Engineering vascularized and innervated bone biomaterials for improved skeletal tissue regeneration

Blood vessels and nerve fibers are distributed throughout the entirety of skeletal tissue, and play important roles during bone development and fracture healing by supplying oxygen, nutrients, and cells. However, despite the successful development of bone mimetic materials that can replace damaged bone from a structural point of view, most of the available bone biomaterials often do not induce sufficient formation of blood vessels and nerves. In part, this is due to the difficulty of integrating and regulating multiple tissue types within artificial materials, which causes a gap between native skeletal tissues. Therefore, understanding the anatomy and underlying interaction mechanisms of blood vessels and nerve fibers in skeletal tissue is important to develop biomaterials that can recapitulate its complex microenvironment. In this perspective, we highlight the structure and osteogenic functions of the vascular and nervous systems in bone, in a coupled manner. In addition, we discuss important design criteria for engineering vascularized, innervated, and neurovascularized bone implant materials, as well as recent advances in the development of such biomaterials. We expect that bone implant materials with neurovascularized networks can more accurately mimic native skeletal tissue and improve the regeneration of bone tissue.     

Bone‐Like Elastomer‐Toughened Scaffolds with Degradability Kinetics Matching Healing Rates of Injured Bone

Biomaterial scaffolds provide a potentially powerful means of creating precisely engineered bone tissue substitutes with appropriate architecture and mechanical properties. Despite many efforts, there are few satisfactory products available for clinical use, and significant challenges remain in the design of smart constructs, especially for mechanically functional scaffolds. For successful long‐term repair of bone, a scaffold must be strong yet have degradation kinetics matching the healing rate of remodeling bone. Here we report a new family of elastomer‐toughened composite scaffolds fabricated from poly(glycerol sebacate) and Bioglass®. These synthetic scaffolds have very similar mechanical properties to that of cancellous bone of the same porosity, and exhibit a mechanically steady state over an extended period in a physiologic environment. The second feature is of great importance to bone tissue engineering, where a lag phase of degradation following implantation is highly desirable in order to provide support to the damaged or fragmented bone tissue. This work shows that a mechanically smart construct with the three‐stage profile (lag, log, and plateau phases) of ideal degradation kinetics in mechanical function is achievable with synthetic biomaterials.     

Development of a novel collagen–GAG nanofibrous scaffold via electrospinning

Collagen and glycosaminoglycan (GAG) are native constituents of human tissues and are widely utilized to fabricate scaffolds serving as an analog of native extracellular matrix (ECM).The development of blended collagen and GAG scaffolds may potentially be used in many soft tissue engineering applications since the scaffolds mimic the structure and biological function of native ECM. In this study, we were able to obtain a novel nanofibrous collagen–GAG scaffold by electrospinning with collagen and chondroitin sulfate (CS), a widely used GAG. The electrospun collagen–GAG scaffold exhibited a uniform fiber structure in nano-scale diameter. By crosslinking with glutaraldehyde vapor, the collagen–GAG scaffolds could resist from collagenase degradation and enhance the biostability of the scaffolds. This led to the increased proliferation of rabbit conjunctiva fibroblast on the scaffolds. Incorporation of CS into collagen nanofibers without crosslinking did not increase the biostability but still promoted cell growth. In conclusion, the electrospun collagen–GAG scaffolds, with high surface-to-volume ratio, may potentially provide a better environment for tissue formation/biosynthesis compared with the traditional scaffolds.     

Electrospun nanofibers of collagen-chitosan and P(LLA-CL) for tissue engineering

Electrospun nanofibers could be used to mimic the nanofibrous structure of the extracellular matrix (ECM) in native tissue.In tissue engineering,the ECM could be used as tissue engineering scaffold to ...     

Glycosaminoglycan‐Based Biohybrid Hydrogels: A Sweet and Smart Choice for Multifunctional Biomaterials

Glycosaminoglycans (GAGs) govern important functional characteristics of the extracellular matrix (ECM) in living tissues. Incorporation of GAGs into biomaterials opens up new routes for the presentation of signaling molecules, providing control over development, homeostasis, inflammation, and tumor formation and progression. Recent approaches to GAG‐based materials are reviewed, highlighting the formation of modular, tunable biohybrid hydrogels by covalent and non‐covalent conjugation schemes, including both theory‐driven design concepts and advanced processing technologies. Examples of the application of the resulting materials in biomedical studies are provided. For perspective, solid‐phase and chemoenzymatic oligosaccharide synthesis methods for GAG‐derived motifs, rational and high‐throughput design strategies for GAG‐based materials, and the utilization of the factor‐scavenging characteristics of GAGs are highlighted.     

A Thin Carbon‐Fiber Web as a Scaffold for Bone‐Tissue Regeneration

Due to the rapid progress being made in tissue regeneration therapy, biomaterials used as scaffolds are expected to play an important role in future clinical application. We report the development of a 3D web (sheet) consisting of high‐purity carbon fibers in a nanoscale structure. When the thin carbon‐fiber web (TCFW) and recombinant human bone morphogenetic protein 2 (rhBMP‐2) composite is implanted in the murine back muscle, new ectopic bone is formed, and the values of the bone mineral content and bone mineral density are significantly higher than those obtained with a collagen sheet. Observation of the interface between the carbon fibers and bone matrix reveal that the fibers are directly integrated into the bone matrix, indicating high bone‐tissue compatibility. Further, the rhBMP‐2/TCFW composite repairs a critical‐size bone defect within a short time period. These results suggest that the TCFW functions as an effective scaffold material and will play an important role in tissue regeneration in the future.     

Nanoreinforced Hydrogels for Tissue Engineering: Biomaterials that are Compatible with Load‐Bearing and Electroactive Tissues

Given their highly porous nature and excellent water retention, hydrogel‐based biomaterials can mimic critical properties of the native cellular environment. However, their potential to emulate the electromechanical milieu of native tissues or conform well with the curved topology of human organs needs to be further explored to address a broad range of physiological demands of the body. In this regard, the incorporation of nanomaterials within hydrogels has shown great promise, as a simple one‐step approach, to generate multifunctional scaffolds with previously unattainable biological, mechanical, and electrical properties. Here, recent advances in the fabrication and application of nanocomposite hydrogels in tissue engineering applications are described, with specific attention toward skeletal and electroactive tissues, such as cardiac, nerve, bone, cartilage, and skeletal muscle. Additionally, some potential uses of nanoreinforced hydrogels within the emerging disciplines of cyborganics, bionics, and soft biorobotics are highlighted.     

The fabrication of biomineralized fiber-aligned PLGA scaffolds and their effect on enhancing osteogenic differentiation of UCMSC cells

The key factor of scaffold design for bone tissue engineering is to mimic the microenvironment of natural bone extracellular matrix (ECM) and guide cell osteogenic differentiation. The biomineralized fiber-aligned PLGA scaffolds (a-PLGA/CaPs) was developed in this study by mimicking the structure and composition of native bone ECM. The aligned PLGA fibers was prepared by wet spinning and then biomineralized via an alternate immersion method. Introduction of a bioceramic component CaP onto the PLGA fibers led to changes in surface roughness and hydrophilicity, which showed to modulate cell adhesion and cell morphology of umbilical cord mesenchymal stem cells (UCMSCs). It was found that organized actin filaments of UCMSCs cultured on both a-PLGA and a-PLGA/CaP scaffolds appeared to follow contact guidance along the aligned fibers, and those cells grown on a-PLGA/CaP scaffolds exhibited a more polarized cellular morphology. The a-PLGA/CaP scaffold with multicycles of mineralization facilitated the cell attachment on the fiber surfaces and then supported better cell adhesion and contact guidance, leading to enhancement in following proliferation and osteogenic differentiation of UCMSCs. Our results give some insights into the regulation of cell behaviors through design of ECM-mimicking structure and composition and provide an alternative wet-spun fiber-aligned scaffold with HA-mineralized layer for bone tissue engineering application.     

Self‐Assembled Collagen Microparticles by Aerosol as a Versatile Platform for Injectable Anisotropic Materials

Extracellular matrices (ECM) rich in type I collagen exhibit characteristic anisotropic ultrastructures. Nevertheless, working in vitro with this biomacromolecule remains challenging. When processed, denaturation of the collagen molecule is easily induced in vitro avoiding proper fibril self‐assembly and further hierarchical order. Here, an innovative approach enables the production of highly concentrated injectable collagen microparticles, based on collagen molecules self‐assembly, thanks to the use of spray‐drying process. The versatility of the process is shown by performing encapsulation of secretion products of gingival mesenchymal stem cells (gMSCs), which are chosen as a bioactive therapeutic product for their potential efficiency in stimulating the regeneration of a damaged ECM. The injection of collagen microparticles in a cell culture medium results in a locally organized fibrillar matrix. The efficiency of this approach for making easily handleable collagen microparticles for encapsulation and injection opens perspectives in active tissue regeneration and 3D bioprinted scaffolds.     

Bioactive materials for tissue engineering, regeneration and repair

Tissue engineering is an interdisciplinary field which applies the principles of engineering and the life sciences to the design, construction, modification, growth and maintenance of living tissues [1, 2]. One of two approaches can be taken: (1) in vitro construction of bioartificial tissues from cells seeded onto a resorbable scaffold or (2) in vivo modification of cell growth and function to stimulate tissue regeneration [2, 3]. This concept represents a shift in emphasis from replacement to regeneration of diseased or damaged tissues, in which the development of bioactive materials has played a significant role.This paper will begin with an overview of the use of biomaterials as implants and their limitations, leading to the reasons for the dramatic shift in focus regarding the approach to repairing damaged tissues. The majority of the paper will discuss the ways in which biomaterials can be developed to implement the concept of tissue engineering. Finally, the implications of these developments for future treatment of damaged or diseased tissues will be considered.     

2D‐Black‐Phosphorus‐Reinforced 3D‐Printed Scaffolds:A Stepwise Countermeasure for Osteosarcoma

With the ever‐deeper understanding of nano–bio interactions and the development of fabrication methodologies of nanomaterials, various therapeutic platforms based on nanomaterials have been developed for next‐generation oncological applications, such as osteosarcoma therapy. In this work, a black phosphorus (BP) reinforced 3D‐printed scaffold is designed and prepared to provide a feasible countermeasure for the efficient localized treatment of osteosarcoma. The in situ phosphorus‐driven, calcium‐extracted biomineralization of the intra‐scaffold BP nanosheets enables both photothermal ablation of osteosarcoma and the subsequent material‐guided bone regeneration in physiological microenvironment, and in the meantime endows the scaffolds with unique physicochemical properties favoring the whole stepwise therapeutic process. Additionally, a corrugated structure analogous to Haversian canals is found on newborn cranial bone tissue of Sprague–Dawley rats, which may provide much inspiration for the future research of bone‐tissue engineering.     

3D Biomaterial Microarrays for Regenerative Medicine: Current State‐of‐the‐Art,Emerging Directions and Future Trends

Three dimensional (3D) biomaterial microarrays hold enormous promise for regenerative medicine because of their ability to accelerate the design and fabrication of biomimetic materials. Such tissue‐like biomaterials can provide an appropriate microenvironment for stimulating and controlling stem cell differentiation into tissue‐specific lineages. The use of 3D biomaterial microarrays can, if optimized correctly, result in a more than 1000‐fold reduction in biomaterials and cells consumption when engineering optimal materials combinations, which makes these miniaturized systems very attractive for tissue engineering and drug screening applications.     

Tailoring material properties of a nanofibrous extracellular matrix derived hydrogel

In the native tissue, the interaction between cells and the extracellular matrix (ECM) is essential for cell migration, proliferation, differentiation, mechanical stability, and signaling. It has been shown that decellularized ECMs can be processed into injectable formulations, thereby allowing for minimally invasive delivery. Upon injection and increase in temperature, these materials self-assemble into porous gels forming a complex network of fibers with nanoscale structure. In this study we aimed to examine and tailor the material properties of a self-assembling ECM hydrogel derived from porcine myocardial tissue, which was developed as a tissue specific injectable scaffold for cardiac tissue engineering. The impact of gelation parameters on ECM hydrogels has not previously been explored. We examined how modulating pH, temperature, ionic strength, and concentration affected the nanoscale architecture, mechanical properties, and gelation kinetics. These material characteristics were assessed using scanning electron microscopy, rheometry, and spectrophotometry, respectively. Since the main component of the myocardial matrix is collagen, many similarities between the ECM hydrogel and collagen gels were observed in terms of the nanofibrous structure and modulation of properties by altering ionic strength. However, variation from collagen gels was noted for the gelation temperature along with varied times and rates of gelation. These discrepancies when compared to collagen are likely due to the presence of other ECM components in the decellularized ECM based hydrogel. These results demonstrate how the material properties of ECM hydrogels could be tailored for future in vitro and in vivo applications.     

Synthesis of Photopolymerizable Hydrophilic Macromers and Evaluation of Their Applicability as Reactive Resin Components for the Fabrication of Three‐Dimensionally Structured Hydrogel Matrices by 2‐Photon‐Polymerization

Two‐photon polymerization (2‐PP) is a promising new photolithographic technique to fabricate three‐dimensional (3D), micro‐ and nano‐structured tissue engineering scaffolds from photopolymerizable monomers. Although various photo resins are known for the use in 2‐PP, there is currently a need for photo‐curable monomers processable by 2‐PP to generate biocompatible 3D‐structured hydrogel materials for soft or cartilage tissue regeneration. In the present work hydrophilic methacrylate monomers and macromers based on synthetic poly(glycerine) and poly(ethylene glycol) urethanes as well as on the biopolymers dextran and hyaluronan is prepared. The photopolymerization behavior of these substances are investigated and formed hydrogel networks are studied with regard to their mechanical properties, cytocompatibility, and hydrolytic degradation. Based on these examinations simple 3D model structures are fabricated from these photo‐curable monomers and macromers by 2‐PP. It is shown that both the synthetic monomers and the dextran methacrylate macromer are efficient 2‐PP starting materials whereas the hyaluronan methacrylate can be used for 2‐PP only in combination with suitable water‐soluble co‐monomers. No cytotoxic effects are found in preliminary chondrocyte cultivation experiments on 2‐PP‐fabricated scaffolds but initial cell adhesion on the hydrophilic scaffold surfaces is rather low and has to be further improved to apply these structures in tissue engineering.     

Surface Micro‐ and Nanoengineering: Applications of Layer‐by‐Layer Technology as a Versatile Tool to Control Cellular Behavior

Extracellular matrix (ECM) cues have been widely investigated for their impact on cellular behavior. Among mechanics, physics, chemistry, and topography, different ECM properties have been discovered as important parameters to modulate cell functions, activating mechanotransduction pathways that can influence gene expression, proliferation or even differentiation. Particularly, ECM topography has been gaining more and more interest based on the evidence that these physical cues can tailor cell behavior. Here, an overview of bottom‐up and top‐down approaches reported to produce materials capable of mimicking the ECM topography and being applied for biomedical purposes is provided. Moreover, the increasing motivation of using the layer‐by‐layer (LbL) technique to reproduce these topographical cues is highlighted. LbL assembly is a versatile methodology used to coat materials with a nanoscale fidelity to the geometry of the template or to produce multilayer thin films composed of polymers, proteins, colloids, or even cells. Different geometries, sizes, or shapes on surface topography can imply different behaviors: effects on the cell adhesion, proliferation, morphology, alignment, migration, gene expression, and even differentiation are considered. Finally, the importance of LbL assembly to produce defined topographical cues on materials is discussed, highlighting the potential of micro‐ and nanoengineered materials to modulate cell function and fate.     

A Mussel‐Inspired Persistent ROS‐Scavenging,Electroactive, and Osteoinductive Scaffold Based on Electrochemical‐Driven In Situ Nanoassembly

Conductive polymers are promising for bone regeneration because they can regulate cell behavior through electrical stimulation; moreover, they are antioxidative agents that can be used to protect cells and tissues from damage originating from reactive oxygen species (ROS). However, conductive polymers lack affinity to cells and osteoinductivity, which limits their application in tissue engineering. Herein, an electroactive, cell affinitive, persistent ROS‐scavenging, and osteoinductive porous Ti scaffold is prepared by the on‐surface in situ assembly of a polypyrrole‐polydopamine‐hydroxyapatite (PPy‐PDA‐HA) film through a layer‐by‐layer pulse electrodeposition (LBL‐PED) method. During LBL‐PED, the PPy‐PDA nanoparticles (NPs) and HA NPs are in situ synthesized and uniformly coated on a porous scaffold from inside to outside. PDA is entangled with and doped into PPy to enhance the ROS scavenging rate of the scaffold and realize repeatable, efficient ROS scavenging over a long period of time. HA and electrical stimulation synergistically promote osteogenic cell differentiation on PPy‐PDA‐HA films. Ultimately, the PPy‐PDA‐HA porous scaffold provides excellent bone regeneration through the synergistic effects of electroactivity, cell affinity, and antioxidative activity of the PPy‐PDA NPs and the osteoinductivity of HA NPs. This study provides a new strategy for functionalizing porous scaffolds that show great promise as implants for tissue regeneration.     

Long-term viability of coronary artery smooth muscle cells on poly(L-lactide-co-epsilon-caprolactone) nanofibrous scaffold indicates its potential for blood vessel tissue engineering.

Biodegradable polymer nanofibres have been extensively studied as cell culture scaffolds in tissue engineering. However, long-term in vitro studies of cell-nanofibre interactions were rarely reported and successful organ regeneration using tissue engineering techniques may take months (e.g. blood vessel tissue engineering). Understanding the long-term interaction between cells and nanofibrous scaffolds (NFS) is crucial in material selection, design and processing of the tissue engineering scaffolds. In this study, poly(L-lactide-co-epsilon-caprolactone) [P(LLA-CL)] (70:30) copolymer NFS were produced by electrospinning. Porcine coronary artery smooth muscle cells (PCASMCs) were seeded and cultured on the scaffold to evaluate cell-nanofibre interactions for up to 105 days. A favourable interaction between this scaffold and PCASMCs was demonstrated by cell viability assay, scanning electron microscopy, histological staining and extracellular matrix (ECM) secretion. Degradation behaviours of the scaffolds with or without PCASMC culture were determined by mechanical testing and gel permeation chromatography (GPC). The results showed that the PCASMCs attached and proliferated well on the P(LLA-CL) NFS. Large amount of ECM protein secretion was observed after 50 days of culture. Multilayers of aligned oriented PCASMCs were formed on the scaffold after two months of in vitro culture. In the degradation study, the PCASMCs were not shown to significantly increase the degradation rate of the scaffolds for up to 105 days of culture. The in vitro degradation time of the scaffold could be as long as eight months by extrapolating the results from GPC. These observations further supported the potential use of the P(LLA-CL) nanofibre in blood vessel tissue engineering.     

Flexible Fabrication of Shape‐Controlled Collagen Building Blocks for Self‐Assembly of 3D Microtissues

Creating artificial tissue‐like structures that possess the functionality, specificity, and architecture of native tissues remains a big challenge. A new and straightforward strategy for generating shape‐controlled collagen building blocks with a well‐defined architecture is presented, which can be used for self‐assembly of complex 3D microtissues. Collagen blocks with tunable geometries are controllably produced and released via a membrane‐templated microdevice. The formation of functional microtissues by embedding tissue‐specific cells into collagen blocks with expression of specific proteins is described. The spontaneous self‐assembly of cell‐laden collagen blocks into organized tissue constructs with predetermined configurations is demonstrated, which are largely driven by the synergistic effects of cell–cell and cell–matrix interactions. This new strategy would open up new avenues for the study of tissue/organ morphogenesis, and tissue engineering applications.     

骨组织工程支架材料的研究进展

骨组织工程的发展,要求充分结合材料工程与生物工程相关知识,对植入材料进行分子及细胞水平的设计.细胞外基质材料(支架材料)的选择与制备是骨组织工程的一项重要而关键的任务.如何找到能促进并指导细胞黏附、增殖的支架材料是目前骨组织工程研究的热点之一.介绍了骨组织工程相关原理,并综述了几种支架材料的发展研究现状.     

Natural‐Based Nanocomposites for Bone Tissue Engineering and Regenerative Medicine: A Review

Tissue engineering and regenerative medicine has been providing exciting technologies for the development of functional substitutes aimed to repair and regenerate damaged tissues and organs. Inspired by the hierarchical nature of bone, nanostructured biomaterials are gaining a singular attention for tissue engineering, owing their ability to promote cell adhesion and proliferation, and hence new bone growth, compared with conventional microsized materials. Of particular interest are nanocomposites involving biopolymeric matrices and bioactive nanosized fillers. Biodegradability, high mechanical strength, and osteointegration and formation of ligamentous tissue are properties required for such materials. Biopolymers are advantageous due to their similarities with extracellular matrices, specific degradation rates, and good biological performance. By its turn, calcium phosphates possess favorable osteoconductivity, resorbability, and biocompatibility. Herein, an overview on the available natural polymer/calcium phosphate nanocomposite materials, their design, and properties is presented. Scaffolds, hydrogels, and fibers as biomimetic strategies for tissue engineering, and processing methodologies are described. The specific biological properties of the nanocomposites, as well as their interaction with cells, including the use of bioactive molecules, are highlighted. Nanocomposites in vivo studies using animal models are also reviewed and discussed.