Characterization Of Scaffolds Research Articles

Investigation of scaffold manufacturing conditions for 3-dimensional culture of myogenic cell line derived from black sea bream (Acanthopagrus schlegelii).

Culturing fish myogenic cells in vitro holds significant potential to revolutionize aquaculture practices and support sustainable food production. However, advancement in in vitro culture technologies for skeletal muscle-derived myogenic cells have predominantly focused on mammals, with limited studies on fish. Scaffold-based three-dimensional (3D) culture systems for fish myogenic cells remain underexplored, highlighting a critical research gap compared to mammalian systems. This study evaluated the effects of scaffold composition and manufacturing methods on cellular growth in the 3D culture of black sea bream (Acanthopagrus schlegelii) myogenic cells. Scaffolds were manufactured using three natural polymers: black sea bream-derived extracellular matrix (ECM), sodium alginate, and gelatin. Two scaffold types were tested: "cell-laden scaffolds" prepared by mixing cells into the pre-scaffold solution followed by gelation, and "cell-seeding scaffolds" produced by freezing, gelation, and lyophilization before cell inoculation. Scaffold characteristics, including pore size, porosity, swelling ratio, and degradation rate, were assessed. Cell-seeding scaffolds exhibited relatively larger pore size, higher porosity, and higher degradation rate, while cell-laden scaffolds had higher swelling ratios. When black sea bream myogenic cells were cultured in these scaffolds, cell-seeding scaffolds supported cellular growth, particularly when composed of 3% sodium alginate and 4% gelatin with any concentration of ECM. In contrast, cell-laden scaffolds did not support cellular growth regardless of their composition. These findings provide fundamental insights for optimizing scaffold properties to develop more optimized conditions for 3D culture of fish muscle lineage cells.

Enhancing the biological characteristics of aminolysis surface-modified 3D printed nanocomposite polycaprolactone/nanohydroxyapatite scaffold via gelatin biomacromolecule immobilization: An in vitro and in vivo study.

The surface characteristics of scaffolds utilized in bone tissue engineering profoundly influence subsequent cellular response. This study investigated the efficacy of applying a gelatin coat to the surface of aminolysis surface-modified scaffolds fabricated through 3D printing with a polycaprolactone/hydroxyapatite nanocomposite, employing the hot-melt extrusion FDM technique. Initially, aminolysis surface modification using hexamethylenediamine enhanced surface hydrophilicity by introducing amine functional groups. Subsequently, gelatin solutions were applied to the scaffolds, and crosslinking with EDC/NHS was performed to increase coating strength. Contact angle measurements revealed a significantly increased surface hydrophilicity post-aminolysis. Aminolysis facilitated uniform gelatin coating formation and distribution. Subsequently, crosslinking enhanced coating durability. The addition of gelatin coating resulted in a notable 20 % increase in scaffold mechanical strength and more than 50 % rise in Young's modulus and exhibited enhancement of biodegradability and bioactivity. Gelatin coated scaffolds also demonstrated improved cell viability and adhesion and over two times higher expression of OPN and ALP genes, suggesting improved biological properties. In addition, in vivo bone formation studies verified the biological enhancement of scaffolds. Utilizing an immobilized crosslinked gelatin biomacromolecule coating effectively enhanced the biological characteristics of 3D printed scaffolds and their potential applications as bone tissue engineering scaffolds.

Influence of the graphene oxide on heat conduction characteristics of cement-based honeycomb scaffolds

Influence of the graphene oxide on heat conduction characteristics of cement-based honeycomb scaffolds

Advancing mechanical and biological characteristics of polymer-ceramic nanocomposite scaffolds for sport injuries and bone tissue engineering: A comprehensive investigation applying finite element analysis and artificial neural network

Advancing mechanical and biological characteristics of polymer-ceramic nanocomposite scaffolds for sport injuries and bone tissue engineering: A comprehensive investigation applying finite element analysis and artificial neural network

Nanoparticles' Perspective in Skin Tissue Engineering: Current Concepts and Future Outlook.

Nanotechnology seems to provide solutions to the unresolved complications in skin tissue engineering. According to the broad function of nanoparticles, this review article is intended to build a perspective for future success in skin tissue engineering. In the present review, recent studies were reviewed, and essential benefits and challenging issues regarding the application of nanoparticles in skin tissue engineering were summarized. Previous studies indicated that nanoparticles can play essential roles in the improvement of engineered skin. Bio-inspired design of an engineered skin structure first needs to understand the native tissue and mimic that in laboratory conditions. Moreover, a fundamental comprehension of the nanoparticles and their related effects on the final structure can guide researchers in recruiting appropriate nanoparticles. Attention to essential details, including the designation of nanoparticle type according to the scaffold, how to prepare the nanoparticles, and what concentration to use, is critical for the application of nanoparticles to become a reality. In conclusion, nanoparticles were applied to promote scaffold characteristics and angiogenesis, improve cell behavior, provide antimicrobial conditions, and cell tracking.

Cannabidiol-Loaded Lipid Nanoparticles Incorporated in Polyvinyl Alcohol and Sodium Alginate Hydrogel Scaffold for Enhancing Cell Migration and Accelerating Wound Healing.

Chronic wounds represent a persistent clinical challenge due to prolonged inflammation and impaired tissue repair mechanisms. Cannabidiol (CBD), recognized for its anti-inflammatory and pro-healing properties, shows therapeutic promise in wound care. However, its delivery via lipid nanoparticles (LNPs) remains challenging due to CBD's inherent instability and low bioavailability. This study developed and characterized a novel hydrogel scaffold composed of CBD-loaded LNPs (CBD/LNPs) integrated into a polyvinyl alcohol (PVA) and sodium alginate (SA) matrix, designed to enhance wound repair and mitigate inflammation. The characteristics of the hydrogel scaffold were observed including the degree of swelling and LNPs' release profiles. Furthermore, in the results, CBD/LNPs displayed enhanced stability and reduced cytotoxicity compared to unencapsulated CBD. In vitro assays demonstrated that CBD/LNPs significantly promoted fibroblast migration in gap-closure wound models and reduced intracellular reactive oxygen species, supporting their potential as a biocompatible and efficacious agent for cellular repair and oxidative stress attenuation. In vivo experiments using adult male Wistar rats with aseptic cutaneous wounds revealed that treatment with CBD/LNP-PVA/SA hydrogel scaffold significantly accelerated wound closure relative to blank hydrogel controls, demonstrating a substantial reduction in the wound area over time. Histological analysis confirms notable improvements in skin morphology in wounds treated with CBD/LNP-PVA/SA hydrogel scaffold with evidence of accelerated epithelialization, enhanced collagen deposition, and increased dermal thickness and vascularization. Additionally, skin histology showed a more organized epidermal layer and reduced inflammatory cell infiltration in CBD/LNP-PVA/SA hydrogel scaffold-treated wounds, corresponding to a 35% increase in the wound closure rate by day 28 post-treatment. These findings suggest that CBD/LNP-PVA/SA hydrogel scaffolds facilitate inflammation resolution and structural wound healing through localized, sustained CBD delivery. The dual anti-inflammatory and wound-healing effects position CBD/LNP-PVA/SA hydrogel scaffold as a promising approach for chronic wound management. Future investigations are warranted to elucidate the mechanistic pathways by which CBD modulates the skin architecture and to explore its translational applications in clinical wound care.

In vitro study of dimethyl glutamate incorporated chitosan/microfibrillated cellulose based matrix in addition of H and Zr on osteoblast cells.

Tissue engineering techniques can be utilized to repair or regenerate damaged tissue by promoting the proliferation and differentiation of cells in bone regeneration. A critical component of this process is the scaffold employed, which should ideally support consistent tissue development during bone regeneration. The aim of this study was to evaluate the morphological, physicochemical, and biological characteristics of various scaffolds: S1 (C/MFC), S2 (C/H/MFC), S3 (C/MFC/Zr), S4 (C/MFC/PCL), S5 (C/H/MFC/PCL), S6 (C/PCL/MFC/Zr), and S7 (C/H/MFC/Zr), which are intended for application in bone regeneration. The scaffolds containing microfibrillated cellulose, chitosan, polycaprolactone, zirconium, and hydroxyapatite were fabricated by the freeze-drying method. Conventional methods, including scanning electron microscopy (SEM) and Fourier transform infrared spectroscopy (FTIR) analysis, were used to evaluate morphological and physicochemical properties of composite scaffolds. The fabricated scaffolds (S1-S7) had spongy properties that all functional groups were present in the sponge. Biological properties for cell survival were evaluated by the MTT assay, ALP, and ARS activities, respectively. In physicochemical studies, scaffolds showed tunable water absorption, swelling studies, degradation, sustained drug release, and mechanical properties. In biological studies, the cell proliferation and attachment were shown to significantly increase in scaffolds on MG63 cells. After 7 days of cell culture, ALP and ARS activity indicated the enhancement of extracellular calcium deposition of the MG63 cells on the treated scaffolds. In summary, the scaffolds S7 (C/H/MFC/Zr) treated with dimethyl glutamate revealed favorable effects on bone tissues, implying a potential towards the treatment of bone defects and drug delivery.

Characteristics of Chitosan Gelatine Limestone-Based Carbonate Hydroxyapatite Composite Scaffold After Crosslinking

Bone damage is one of the most common cases in dentistry. Tissue engineering is advancing in biotechnology to aid bone regeneration using scaffolds. Scaffolds need to be biocompatible, bioactive, and bioresorbable. Purpose: Analyzing the characteristics of scaffold C-G:CHA after crosslinking with 0.25% glutaraldehyde. Methods: The scaffold is synthesized from C–G:CHA in ratios of 40:60, 30:70, and 20:80 (w/w) using a freeze-drying technique and crosslinked with 0.25% glutaraldehyde. Compressive strengths are tested with a Universal Testing Machine Mini Autograph. FTIR, XRD, and SEM EDX were used to identify the most optimal base of each ratio. Data are analyzed with a one-way ANOVA parametric test. Results: The FTIR test showed that adding 0.25% glutaraldehyde formed a new chemical group. The XRD test indicated the use of 0.25% glutaraldehyde as a crosslinking agent contributed to the scaffold having an amorphous form. The SEM test results of the porosity of the C-G:CHA were 88.41% to 91.14%. After crosslinking the porosities slightly decreased. The EDX analysis showed that the Ca/P ratio in the C-G:CHA scaffold is 1.79 to 2.07. The average compressive strength of the C-G:CHA scaffold increases after being crosslinked with glutaraldehyde. Conclusion: Scaffold C-G:CHA crosslinked with 0.25% glutaraldehyde effective to increase compressive strength. The 30:70 ratio is ideal because it has a Ca/P ratio and average pore size closest to bone.

Biological and structural properties of curcumin-loaded graphene oxide incorporated collagen as composite scaffold for bone regeneration.

To address the challenges related to bone defects, including osteoinductivity deficiency and post-implantation infection risk, this study developed the collagen composite scaffolds (CUR-GO-COL) with multifunctionality by integrating the curcumin-loaded graphene oxide with collagen through a freeze-drying-cross-linking process. The morphological and structural characteristics of the composite scaffolds were analyzed, along with their physicochemical properties, including water absorption capacity, water retention rate, porosity, in vitro degradation, and curcumin release. To evaluate the biocompatibility, cell viability, proliferation, and adhesion capabilities of the composite scaffolds, as well as their osteogenic and antimicrobial properties, in vitro cell and bacterial assays were conducted. These assays were designed to assess the impact of the composite scaffolds on cell behavior and bacterial growth, thereby providing insights into their potential for promoting osteogenesis and inhibiting infection. The CUR-GO-COL composite scaffold with a CUR-GO concentration of 0.05% (w/v) exhibits optimal biological compatibility and stable and slow curcumin release rate. Furthermore, in vitro cell and bacterial tests demonstrated that the prepared CUR-GO-COL composite scaffolds enhance cell viability, proliferation and adhesion, and offer superior osteogenic and antimicrobial properties compared with the CUR-GO composite scaffold, confirming the osteogenesis promotion and antimicrobial effects. The introduction of CUR-GO into collagen scaffold creates a bone-friendly microenvironment, and offers a theoretical foundation for the design, investigation and utilization of multifunctional bone tissue biomaterials.

Structural, mechanical, and cytocompatibility characteristics of hybrid scaffolds from chitosan/decellularized testicular ECM

Tissue engineering has facilitated the development of novel therapeutic strategies for male reproductive disorders. Decellularized extracellular matrix (ECM) scaffolds provide a wide range of functional components that promote cellular behavior. This research aimed to develop reinforced scaffolds for testicular tissue engineering by combining testicular ECM (TE) derived pre-gel with chitosan (CS) solution at varying ratios (TE25/CS75, TE50/CS50, and TE75/CS25). To determine the optimum ratio of TE to CS solution, final scaffold properties were investigated including pore size, porosity, mechanical strength, swelling ratio, degradation rate followed by in-vitro biological evaluations. All groups revealed an interconnected porous structure with high porosity (from 76.6 % to 90.9 %) and adequate pore sizes (between 50 and 226 μm), while the pores of TE50/CS50 scaffold were distributed more uniformly. The mechanical properties of scaffolds were enhanced by combining CS with TE, whereas their swelling ratio decreased. It was observed that the scaffolds' degradation rate rose substantially as the ratio of TE to CS increased. The MTT assay revealed that none of the scaffolds exhibited cytotoxic properties. The results of this study demonstrated that all fabricated hybrid scaffolds, especially the TE50/CS50, have potential for testicular tissue engineering applications.

Enhanced osteogenic potential of spider silk fibroin-based composite scaffolds incorporating carboxymethyl cellulose for bone tissue engineering

This study aimed to investigate the characteristics of composite scaffolds that combine fibroin derived from spider silk and carboxymethyl cellulose (CMC) in the field of bone tissue engineering. Fibroin, obtained from spider silk, serves as a valuable biomaterial and constitutes the primary component of fibrous protein-based spider silk threads. To enhance the binding efficiency in bone formation after scaffold implantation, CMC was integrated into fibroin, aiming to improve the injectability properties of the scaffold in bone substitutes. For bone marrow mesenchymal stem cell (BMSC) tissue engineering, BMSCs isolated from mice were seeded onto the scaffold, and the rate of cell proliferation was assessed. The composite scaffold, with the addition of CMC to fibroin, exhibited superior characteristics compared to scaffolds containing only silks, including porous morphology, porosity, surface wettability, water absorption, and thermal properties. Alkaline phosphatase activity in BMSCs was significantly higher in the CMC-containing scaffold compared to the silk-only scaffold, and the CMC-containing scaffold demonstrated increased expression of osteocyte marker genes and proteins. In conclusion, the biocompatibility and hydrophilicity of CMC-containing scaffolds play essential roles in the growth and proliferation of osteocytes. Furthermore, the CMC-containing scaffold design proposed in this study is expected to have a substantial impact on promoting ossification of BMSCs.

Improving the processability and mechanical strength of self-hardening robocasted hydroxyapatite scaffolds with silane coupling agents

Bone cements are the subject of intensive research, primarily due to their versatility and the increasing importance for personalized medicine. In this study, novel hybrid self-setting scaffolds, based on calcium phosphates and natural polymers, were fabricated using the robocasting technique. Additionally, the influence of two different silane coupling agents, tetraethyl orthosilicate (TEOS) and 3-glycidoxypropyltrimethoxysilane (GPTMS), on the physicochemical and biological properties of the obtained materials was thoroughly investigated. The chemical and phase compositions (XRF, XRD, FTIR), setting process, rheological properties, mechanical strength, microstructure (SEM), and chemical stability in vitro were comprehensively examined. The use of silane coupling agents improved compressive strength of the scaffolds from 5.20 to 9.26 MPa. The incorporation of citrus pectin into the liquid phase of the materials, along with the use of a hybrid hydroxyapatite-chitosan powder, not only facilitated the development of printable pastes suitable for robocasting but also enhanced the physicochemical properties of the robocasted scaffolds. The results presented in this study underscore the beneficial influence of silane coupling agents on the characteristics of calcium phosphate-based bone scaffolds. Developed robocasted scaffolds hold great potential for applications in the field of bone tissue engineering and personalized medicine. Further in vitro and in vivo studies are necessary to validate their suitability for clinical applications.

Osteoinduction and Osteoconduction Evaluation of Biodegradable Magnesium Alloy Scaffolds in Repairing Large Segmental Defects in Long Bones of Rabbit Models.

Magnesium (Mg) alloy scaffolds demonstrate promising potential for clinical applications in the repair of segmental bone defects. However, the specific mechanisms of osteoconduction and osteoinduction facilitated by these scaffolds themselves during the bone reconstruction process remain inadequately defined. This investigation systematically assesses the properties of MAO-coated Mg base implants both in vitro and in vivo. Furthermore, it elucidates the correlation between scaffold characteristics and bone regeneration in the repair of extensive long-bone defects, measuring up to 20 mm, without the use of additional bone graft materials. Electrochemical measurements and immersion tests conducted in vitro indicate that the MAO coating substantially enhances the corrosion resistance of the underlying Mg alloy. Meanwhile, the application of MAO coatings has been shown to significantly improve cytocompatibility, cellular adhesion, and osteogenic differentiation, as evidenced by the CCK-8 assays, ALP activity measurements, Western blot, and RT-qPCR in vitro. At 24 weeks postimplantation with the MAO-coated Mg alloy scaffold, the large segmental defects were effectively repaired concerning both integrity and continuity. The Micro-CT gradual replacement of old bone with new bone on the implant surface was observed by X-ray and Micro-CT. Meanwhile, the histological results indicated that the MAO-coated Mg alloy scaffold maintained a robust osteogenic response. In summary, the MAO-coated Mg alloy scaffold independently exhibits effective osteoconduction and osteoinduction, playing a significant role in bone repair function without the need for additional bone graft materials.

Periosteum-bone inspired hierarchical scaffold with endogenous piezoelectricity for neuro-vascularized bone regeneration

The development of scaffolds for repairing critical-sized bone defects heavily relies on establishing a neuro-vascularized network for proper penetration of nerves and blood vessels. Despite significant advancements in using artificial bone-like scaffolds infused with various agents, challenges remain. Natural bone tissue consists of a porous bone matrix surrounded by a neuro-vascularized periosteum, with unique piezoelectric properties essential for bone growth. Drawing inspiration from this assembly, we developed a periosteum-bone-mimicking bilayer scaffold with piezoelectric properties for regeneration of critical-sized bone defects. The periosteum-like layer of this scaffold features a double network hydrogel composed of chelated alginate, gelatin methacrylate, and sintered whitlockite nanoparticles, emulating the viscoelastic and piezoelectric properties of the natural periosteum. The bone-like layer is composed of a porous structure of chitosan and bioactive hydroxyapatite created through a biomineralization process. Unlike conventional bone-like scaffolds, this bioinspired bilayer scaffold significantly enhances osteogenesis, angiogenesis, and neurogenesis combined with low-intensity pulsed ultrasound-assisted piezoelectric stimulation. Such a scheme enhances neuro-vascularized bone regeneration in vivo. The results suggest that the bilayer scaffold could serve as an effective self-powered electrical stimulator to expedite bone regeneration under dynamic physical stimulation.

The comparison of eight different common in vitro and ex vivo environments with in vivo conditions applying model collagen samples: Correlation possibilities and their limits

New biomaterials are routinely evaluated for their degradation behaviour in the real body environment. Following the 3R strategy, in vitro simulated body conditions are often preferred. No studies that simultaneously compare such conditions with the real body environment have been conducted to date. Model porous collagen scaffolds were exposed for 21 days to eight different environments: simple salt-based and enzymatic media, human blood plasma, cell culture media with and without human fibroblasts and ex vivo model cortical bone, and subsequently compared with an in vivo environment represented by a pig peritoneum. The mechanical properties of the scaffolds were then determined via uniaxial compression testing, and the structural properties via the micro-CT, weight loss, infrared spectroscopy, X-ray diffraction and histological methods. Interestingly, the various analysed simulated body conditions caused differing alterations in the collagen scaffold characteristics when compared with the real body environment. The mechanical properties were similar during the first 7 days of incubation but diverged after 14 and 21 days. The structural properties varied significantly after just 7 days of incubation. The histological evaluation of the scaffolds exposed to the cellular, ex vivo and in vivo conditions revealed the poor ability of cells to completely populate the scaffolds, accompanied by the massive ingrowth of connective tissue into the in vivo exposed scaffolds, which resulted in their variable global behaviour. In conclusion, the value of in vitro simulated body environments lies in their screening capacity and feasibility; however, direct extrapolation to real body conditions needs to be verified going forward.

Cotton-like antibacterial polyacrylonitrile nanofiber-reinforced chitosan scaffold: Physicochemical, mechanical, antibacterial, and MC3T3-E1 cell viability study

Bio-scaffolds, while mimicking the morphology of native tissue and demonstrating suitable mechanical strength, enhanced cell adhesion, proliferation, infiltration, and differentiation, are often prone to failure due to microbial infections. As a result, tissue engineers are seeking ideal scaffolds with antibacterial properties. In this study, silver nanoparticles (AgNPs) were integrated into cotton-like polyacrylonitrile nanofibers via a polydopamine (PDA) interlayer (Ag@p-PAN). These Ag@p-PAN nanofibers were then incorporated into the chitosan (CS) matrix, developing an antibacterial CS/Ag@p-PAN composite scaffold. The composite scaffold features an interconnected porous morphology with fiber-infused pore walls, improved water absorption and swelling properties, a controlled degradation profile, enhanced porosity, better mechanical strength, strong antibacterial properties, and excellent MC3T3-E1 cell viability, adhesion, proliferation, and infiltration. This study presents a novel method for reinforcing CS-based scaffolds by incorporating bioactive nanofibers, offering potential applications in tissue engineering and other biomedical fields.

Ultrasonic signatures of multiphase architectured media: Application to biomechanics

Assessing the mechanical characteristics of periodic architectured scaffolds employed in bone tissue repair, while preserving their structural integrity, poses a significant challenge from both theoretical and experimental viewpoints. By leveraging concepts arising from the phononic crystals community, here we investigate the reflection and transmission of elastic waves propagating through water-immersed biphasic architectured samples in the MHz regime, which exhibit a periodic organization at a length scale of a few hundred micrometers. Experimental outcomes are systematically compared with modeled predictions, spanning a range of scaffold-like samples engineered to mimic the expected variations that take place in a biological environment. A particular attention is given to critical modeling considerations such as the behavior in reflection, the interaction of Bloch waves with the viscoelastic properties of the constituent phases, as well as the role of modal conversion at the interface between the homogeneous incident medium and the architectured one. Altogether, the reported results suggest that specific ultrasonic signatures associated with elastic wave propagation in periodic media, encompassing phenomena such as bandgaps and dispersion, offer valuable insights towards the nondestructive monitoring of micro-architectured media like scaffolds.

Development of artificial photosystems based on designed proteins for mechanistic insights into photosynthesis.

This review aims to provide an overview of the progress in protein-based artificial photosystem design and their potential to uncover the underlying principles governing light-harvesting in photosynthesis. While significant advances have been made in this area, a gap persists in reviewing these advances. This review provides a perspective of the field, pinpointing knowledge gaps and unresolved challenges that warrant further inquiry. In particular, it delves into the key considerations when designing photosystems based on the chromophore and protein scaffold characteristics, presents the established strategies for artificial photosystems engineering with their advantages and disadvantages, and underscores the recent breakthroughs in understanding the molecular mechanisms governing light-harvesting, charge separation, and the role of the protein motions in the chromophore's excited state relaxation. By disseminating this knowledge, this article provides a foundational resource for defining the field of bio-hybrid photosystems and aims to inspire the continued exploration of artificial photosystems using protein design.

Structural Variance of Doxorubicin and Anthracycline Analogues as Topoisomerase Alpha and Beta (Top2a and Top2b) Inhibitors. Potential Design of Analogue Candidates of Less Side Effects on Cardiomyocytes.

Doxorubicin that is on WHO's list of essential medicines and other anthracycline analogues, in general, are natural metabolites isolated from Streptomycetaceae, or semi-synthetized derivatives stated as first-generation anticancer agents. The tetracyclic scaffold attached mostly to amino sugar is known to be effective against solid tumors compared to other anticancer agents. The mechanism had been stated as intercalating agent at the minor groove of DNA strands during the step of releasing supercoiled DNA. Along with their anticancer activity, anthracyclines possess antimicrobial effects of notable MIC values. Cardiotoxicity represents the main challenge for both of medical care for treatment of cancers and drug discoverers. This exertion deals with careful structural investigation of the three-dimensional, fully optimized drugs in use. Drug-candidates in clinical studies, and leads failed in last developments. The aim is to find a structural gate to guard against or reduce the cardiac side effects. It deals also, with the topological features differentiating between antibacterial and anticancer agents bearing the tetracyclic scaffold features as well as between the topoisomerases as target molecules.

Advances in reversible covalent kinase inhibitors.

Reversible covalent kinase inhibitors (RCKIs) are a class of novel kinase inhibitors attracting increasing attention because they simultaneously show the selectivity of covalent kinase inhibitors yet avoid permanent protein-modification-induced adverse effects. Over the last decade, RCKIs have been reported to target different kinases, including Atypical group of kinases. Currently, three RCKIs are undergoing clinical trials. Here, advances in RCKIs are reviewed to systematically summarize the characteristics of electrophilic groups, chemical scaffolds, nucleophilic residues, and binding modes. In so doing, we integrate key insights into privileged electrophiles, the distribution of nucleophiles, and hence effective design strategies for the development of RCKIs. Finally, we provide a further perspective on future design strategies for RCKIs, including those that target proteins other than kinases.