologies capable of providing valuable readouts. In this regard, the combination of microfluidics technology with miniaturized biosensors offers unprecedented advantages to accelerate the development of drugs. This integrated technology have the potential to unravel the key pathophysiological processes of cancer progression and metastasis, overcoming the existing gap on in vitro predictive platforms and in vivo model systems. Herein, we discuss how this combination may boost the field of cancer theranostics and drug discovery/screening toward more precise devices with clinical relevance.

Coupling Micro-Physiological Systems and Biosensors for Improving Cancer Biomarkers Detection.

Early cancer detection is still a major clinical challenge. The development of innovative and noninvasive screening approaches for the detection of predictive biomarkers indicating the stage of the disease could save many lives. Traditional in vitro and in vivo models are not adequate to copycat the native tumor microenvironment and for the discovery of new biomarkers. Recent advances in microfluidics, biosensors, and 3D cell biology speed up the development of micro-physiological bioengineered systems that improve the discovery of new potential cancer biomarkers. This can accelerate the individualization of cancer treatments leading to precision medicine-oriented approaches that could improve patient prognosis. For this reason, it is necessary to develop point-of-care diagnostic tools that can be user-friendly, miniaturized, and easily translated into clinical practice. This chapter describes how far this new generation of cutting-edge technologies, such as microfluidics, label-free detection systems, and molecular diagnostics, are from being applied in the current clinical practice.

From Exosomes to Circulating Tumor Cells: Using Microfluidics to Detect High Predictive Cancer Biomarkers.

Early cancer screening and effective diagnosis is the most effective form to diminish the number of cancer-related deaths. Liquid biopsy constitutes an attractive alternative to tumor biopsy due to its non-invasive nature and sample accessibility, which permits effective screening and patient monitoring. Within the plethora of biomarkers present in circulation, liquid biopsy has mainly been performed by analyzing circulating tumor cells, and more recently, extracellular vesicles. Tracking these biological particles could provide valuable insights into cancer origin, progression, treatment efficacy, and patient prognosis. Microfluidic devices have emerged as viable solutions for point-of-care cancer screening and monitoring due to their user-friendly operation, low operation costs, and capability of processing, quantifying, and analyzing these bioparticles in a single device. However, the size difference between cells and exosomes (micrometer vs nanometer) requires an adaptation of microfluidic isolation approaches, particularly in label-free methodologies governed by particle and fluid mechanics. This chapter will explore the theory behind particle isolation and sorting in different microfluidic techniques necessary to guide researchers into the design and development of such devices.

Emerging Microfluidic and Biosensor Technologies for Improved Cancer Theranostics.

Microfluidics and biosensors have already demonstrated their potential in cancer research. Typical applications of microfluidic devices include the realistic modeling of the tumor microenvironment for mechanistic investigations or the real-time monitoring/screening of drug efficacy. Similarly, point-of-care biosensing platforms are instrumental for the early detection of predictive biomarkers and their accurate quantification. The combination of both technologies offers unprecedented advantages for the management of the disease, with an enormous potential to contribute to improving patient prognosis. Despite their high performance, these methodologies are still encountering obstacles for being adopted by the healthcare market, such as a lack of standardization, reproducibility, or high technical complexity. Therefore, the cancer research community is demanding better tools capable of boosting the efficiency of cancer diagnosis and therapy. During the last years, innovative microfluidic and biosensing technologies, both individually and combined, have emerged to improve cancer theranostics. In this chapter, we discuss how these emerging-and in some cases unconventional-microfluidics and biosensor technologies, tools, and concepts can enhance the predictive power of point-of-care devices and the development of more efficient cancer therapies.

Engineering Patient-on-a-Chip Models for Personalized Cancer Medicine.

Traditional in vitro and in vivo models typically used in cancer research have demonstrated a low predictive power for human response. This leads to high attrition rates of new drugs in clinical trials, which threaten cancer patient prognosis. Tremendous efforts have been directed towards the development of a new generation of highly predictable pre-clinical models capable to reproduce in vitro the biological complexity of the human body. Recent advances in nanotechnology and tissue engineering have enabled the development of predictive organs-on-a-chip models of cancer with advanced capabilities. These models can reproduce in vitro the complex three-dimensional physiology and interactions that occur between organs and tissues in vivo, offering multiple advantages when compared to traditional models. Importantly, these models can be tailored to the biological complexity of individual cancer patients resulting into biomimetic and personalized cancer patient-on-a-chip platforms. The individualized models provide a more accurate and physiological environment to predict tumor progression on patients and their response to drugs. In this chapter, we describe the latest advances in the field of cancer patient-on-a-chip, and discuss about their main applications and current challenges. Overall, we anticipate that this new paradigm in cancer in vitro models may open up new avenues in the field of personalized - cancer - medicine, which may allow pharmaceutical companies to develop more efficient drugs, and clinicians to apply patient-specific therapies.

3D Protein-Based Bilayer Artificial Skin for the Guided Scarless Healing of Third-Degree Burn Wounds in Vivo.

Severe burn injuries can lead to delays in healing and devastating scar formation. Attempts have been made to develop a suitable skin substitute for the scarless healing of such skin wounds. Currently, there is no effective strategy for completely scarless healing after the thermal injuries. In our recent work, we fabricated and evaluated a 3D protein-based artificial skin made from decellularized human amniotic membrane (AM) and electrospun nanofibrous silk fibroin (ESF) in vitro. We also characterized both biophysical and cell culture investigation to establish in vitro performance of the developed bilayer scaffolds. In this report, we evaluate the appropriate utility of this fabricated bilayered artificial skin in vivo with particular emphasis on healing and scar formation due to the biochemical and biomechanical complexity of the skin. For this work, AM and AM/ESF membranes alone or seeded with adipose-tissue-derived mesenchymal stem cells (AT-MSCs) are implanted on full-thickness burn wounds in mice. The healing efficacy and scar formation are evaluated at 7, 14, and 28 days post-implantation in vivo. Our data reveal that ESF accelerates the wound-healing process through the early recruitment of inflammatory cells such as macrophages into the defective site as well as the up-regulation of angiogenic factors from the AT-MSCs and the facilitation of the remodeling phase. In vivo application of the prepared AM/ESF membrane seeded with the AT-MSCs reduces significantly the post-burn scars. The in vivo data suggest that the potential applications of the AM/ESF bilayered artificial skin may be considered a clinical translational product with stem cells to guide the scarless healing of severe burn injuries.

Gold nanoparticle-embedded silk protein-ZnO nanorod hybrids for flexible bio-photonic devices.

Silk protein has been used as a biopolymer substrate for flexible photonic devices. Here, we demonstrate ZnO nanorod array hybrid photodetectors on Au nanoparticle-embedded silk protein for flexible optoelectronics. Hybrid samples exhibit optical absorption at the band edge of ZnO as well as plasmonic energy due to Au nanoparticles, making them attractive for selective UV and visible wavelength detection. The device prepared on Au-silk protein shows a much lower dark current and a higher photo to dark-current ratio of ∼105 as compared to the control sample without Au nanoparticles. The hybrid device also exhibits a higher specific detectivity due to higher responsivity arising from the photo-generated hole trapping by Au nanoparticles. Sharp pulses in the transient photocurrent have been observed in devices prepared on glass and Au-silk protein substrates due to the light induced pyroelectric effect of ZnO, enabling the demonstration of self-powered photodetectors at zero bias. Flexible hybrid detectors have been demonstrated on Au-silk/polyethylene terephthalate substrates, exhibiting characteristics similar to those fabricated on rigid glass substrates. A study of the performance of photodetectors with different bending angles indicates very good mechanical stability of silk protein based flexible devices. This novel concept of ZnO nanorod array photodetectors on a natural silk protein platform provides an opportunity to realize integrated flexible and self-powered bio-photonic devices for medical applications in near future.

Hydroxyapatite reinforced inherent RGD containing silk fibroin composite scaffolds: Promising platform for bone tissue engineering.

Replacement and repair of ectopic bone defects and traumatized bone tissues are done using porous scaffolds and composites. The prerequisites for such scaffolds include high mechanical strength, osseoconductivity and cytocompatibility. The present work is designed to address such requirements by fabricating a reinforced cytocompatible scaffold. Biocompatible silk protein fibroin collected from tropical non-mulberry tasar silkworm (Antheraea mylitta) is used to fabricate fibroin-hydroxyapatite (HAp) nanocomposite particles using chemical precipitation method. In situ reinforcement of fibroin-HAp nanocomposite and external deposition of HAp particles on fibroin scaffold is carried out for comparative evaluations of bio-physical and biochemical characteristics. HAp deposited fibroin scaffolds provide greater mechanical strength and cytocompatibility, when compared with fibroin-HAp nanoparticles reinforced fibroin scaffolds. Minimal immune responses of both types of composite scaffolds are observed using osteoblast-macrophage co-culture model. Nanocomposite reinforced fibroin scaffold can be tailored further to accommodate different requirements depending on bone type or bone regeneration period.

Silk fibroin nanoparticles support in vitro sustained antibiotic release and osteogenesis on titanium surface.

UNLABELLED: Increasing amounts of metal-based implants are used for orthopedic or dental surgeries throughout the world. Still several implant-related problems such as inflammation, loosening and bacterial infection are prevalent. These problems stem from the immediate microbial contamination and failure of initial osteoblast adhesion. Additionally, bacterial infections can cause serious and life-threatening conditions such as osteomyelitis. Here, antibiotic (gentamicin)-loaded silk protein fibroin (non-mulberry silkworm, Antheraea mylitta) nanoparticles are fabricated and deposited over the titanium surface to achieve sustained drug release in vitro and to alter the surface nano-roughness. Based on the altered surface topography, chemistry and antibacterial activity, we conclude that the nanoparticle-deposited surfaces are superior for osteoblast adhesion, proliferation and differentiation in comparison to bare Ti. This method can be utilized as a cost-effective approach in implant modification. FROM THE CLINICAL EDITOR: Titanium-based implants are commonly used in the field of orthopedics or dentistry. Surface modification of an implant is vital to ensure osseointegration. In this article, the author investigated the use of silk protein fibroins for metal surface modification and also for drug delivery against bacteria. The encouraging data should provide a new method in terms of nanotechnology in the respective clinical fields.

Natural Non-Mulberry Silk Nanoparticles for Potential-Controlled Drug Release.

Natural silk protein nanoparticles are a promising biomaterial for drug delivery due to their pleiotropic properties, including biocompatibility, high bioavailability, and biodegradability. Chinese oak tasar Antheraea pernyi silk fibroin (ApF) nanoparticles are easily obtained using cations as reagents under mild conditions. The mild conditions are potentially advantageous for the encapsulation of sensitive drugs and therapeutic molecules. In the present study, silk fibroin protein nanoparticles are loaded with differently-charged small-molecule drugs, such as doxorubicin hydrochloride, ibuprofen, and ibuprofen-Na, by simple absorption based on electrostatic interactions. The structure, morphology and biocompatibility of the silk nanoparticles in vitro are investigated. In vitro release of the drugs from the nanoparticles depends on charge-charge interactions between the drugs and the nanoparticles. The release behavior of the compounds from the nanoparticles demonstrates that positively-charged molecules are released in a more prolonged or sustained manner. Cell viability studies with L929 demonstrated that the ApF nanoparticles significantly promoted cell growth. The results suggest that Chinese oak tasar Antheraea pernyi silk fibroin nanoparticles can be used as an alternative matrix for drug carrying and controlled release in diverse biomedical applications.

Nanofibrous nonmulberry silk/PVA scaffold for osteoinduction and osseointegration.

Poly-vinyl alcohol and nonmulberry tasar silk fibroin of Antheraea mylitta are blended to fabricate nanofibrous scaffolds for bone regeneration. Nanofibrous matrices are prepared by electrospinning the equal volume ratio blends of silk fibroin (2 and 4 wt%) with poly-vinyl alcohol solution (10 wt%) and designated as 2SF/PVA and 4SF/PVA, respectively with average nanofiber diameters of 177 ± 13 nm (2SF/PVA) and 193 ± 17 nm (4SF/PVA). Fourier transform infrared spectroscopy confirms retention of the secondary structure of fibroin in blends indicating the structural stability of neo-matrix. Both thermal stability and contact angle of the blends decrease with increasing fibroin percentage. Conversely, fibroin imparts mechanical stability to the blends; greater tensile strength is observed with increasing fibroin concentration. Blended scaffolds are biodegradable and support well the neo-bone matrix synthesis by human osteoblast like cells. The findings indicate the potentiality of nanofibrous scaffolds of nonmulberry fibroin as bone scaffolding material.

Potential mode of protection of silkworm pupae from environmental stress by harboring the bacterial biofilm on the surfaces of silk cocoons.

The silkworm forms cocoon to protect its pupa that survives for months inside the cocoon without being affected by various environmental stresses. To understand the possible mode of pupal survival within the cocoon encasement, we investigate the cause that protects the cocoon. During the end of the spinning process, we have isolated different bacterial species from the cocoon surface. These are identified using molecular techniques and checked for their abilities to form biofilm in vitro. The bacteria are able to form biofilm either individually or in consortia. Of which, Bacillus and Erwinia species are prominent biofilm formers. Interestingly, these bacteria have the ability to form biofilm on the cocoon mimetic surface of the silk protein Sericin Hope that contains only sericin. The origin and the behavior of the bacteria lead us to hypothesize the possible role of biofilm layer on the cocoon surface, which provides protection from adverse environmental conditions.

Cytotoxicity and sustained release of modified divinylsulfone from silk based 3D construct.

Monovinylsulfones have been extensively studied for its biological activities but modified divinylsulfones (mDVS2) were largely neglected due to the non-availability of appropriate synthetic routes. The present report describes the potential of a unique derivative of divinylsulfone as a remedial molecule. The mDVS2, available in reasonably large amount through an easy synthesis route, incites necrosis in invasive and non-invasive breast cancer cells in a time and concentration dependent manner. This molecule is further used to fabricate mDVS2 embedded silk based 3D scaffolds in order to achieve sustained release. The entrapped molecules retain their activity over time, as 100% cell death is observed within 7 days. The findings demonstrate the cytotoxic property of mDVS and highlight the importance of under utilized mDVSs as potential therapeutic agents.

One-step synthesis of natural silk sericin-based microcapsules with bionic structures.

Different techniques are being developed for fabricating microcapsules; it is still a challenge to fabricate them in an efficient and environment-friendly process. Here, a one-step green route to synthesize silk protein sericin-based microcapsules without any assistance of organic solvents is reported. By carefully changing the concentration of calcium ions accompanied with stirring, the morphology of the microcapsules can easily be regulated to form either discoidal, biconcave, cocoon-like, or tubular structures. The chelation of Ca(2+) and shearing force from agitation may induce the conformational transformation of sericin, which possibly results in the formation of microcapsules through the self-assembly of the protein subsequently. The as-prepared cocoon-like microcapsules exhibit pH-dependent stability. A potential application of microcapsules being fabricated from natural water-soluble silk protein sericin for controlled bioactive molecules loading and release system by a pH-triggered manner is quite feasible.

Nanomaterials-Based Hybrid Bioink Platforms in Advancing 3D Bioprinting Technologies for Regenerative Medicine.

3D bioprinting is recognized as the ultimate additive biomanufacturing technology in tissue engineering and regeneration, augmented with intelligent bioinks and bioprinters to construct tissues or organs, thereby eliminating the stipulation for artificial organs. For 3D bioprinting of soft tissues, such as kidneys, hearts, and other human body parts, formulations of bioink with enhanced bioinspired rheological and mechanical properties were essential. Nanomaterials-based hybrid bioinks have the potential to overcome the above-mentioned problem and require much attention among researchers. Natural and synthetic nanomaterials such as carbon nanotubes, graphene oxides, titanium oxides, nanosilicates, nanoclay, nanocellulose, etc. and their blended have been used in various 3D bioprinters as bioinks and benefitted enhanced bioprintability, biocompatibility, and biodegradability. A limited number of articles were published, and the above-mentioned requirement pushed us to write this review. We reviewed, explored, and discussed the nanomaterials and nanocomposite-based hybrid bioinks for the 3D bioprinting technology, 3D bioprinters properties, natural, synthetic, and nanomaterial-based hybrid bioinks, including applications with challenges, limitations, ethical considerations, potential solution for future perspective, and technological advancement of efficient and cost-effective 3D bioprinting methods in tissue regeneration and healthcare.