Biomimetic natural biomaterials for tissue engineering and regenerative medicine:new biosynthesis methods,recent advances,and emerging applications

Biomimetic materials have emerged as attractive and competitive alternatives for tissue engineering(TE)and regenerative medicine.In contrast to conventional biomaterials or synthetic materials,biomimetic scaffolds based on natural biomaterial can offer cells a broad spectrum of biochemical and biophysical cues that mimic the in vivo extracellular matrix(ECM).Additionally,such materials have mechanical adaptability,micro-structure interconnectivity,and inherent bioactivity,making them ideal for the design of living implants for specific applications in TE and regenerative medicine.This paper provides an overview for recent progress of biomimetic natural biomaterials(BNBMs),including advances in their preparation,functionality,potential applications and future challenges.We highlight recent advances in the fabrication of BNBMs and outline general strategies for functionalizing and tailoring the BNBMs with various biological and physicochemical characteristics of native ECM.Moreover,we offer an overview of recent key advances in the functionalization and applications of versatile BNBMs for TE applications.Finally,we conclude by offering our perspective on open challenges and future developments in this rapidly-evolving field.

A Review on Silk Fibroin as a Biomaterial in Tissue Engineering

Regenerative medicine progress is based on the development of cell and tissue bioengineering. One of the aims of tissue engineering is the development of scaffolds, which should substitute the functions of the replaced organ after their implantation into the body. The tissue engineering material must meet a range of requirements, including biocompatibility, mechanical strength, and elasticity. Furthermore, the materials have to be attractive for cell growth: stimulate cell adhesion, migration, proliferation and differentiation. One of the natural biomaterials is silk and its component (silk fibroin). An increasing number of scientists in the world are studying silk and silk fibroin. The purpose of this review article is to provide information about the properties of natural silk (silk fibroin), as well as its manufacture and clinical application of each configuration of silk fibroin in medicine. Materials and research methods. Actual publications of foreign authors on resources PubMed, Medline, E-library have been analyzed. The selection criteria were materials containing information about the structure and components of silk, methods of its production in nature. This article placed strong emphasis on silk fibroin, the ways of artificial modification of it for use in various sphere of medicine.

Bone tissue engineering via nanostructured calcium phosphate biomaterials and stem cells

Tissue engineering is promising to meet the increasing need for bone regeneration. Nanostructured calcium phosphate （CAP） biomaterials/scaffolds are of special interest as they share chemical/crystallographic similarities to inorganic components of bone. Three applications of nano-CaP are discussed in this review： nanostructured calcium phosphate cement （CPC）; nano-CaP composites; and nano-CaP coatings. The interactions between stem cells and nano-CaP are highlighted, including cell attachment, orientation/ morphology, differentiation and in vivo bone regeneration. Several trends can be seen： （i） nano-CaP biomaterials support stem cell attachment/proliferation and induce osteogenic differentiation, in some cases even without osteogenic supplements; （ii） the influence of nano-CaP surface patterns on cell alignment is not prominent due to non-uniform distribution of nano-crystals; （iii） nano-CaP can achieve better bone regeneration than conventional CaP biomaterials; （iv） combining stem cells with nano-CaP accelerates bone regeneration, the effect of which can be further enhanced by growth factors; and （v） cell microencapsulation in nano-CaP scaffolds is promising for bone tissue engineering. These understandings would help researchers to further uncover the underlying mechanisms and interactions in nano-CaP stem cell constructs in vitro and in vivo, tailor nano-CaP composite construct design and stem cell type selection to enhance cell function and bone regeneration, and translate laboratory findings to clinical treatments.

Green Electrospun Silk Fibroin/Galactose Chitosan Composite Nanofibrous Scaffolds for Hepatic Tissue Engineering

The electrospun nanofibrous scaffolds made of proteins and polysaccharides were thought to be able to simulate the structure of natural extracellular matrix well.Silk fibroin(SF)and chitosan(CS)are probably the most widely used natural materials in biomedical fields including liver tissue engineering for their good properties and wide variety of sources.The asialoglycoprotein receptors of hepatocyte were reported to specifically recognize and interact with galactose.In this work,a green electrospun SF/galactosylated chitosan(GC)composite nanofibrous scaffold was fabricated and characterized.The data indicated that the addition of GC greatly influenced the spinning effect of SF aqueous solution,and the average diameter of the composite nanofibers was about 520nm.Moreover,the green electrospun SF/GC nanofibrous scaffolds were demonstrated significantly enhancing the adhesion and proliferation of hepatocyte(RH35)according to our data.The present study did a useful exploration on constructing scaffolds for liver regeneration by green electrospinning,and also laid a good foundation for the further applicative research of this green electrospun scaffolds in liver tissue engineering.

Recent advancements in applications of chitosan-based biomaterials for skin tissue engineering

The use of polymer based composites in the treatment of skin tissue damages,has got huge attention in clinical demand,which enforced the scientists to improve the methods of biopolymer designing in order to obtain highly efficient system for complete restoration of damaged tissue.In last few decades,chitosan-based biomaterials have major applications in skin tissue engineering due to its biocompatible,hemostatic,antimicrobial and biodegradable capabilities.This article overviewed the promising biological properties of chitosan and further discussed the various preparation methods involved in chitosan-based biomaterials.In addition,this review also gave a comprehensive discussion of different forms of chitosan-based biomaterials including membrane,sponge,nanofiber and hydrogel that were extensively employed in skin tissue engineering.This review will help to form a base for the advanced applications of chitosan-based biomaterials in treatment of skin tissue damages.

Vital roles of stem cells and biomaterials in skin tissue engineering

Tissue engineering essentially refers to technology for growing new human tissue and is distinct from regenerative medicine. Currently, pieces of skin are already being fabricated for clinical use and many other tissue types may be fabricated in the future.Tissue engineering was first defined in 1987 by the United States National Science Foundation which critically discussed the future targets of bioengineering research and its consequences. The principles of tissue engineering are to initiate cell cultures in vitro, grow them on scaffolds in situ and transplant the composite into a recipient in vivo. From the beginning, scaffolds have been necessary in tissue engineering applications. Regardless, the latest technology has redirected established approaches by omitting scaffolds. Currently, scientists from diverse research institutes are engineering skin without scaffolds. Due to their advantageous properties, stem cells have robustly transformed the tissue engineering field as part of an engineered bilayered skin substitute that will later be discussed in detail. Additionally, utilizing biomaterials or skin replacement products in skin tissue engineering as strategy to successfully direct cell proliferation and differentiation as well as to optimize the safety of handling during grafting is beneficial. This approach has also led to the cells' application in developing the novel skin substitute that will be briefly explained in this review.

Constructing a biofunctionalized 3D-printed gelatin/sodium alginate/chitosan tri-polymer complex scaffold with improvised biological andmechanical properties for bone-tissue engineering

Sodium alginate(SA)/chitosan(CH)polyelectrolyte scaffold is a suitable substrate for tissue-engineering application.The present study deals with further improvement in the tensile strength and biological properties of this type of scaffold to make it a potential template for bone-tissue regeneration.We experimented with adding 0%–15%(volume fraction)gelatin(GE),a protein-based biopolymer known to promote cell adhesion,proliferation,and differentiation.The resulting tri-polymer complex was used as bioink to fabricate SA/CH/GEmatrices by three-dimensional(3D)printing.Morphological studies using scanning electron microscopy revealed the microfibrous porous architecture of all the structures,which had a pore size range of 383–419μm.X-ray diffraction and Fourier-transform infrared spectroscopy analyses revealed the amorphous nature of the scaffold and the strong electrostatic interactions among the functional groups of the polymers,thereby forming polyelectrolyte complexes which were found to improve mechanical properties and structural stability.The scaffolds exhibited a desirable degradation rate,controlled swelling,and hydrophilic characteristics which are favorable for bone-tissue engineering.The tensile strength improved from(386±15)to(693±15)kPa due to the increased stiffness of SA/CH scaffolds upon addition of gelatin.The enhanced protein adsorption and in vitro bioactivity(forming an apatite layer)confirmed the ability of the SA/CH/GE scaffold to offer higher cellular adhesion and a bone-like environment to cells during the process of tissue regeneration.In vitro biological evaluation including the MTT assay,confocal microscopy analysis,and alizarin red S assay showed a significant increase in cell attachment,cell viability,and cell proliferation,which further improved biomineralization over the scaffold surface.In addition,SA/CH containing 15%gelatin designated as SA/CH/GE15 showed superior performance to the other fabricated 3D structures,demonstrating its potential for use in bone-tissue engineering.

Biomaterials and tissue engineering in traumatic brain injury:novel perspectives on promoting neural regeneration

Traumatic brain injury is a serious medical condition that can be attributed to falls, motor vehicle accidents, sports injuries and acts of violence, causing a series of neural injuries and neuropsychiatric symptoms. However, limited accessibility to the injury sites, complicated histological and anatomical structure, intricate cellular and extracellular milieu, lack of regenerative capacity in the native cells, vast variety of damage routes, and the insufficient time available for treatment have restricted the widespread application of several therapeutic methods in cases of central nervous system injury. Tissue engineering and regenerative medicine have emerged as innovative approaches in the field of nerve regeneration. By combining biomaterials, stem cells, and growth factors, these approaches have provided a platform for developing effective treatments for neural injuries, which can offer the potential to restore neural function, improve patient outcomes, and reduce the need for drugs and invasive surgical procedures. Biomaterials have shown advantages in promoting neural development, inhibiting glial scar formation, and providing a suitable biomimetic neural microenvironment, which makes their application promising in the field of neural regeneration. For instance, bioactive scaffolds loaded with stem cells can provide a biocompatible and biodegradable milieu. Furthermore, stem cells-derived exosomes combine the advantages of stem cells, avoid the risk of immune rejection, cooperate with biomaterials to enhance their biological functions, and exert stable functions, thereby inducing angiogenesis and neural regeneration in patients with traumatic brain injury and promoting the recovery of brain function. Unfortunately, biomaterials have shown positive effects in the laboratory, but when similar materials are used in clinical studies of human central nervous system regeneration, their efficacy is unsatisfactory. Here, we review the characteristics and properties of various bioactive materials, followed by the introduction of applications based on biochemistry and cell molecules, and discuss the emerging role of biomaterials in promoting neural regeneration. Further, we summarize the adaptive biomaterials infused with exosomes produced from stem cells and stem cells themselves for the treatment of traumatic brain injury. Finally, we present the main limitations of biomaterials for the treatment of traumatic brain injury and offer insights into their future potential.

The application of ECM-derived biomaterials in cartilage tissue engineering

Given the tremendous increase in the risks of cartilage defects in the sports and aging population,current treatments are limited,and new repair strategies are needed.Cartilage tissue engineering(CTE)is a promising approach to handle this burden and several fabrication technologies and biomaterials have been developed these years.The extracellular matrix(ECM)of cartilage consists of a tissue-specific 3D microenvironment with excellent biomechanical and biochemical properties,which regulates cell proliferation,adhesion,migration,and differentiation,thus attracting a great deal of attention to the rapid development of CTE based on ECM components.New generations of biomaterials are being developed rapidly for use as scaffolds to mimic the natural ECM environment.In this review,we discuss such CTE scaffolds based on ECM-derived biomaterials by reviewing the biomaterials for CTE,the applications in different scaffolds and their processing approaches,as well as the current clinical applications of those ECM-based CTE scaffolds.

Innovations in 3D bioprinting and biomaterials for liver tissue engineering:Paving the way for tissue-engineered liver

The liver is a pivotal organ that maintains internal homeostasis and actively participates in multiple physiological processes.Liver tissue engineering(LTE),by which in vitro biomimetic liver models are constructed,serves as a platform for disease research,drug screening,and cell replacement therapies.3D bioprinting is used in tissue engineering to create microenvironments that closely mimic authentic tissues with carefully selected functional biomaterials.Ideal functional biomaterials exhibit characteristics such as high biocompatibility,mechanical strength,flexibility,processability,and tunable degradability.Biomaterials can be categorized into natural and synthetic biomaterials,each with its own advantages and limitations,and their combinations serve as a primary source of 3D bioprinting materials.It is noteworthy that the liver decellularized extracellular matrix(dECM),obtained by removing cellular components from tissues,possesses traits such as bioactivity,biocompatibility,and non-immunogenicity,making it a common choice among functional biomaterials.Furthermore,crosslinking of biomaterials significantly impacts the mechanical strength,physicochemical properties,and cellular behavior of the printed structures.This review covers the current utilization of biomaterials in LTE,focusing on natural and synthetic biomaterials as well as the selection and application of crosslinking methods.The aim is to enhance the fidelity of in vitro liver tissue models by providing a comprehensive coverage of functional biomaterials,thereby establishing a versatile platform for tissue-engineered livers.

Biomaterial–Related Cell Microenvironment in Tissue Engineering and Regenerative Medicine

An appropriate cell microenvironment is key to tissue engineering and regenerative medicine.Revealing the factors that influence the cell microenvironment is a fundamental research topic in the fields of cell biology,biomaterials,tissue engineering,and regenerative medicine.The cell microenvironment consists of not only its surrounding cells and soluble factors,but also its extracellular matrix(ECM)or nearby external biomaterials in tissue engineering and regeneration.This review focuses on six aspects of bioma-terial-related cell microenvironments:①chemical composition of materials,②material dimensions and architecture,③material-controlled cell geometry,④effects of material charges on cells,⑤matrix stiff-ness and biomechanical microenvironment,and⑥surface modification of materials.The present chal-lenges in tissue engineering are also mentioned,and eight perspectives are predicted.

Dental-derived stem cells in tissue engineering:the role of biomaterials and host response

Dental-derived stem cells(DSCs)are attractive cell sources due to their easy access,superior growth capacity and low immunogenicity.They can respond to multiple extracellular matrix signals,which provide biophysical and biochemical cues to regulate the fate of residing cells.However,the direct transplantation of DSCs suffers from poor proliferation and differentiation toward functional cells and low survival rates due to local inflammation.Recently,elegant advances in the design of novel biomaterials have been made to give promise to the use of biomimetic biomaterials to regulate various cell behaviors,including proliferation,differentiation and migration.Biomaterials could be tailored with multiple functionalities,e.g.,stimuli-responsiveness.There is an emerging need to summarize recent advances in engineered biomaterials-mediated delivery and therapy of DSCs and their potential applications.Herein,we outlined the design of biomaterials for supporting DSCs and the host response to the transplantation.

Molecular Tissue Engineering:Concepts,Status and Challenge

Tissue engineering has confronted many difficulties mainly as follows:1)How to modulate the adherence,proliferation,and oriented differentiation of seed cells, especially that of stemcells. 2) Massive preparation and sustained controllable delivery of tissue inducing factors or plasmid DNA, such as growth factors, angiogenesis stimulators,and so on. 3) Development of 'intelligent biomimetic materials' as extracellular matrix with a good superficial and structural compatibility as well as biological activity to stimulate predictable, controllable and desirable responses under defined conditions.Molecular biology is currently one of the most exciting fields of research across life sciences,and the advances in it also bring a bright future for tissue engineering to overcome these difficulties.In recent years,tissue engineering benefits a lot from molecular biology.Only a comprehensive understanding of the involved ingredients of tissue engineering (cells,tissue inducing factors,genes,biomaterials) and the subtle relationships between them at molecular level can lead to a successful manipulation of reparative processes and a better biological substitute.Molecular tissue engineering,the offspring of the tissue engineering and molecular biology,has gained an increasing importance in recent years.It offers the promise of not simply replacing tissue,but improving the restoration.The studies presented in this article put forward this new concept for the first time and provide an insight into the basic principles,status and challenges of this emerging technology.

Magnesium incorporated chitosan based scaffolds for tissue engineering applications

Chitosan based porous scaffolds are of great interest in biomedical applications especially in tissue engineering because of their excellent biocompatibility in vivo,controllable degradation rate and tailorable mechanical properties.This paper presents a study of the fabrication and characterization of bioactive scaffolds made of chitosan(CS),carboxymethyl chitosan(CMC)and magnesium gluconate(MgG).Scaffolds were fabricated by subsequent freezing-induced phase separation and lyophilization of polyelectrolyte complexes of CS,CMC and MgG.The scaffolds possess uniform porosity with highly interconnected pores of 50-250 μm size range.Compressive strengths up to 400 kPa,and elastic moduli up to 5 MPa were obtained.The scaffolds were found to remain intact,retaining their original threedimensional frameworks while testing in in-vitro conditions.These scaffolds exhibited no cytotoxicity to 3T3 fibroblast and osteoblast cells.These observations demonstrate the efficacy of this new approach to preparing scaffold materials suitable for tissue engineering applications.

Nano-hydroxyapatite formation via co-precipitation with chitosan-g-poly（N-isopropylacrylamide） in coil and globule states for tissue engineering application

With the excellent biocompatibility and osteo- conductivity, nano-hydroxyapatite （nHA） has shown significant prospect in the biomedical applications. Con- trolling the size, crystallinity and surface properties of nHA crystals is a critical challenge in the design of HA based biomaterials. With the graft copolymer of chitosan and poly（N-isopropylacrylamide） in coil and globule states as a template respectively, a novel composite from chitosan-g-poly（N-isopropylacrylamide） and nano-hydro- xyapatite （CS-g-PNIPAM/nHA） was prepared via co- precipitation. Zeta potential analysis, thermogravimetric analysis and X-ray diffraction were used to identify the formation mechanism of the CS-g-PNIPAM/nHA compo- site and its morphology was observed by transmission electron microscopy. The results suggested that the physical aggregation states of the template polymer could induce or control the size, crystallinity and morphology of HA crystals in the CS-g-PNIPAM/nHA composite. The CS-g-PNIPAM/nHA composite was then introduced to chitosan-gelatin （CS-Gel） polyelectronic complex and the cytocompatibility of the resulting CS- Gel/composite hybrid film was evaluated. This hybrid film was proved to be favorable for the proliferation of MC 3T3-E1 cells. Therefore, the CS-g-PNIPAM/nHA compo- site is a potential biomaterial in bone tissue engineering.

Magnetic resonance imaging-three-dimensional printing technology fabricates customized scaffolds for brain tissue engineering

Conventional fabrication methods lack the ability to control both macro- and micro-structures of generated scaffolds. Three-dimensional printing is a solid free-form fabrication method that provides novel ways to create customized scaffolds with high precision and accuracy. In this study, an electrically controlled cortical impactor was used to induce randomized brain tissue defects. The overall shape of scaffolds was designed using rat-specific anatomical data obtained from magnetic resonance imaging, and the internal structure was created by computer- aided design. As the result of limitations arising from insufficient resolution of the manufacturing process, we magnified the size of the cavity model prototype five-fold to successfully fabricate customized collagen-chitosan scaffolds using three-dimensional printing. Results demonstrated that scaffolds have three-dimensional porous structures, high porosity, highly specific surface areas, pore connectivity and good internal characteristics. Neural stem cells co-cultured with scaffolds showed good viability, indicating good biocompatibility and biodegradability. This technique may be a promising new strategy for regenerating complex damaged brain tissues, and helps pave the way toward personalized medicine.

Oxysterols as promising small molecules for bone tissue engineering: Systematic review

BACKGROUND Bone tissue engineering is an area of continued interest within orthopaedic surgery,as it promises to create implantable bone substitute materials that obviate the need for autologous bone graft.Recently,oxysterols–oxygenated derivatives of cholesterol-have been proposed as a novel class of osteoinductive small molecules for bone tissue engineering.Here,we present the first systematic review of the in vivo evidence describing the potential therapeutic utility of oxysterols for bone tissue engineering.AIM To systematically review the available literature examining the effect of oxysterols on in vivo bone formation.METHODS We conducted a systematic review of the literature following PRISMA guidelines.Using the PubMed/MEDLINE,Embase,and Web of Science databases,we queried all publications in the English-language literature investigating the effect of oxysterols on in vivo bone formation.Articles were screened for eligibility using PICOS criteria and assessed for potential bias using an expanded version of the SYRCLE Risk of Bias assessment tool.All full-text articles examining the effect of oxysterols on in vivo bone formation were included.Extracted data included:Animal species,surgical/defect model,description of therapeutic and control treatments,and method for assessing bone growth.Primary outcome was fusion rate for spinal fusion models and percent bone regeneration for critical-sized defect models.Data were tabulated and described by both surgical/defect model and oxysterol employed.Additionally,data from all included studies were aggregated to posit the mechanism by which oxysterols may mediate in vivo bone formation.RESULTS Our search identified 267 unique articles,of which 27 underwent full-text review.Thirteen studies(all preclinical)met our inclusion/exclusion criteria.Of the 13 included studies,5 employed spinal fusion models,2 employed critical-sized alveolar defect models,and 6 employed critical-sized calvarial defect models.Based upon SYRCLE criteria,the included studies were found to possess an overall“unclear risk of bias”;54%of studies reported treatment randomization and 38%reported blinding at any level.Overall,seven unique oxysterols were evaluated:20(S)-hydroxycholesterol,22(R)-hydroxycholesterol,22(S)-hydroxycholesterol,Oxy4/Oxy34,Oxy18,Oxy21/Oxy133,and Oxy49.All had statistically significant in vivo osteoinductive properties,with Oxy4/Oxy34,Oxy21/Oxy133,and Oxy49 showing a dose-dependent effect in some cases.In the eight studies that directly compared oxysterols to rhBMP-2-treated animals,similar rates of bone growth occurred in the two groups.Biochemical investigation of these effects suggests that they may be primarily mediated by direct activation of Smoothened in the Hedgehog signaling pathway.CONCLUSION Present preclinical evidence suggests oxysterols significantly augment in vivo bone formation.However,clinical trials are necessary to determine which have the greatest therapeutic potential for orthopaedic surgery patients.

Evaluation of PLGA/Chitosan/HA Conduits for Nerve Tissue Reconstruction

A micro-envioment for nerve cells and tissue growth were designed and constructed via surface modification of poly（L-lactide-co-glycolide）（PLGA） with chitosan and hydroxyapatire（HA）. The poly（L_lactide-co-glycolide）/chitosan/hydroxyapatite （PLGA/chitosan/HA） conduits were manufactured by a combined solvent casting and particulate leaching technique. The conduits were highly porous with an interconnected pore structure and 76.5% porosity. Micropores with 50-100 micrometer diameter were formed in the conduits. In vivo application of PLGA/chitosan/HA conduits for reconstruction of 10 mm sciatic nerve defect was assessed by the walking track analysis, the quantifying of the wet weight of tibialis anterior muscle and the histological assessment. The conduits in host rats in vivo can not only be an effective in promoting regenerating of nerves but can also lead to favorable nerve functional recovery.

Development and potential of a biomimetic chitosan/type Ⅱ collagen scaffold for cartilage tissue engineering

Damaged articular cartilage has very limited capacity for spontaneous healing. Tissue engineering provides a new hope for functional cartilage repair. Creation of an appropriate cell carrier is one of the critical steps for successful tissue engineering. With the supposition that a biomimetic construct might promise to generate better effects, we developed a novel composite scaffold and investigated its potential for cartilage tissue engineering.

Porous Chitosan Microcarriers for Large Scale Cultivation of Cells for Tissue Engineering: Fabrication and Evaluation

Porous chitosan microspheres with diameters ranging from 180μm to 280 μm were successfully prepared, using an anti-phase suspension method combined with temperature controlled freeze-extraction. The mean pore diameter could be regulated from 5 μm to 60μm by varying the freezing temperature through the cooling rate. Results with in vitro chondrocyte cultures showed that cells could attach, proliferate and spread on these porous microspheres as well as inside the microcarriers. The materials and cell cocultures were characterized using both optical and scanning electron microscopy. These results show that the porous chitosan microspheres are promising candidates for tissue culture for use as an injectable tissue engineering scaffold.