Regeneration Of Complex Tissues Research Articles

Nanofiber Technology for Regenerative Engineering.

Regenerative engineering is powerfully emerging as a successful strategy for the regeneration of complex tissues and biological organs using a convergent approach that integrates several fields of expertise. This innovative and disruptive approach has spurred the demands for more choice of biomaterials with distinctive biological recognition properties. An ideal biomaterial is one that closely mimics the hierarchical architecture and features of the extracellular matrices (ECM) of native tissues. Nanofabrication technology presents an excellent springboard for the development of nanofiber scaffolds that can have positive interactions in the immediate cellular environment and stimulate specific regenerative cascades at the molecular level to yield healthy tissues. This paper systematically reviews the electrospinning process technology and its utility in matrix-based regenerative engineering, focusing mainly on musculoskeletal tissues. It briefly outlines the electrospinning/three-dimensional printing system duality and concludes with a discussion on the technology outlook and future directions of nanofiber matrices.

Three-Dimensional Bioprinting of Perfusable Hierarchical Microchannels with Alginate and Silk Fibroin Double Cross-linked Network.

Vascularization is essential for the regeneration of three-dimensional (3D) bioprinting organs. As a general method to produce microfluidic channels in 3D printing constructs, coaxial extrusion has attracted great attention. However, the biocompatible bioinks are very limited for coaxial extrusion to fabricate microchannels with regular structure and enough mechanical properties. Herein, a hybrid bioink composed of alginate (Alg) and silk fibroin (SF) was proposed for 3D bioprinting of microchannel networks based on coaxial extrusion. The rheological properties of the bioink demonstrated that the hybrid Alg/SF bioink exhibited improved viscosity and shear thinning behavior compared with either pure Alg or SF bioink and had similar storage and loss modulus in a wide range of shear frequency, indicating a sound printability. Using a coaxial extrusion system with calcium ions and Pluronic F127 flowing through the core nozzle as cross-linkers, the Alg/SF bioink could be extruded and deposited to form a 3D scaffold with interconnected microchannels. The regular structure and smooth pore wall of microchannels inside the scaffold were demonstrated by optical coherence tomography. Micropores left by the rinse of F127 were observed by scanning electron microscope, constituting a hierarchical structure together with the microchannels and printed macropores. Fourier transform infrared spectroscopy analysis proved the complete rinse of F127 and the formation of β-sheet SF structure. Thus, Alg/SF could form a double cross-linked network, which was much stronger than the pure Alg network. Moreover, cells in the Alg/SF scaffold showed higher viability and proliferation rate than in the Alg scaffold. Therefore, Alg/SF is a promising bioink for coaxial extrusion-based 3D bioprinting, with the printed microchannel network beneficial for complex tissue and organ regeneration.

3D printable carboxylated cellulose nanocrystal-reinforced hydrogel inks for tissue engineering

To achieve a three-dimensional (3D) microenvironment for complex tissue regeneration is a great challenge when developing biomaterials as artificial extracellular matrix (ECM) with properties similar to that of native tissue. Polysaccharide-based hydrogel shows great potential as ECM in the regeneration of damaged tissues or reconstruction of organs, demonstrating properties similar to those of native ECM. Extrusion 3D printing of cell-free or cell-loaded hydrogel ink has led to a more sophisticated fabrication of the desired compositions and architectures for tissue engineering applications. The development of stable cell-free and cell-loaded hydrogel inks with optimal physicochemical properties and biocompatibility is also a major concern in direct-write extrusion-based 3D printing. In this study, carboxylated cellulose nanocrystals (cCNCs) were prepared using ammonium persulfate, where transmission electron microscopy, Fourier-transform infrared spectroscopy, and x-ray diffraction analyses confirmed their successful preparation. Further, the effect of cCNCs (–COOH) and/or xanthan gum (XG) (–COOH) was evaluated on the rheological behavior of the sodium alginate (SA) hydrogel matrix. The incorporation of cCNCs and XG manipulated the flow and shear-thinning behavior of the hydrogel inks, thereby improving the printing ability. The results showed good rheological properties, post-printing fidelity, and dynamic mechanical properties under compression of the developed hydrogel inks. Furthermore, good viability of the human skin fibroblast (CCD-986Sk) cells on bulk hydrogels (hydrogel inks) was observed, as demonstrated by both qualitative and quantitative cell analyses. The use of cCNCs and XG in SA hydrogel inks provides a primary insight for further improvement in designing 3D bioprintable hydrogel inks.

Predicting the orientation of magnetic microgel rods for soft anisotropic biomimetic hydrogels

Predicting the orientation rate of rod-shaped magnetic microgels to induce unidirectional regeneration of complex and sensitive tissues.

Mechanisms of Complex Tissue Regeneration and the Functional Outcomes of Craniofacial Dysmorphology

Mechanisms of Complex Tissue Regeneration and the Functional Outcomes of Craniofacial Dysmorphology

TGF-β Signaling Promotes Tissue Formation during Cardiac Valve Regeneration in Adult Zebrafish

Cardiac valve disease can lead to severe cardiac dysfunction and is thus a frequent cause of morbidity and mortality. Its main treatment is valve replacement, which is currently greatly limited by the poor recellularization and tissue formation potential of the implanted valves. As we still lack suitable animal models to identify modulators of these processes, here we used adult zebrafish and found that, upon valve decellularization, they initiate a rapid regenerative program that leads to the formation of new functional valves. After injury, endothelial and kidney marrow-derived cells undergo cell cycle re-entry and differentiate into new extracellular matrix-secreting valve cells. The TGF-β signaling pathway promotes the regenerative process by enhancing progenitor cell proliferation as well as valve cell differentiation. These findings reveal a key role for TGF-β signaling in cardiac valve regeneration and establish the zebrafish as a model to identify and test factors promoting cardiac valve recellularization and growth.

Hierarchical porosity inherited by natural sources affects the mechanical and biological behaviour of bone scaffolds

A 3-D porous apatite scaffold (B-HA), recently obtained through biomorphic transformation of a natural wood, is investigated on its multi-scale porous structure determining superior biological and mechanical behaviour. B-HA shows a hierarchically organized architecture with wide aligned channels interconnected with smaller tubules, thus recapitulating in detail the lymphatic network of the original wood template. As induced by its biomimetic architecture, B-HA displays values of tensile/compressive strengths and stiffness, higher than values usually measured in sintered ceramics with isotropic porosity. Furthermore, B-HA shows uncommon toughness for a pure ceramic body, plus a tensile/compressive-strength ratio higher than one, so that the material occupies a zone in the Ashby map where ceramics are normally absent. From biological viewpoint, cell co-culture tests in bioreactor are shown to report encouraging results in enhancing the complex tissue regeneration process, thus making B-HA promising as a scaffold able to promote bone regeneration, particularly for large bone defects.

Extracellular vesicles as a novel therapeutic tool for cell-free regenerative medicine in oral rehabilitation.

Oral maxillofacial defects may always lead to complicated hard and soft tissue loss, including bone, nerve, blood vessels, teeth and skin, which are difficult to restore and severely influence the life quality of patients. Extracellular vesicles (EVs), including exosomes, microvesicles and apoptotic bodies, are emerging as potential solutions for complex tissue regeneration through cell-free therapies. In this review, we highlight the functional roles of EVs in the regenerative medicine for oral maxillofacial rehabilitation, specifically bone, skin, blood vessels, peripheral nerve and tooth-related tissue regeneration. Publications were reviewed by two researchers independently basing on three databases (PubMed, MEDLINE and Web of Science), until 31 December 2018. Basing on current researches, we classified the origin of EVs for regenerative medicine into four categories: related cells in the regenerative niche, mesenchymal stem cells, immune cells and body fluids. The secretome of different cells are distinct, while the same cells secrete different EVs under varied conditions; therefore, the content profiles of EVs and regulatory mechanisms on target cells are compared and emphasised. By unravelling the regulatory mechanisms of EVs in tissue regeneration, modified cells and tailored EVs with specific target may be produced for precision medicine with high efficacy.

3D printing electrospinning fiber-reinforced decellularized extracellular matrix for cartilage regeneration

Cartilage decellularized matrix (CDM) is considered a promising biomaterial for fabricating cartilage tissue engineering scaffolds. An ideal CDM-based scaffold should possess customizable 3D shape for complex tissue regeneration and proper pore size for cell infiltration, as well as provide mechanical support for cell growth. 3D printing is an efficiently technique for preparing customizable 3D scaffolds, however, fabricating CDM-based 3D-printed scaffolds with customizable shapes, proper pore structure and satisfactory mechanical properties remains a challenge. In the current study, to achieve customizable CDM-based 3D scaffolds, CDM was successfully processed into inks suitable for 3D printing. Further, the poor mechanics of CDM-based scaffolds were significantly improved by adding electrospinning fiber into the CDM-based inks for 3D printing. Importantly, the 3D-printed electrospinning fiber-reinforced CDM-based scaffold presented good biocompatibility and can enhance repair articular cartilage defects in rabbits. The current study provides a novel strategy for printing electrospinning fiber-reinforced CDM-based scaffolds for tissue regeneration.

Regulation of Genomic Output and (Pluri)potency in Regeneration.

Regeneration is a remarkable phenomenon that has been the subject of awe and bafflement for hundreds of years. Although regeneration competence is found in highly divergent organisms throughout the animal kingdom, recent advances in tools used for molecular and genomic characterization have uncovered common genes, molecular mechanisms, and genomic features in regenerating animals. In this review we focus on what is known about how genome regulation modulates cellular potency during regeneration. We discuss this regulation in the context of complex tissue regeneration in animals, from Hydra to humans, with reference to ex vivo-cultured cell models of pluripotency when appropriate. We emphasize the importance of a detailed molecular understanding of both the mechanisms that regulate genomic output and the functional assays that assess the biological relevance of such molecular characterizations.

Development: How Tadpoles ROC Tail Regeneration

Specialized epidermal cells are essential for the complex tissue regeneration required to replace tails and limbs, but their exact identities and molecular roles remain murky. Recent work in Xenopus has identified an epidermal cell population critical for tail regeneration, providing intriguing new directions for the field.

Epigenetic inhibitor zebularine activates ear pinna wound closure in the mouse

BackgroundMost studies on regenerative medicine focus on cell-based therapies and transplantations. Small-molecule therapeutics, though proved effective in different medical conditions, have not been extensively investigated in regenerative research. It is known that healing potential decreases with development and developmental changes are driven by epigenetic mechanisms, which suggests epigenetic repression of regenerative capacity. MethodsWe applied zebularine, a nucleoside inhibitor of DNA methyltransferases, to stimulate the regenerative response in a model of ear pinna injury in mice. FindingsWe observed the regeneration of complex tissue that was manifested as improved ear hole repair in mice that received intraperitoneal injections of zebularine. Six weeks after injury, the mean hole area decreased by 83.2 ± 9.4% in zebularine-treated and by 43.6 ± 15.4% in control mice (p < 10−30). Combined delivery of zebularine and retinoic acid potentiated and accelerated this effect, resulting in complete ear hole closure within three weeks after injury. We found a decrease in DNA methylation and transcriptional activation of neurodevelopmental and pluripotency genes in the regenerating tissues. InterpretationThis study is the first to demonstrate an effective induction of complex tissue regeneration in adult mammals using zebularine. We showed that the synergistic action of an epigenetic drug (zebularine) and a transcriptional activator (retinoic acid) could be effectively utilized to induce the regenerative response, thus delineating a novel pharmacological strategy for regeneration. The strategy was effective in the model of ear pinna regeneration in mice, but zebularine acts on different cell types, therefore, a similar approach can be tested in other tissues and organs.

Three-Dimensional Printing of Tissue Engineering Scaffolds with Horizontal Pore and Composition Gradients.

In this study, we report the development of a novel multimaterial segmented three-dimensional printing methodology to fabricate porous scaffolds containing discrete horizontal gradients of composition and porosity. This methodology is particularly beneficial to preparing porous scaffolds with intricate structures and graded compositions for the regeneration of complex tissues. The technique presented is compatible with many commercially available bioprinters commonly used in biofabrication, and can be adapted to better replicate the architectural and compositional requirements of individual tissues compared with traditional scaffold printing methods.

Recombinant Human Bone Morphogenetic Protein 7 Exerts Osteo-Catabolic Effects on Bone Grafts That Outweigh Its Osteo-Anabolic Capacity.

This study aimed to investigate the effects of recombinant human bone morphogenetic protein (rhBMP-7) on human cancellous bone grafts (BGs) while differentiating between anabolic and catabolic events. Human BGs alone or supplemented with rhBMP-7 were harvested 14weeks after subcutaneous implantation into NOD/Scid mice, and studied via micro-CT, histomorphometry, immunohistochemistry and flow cytometry. Immunohistochemical staining for human-specific proteins made it possible to differentiate between grafted human bone and newly formed murine bone. Only BGs implanted with rhBMP-7 formed an ossicle containing a functional hematopoietic compartment. The total ossicle volume in the BMP+ group was higher than in the BMP- group (835mm3 vs. 365mm3, respectively, p < 0.001). The BMP+ group showed larger BM spaces (0.47mm vs. 0.28mm, p = 0.002) and lower bone volume-to-total volume ratio (31% vs. 47%, p = 0.002). Immunohistochemical staining for human-specific proteins confirmed a higher ratio of newly formed bone area (murine) to total area (0.12 vs. 0.001, p < 0.001) in the BMP+ group, while the ratio of grafted bone (human) area to total area was smaller (0.14 vs. 0.34, p = 0.004). The results demonstrate that rhBMP-7 induces BG resorption at a higher rate than new bone formation while creating a haematopoietic niche. Clinicians therefore need to consider the net catabolic effect when rhBMP-7 is used with BGs. Overall, this model indicates its promising application to further decipher BMPs action on BGs and its potential in complex bone tissue regeneration.

Pore-forming bioinks to enable spatio-temporally defined gene delivery in bioprinted tissues

The regeneration of complex tissues and organs remains a major clinical challenge. With a view towards bioprinting such tissues, we developed a new class of pore-forming bioink to spatially and temporally control the presentation of therapeutic genes within bioprinted tissues. By blending sacrificial and stable hydrogels, we were able to produce bioinks whose porosity increased with time following printing. When combined with amphipathic peptide-based plasmid DNA delivery, these bioinks supported enhanced non-viral gene transfer to stem cells in vitro. By modulating the porosity of these bioinks, it was possible to direct either rapid and transient (pore-forming bioinks), or slower and more sustained (solid bioinks) transfection of host or transplanted cells in vivo. To demonstrate the utility of these bioinks for the bioprinting of spatially complex tissues, they were next used to zonally position stem cells and plasmids encoding for either osteogenic (BMP2) or chondrogenic (combination of TGF-β3, BMP2 and SOX9) genes within networks of 3D printed thermoplastic fibers to produce mechanically reinforced, gene activated constructs. In vivo, these bioprinted tissues supported the development of a vascularised, bony tissue overlaid by a layer of stable cartilage. When combined with multiple-tool biofabrication strategies, these gene activated bioinks can enable the bioprinting of a wide range of spatially complex tissues.

Research progress of three-dimensional printing technology in liver surgery and hepatotoxicity evaluation

Three-dimensional (3D) printing, an emerging rapid prototyping technology, has been widely used in biomedical field. 3D printing was originally used to construct the visualization models and molds in medicine. With the development of 3D printing in biomedical field, the technology was gradually applied in complex tissue regeneration and organ reconstruction. Artificial tissues and organs obtained by 3D printing are expected to be used for organ transplantation, new drug development and drug toxicity evaluation in the field of medicine and health care research. This paper describes the individualized application of 3D printing technology in liver surgery and introduces the research progress of 3D bioprinting technology in liver transplantation, drug metabolism and hepatotoxicity evaluation, and prospects its future development trend to provide a reference for further study.

Facile incorporation of REDV into porous silk fibroin scaffolds for enhancing vascularization of thick tissues

Rapid neovascularization within scaffolds is critical for the regeneration of thick complex tissues. The surface immobilization of peptides and other active molecules have been explored to improve the vascularization capacity of implants. However, the rapid degradation of these molecules, the reaction conditions and cross-linking usually result in decreased vascularization capability. Here, we introduced a temperate, all-aqueous process to achieve bulk porous silk fibroin (SF) scaffolds. A temperature controlled process was used to induce the water stable structure by SF self-assembly. Arg-Glu-Asp-Val (REDV) peptides were added into SF solution and fixed within SF scaffolds during the self-assembly process. The results showed that the functionalized scaffolds markedly promoted the adhesion of endothelial cells in vitro. Moreover, the in vivo studies indicated enhanced cell infiltration in the bulk functionalized SF scaffolds and impressive vascularization at 4 weeks post-implantation. The functionalized scaffolds demonstrated excellent vascularization capability, providing an exciting biomaterial option for thick tissue regeneration.

The quest for mechanically and biologically functional soft biomaterials via soft network composites.

Developing multifunctional soft biomaterials capable of addressing all the requirements of the complex tissue regeneration process is a multifaceted problem. In order to tackle the current challenges, recent research efforts are increasingly being directed towards biomimetic design concepts that can be translated into soft biomaterials via advanced manufacturing technologies. Among those, soft network composites consisting of a continuous hydrogel matrix and a reinforcing fibrous network closely resemble native soft biological materials in terms of design and composition as well as physicochemical properties. This article reviews soft network composite systems with a particular emphasis on the design, biomaterial and fabrication aspects within the context of soft tissue engineering and drug delivery applications.

3D Printing of Thermo-Responsive Methylcellulose Hydrogels for Cell-Sheet Engineering.

A possible strategy in regenerative medicine is cell-sheet engineering (CSE), i.e., developing smart cell culture surfaces from which to obtain intact cell sheets (CS). The main goal of this study was to develop 3D printing via extrusion-based bioprinting of methylcellulose (MC)-based hydrogels. Hydrogels were prepared by mixing MC powder in saline solutions (Na2SO4 and PBS). MC-based hydrogels were analyzed to investigate the rheological behavior and thus optimize the printing process parameters. Cells were tested in vitro on ring-shaped printed hydrogels; bulk MC hydrogels were used for comparison. In vitro tests used murine embryonic fibroblasts (NIH/3T3) and endothelial murine cells (MS1), and the resulting cell sheets were characterized analyzing cell viability and immunofluorescence. In terms of CS preparation, 3D printing proved to be an optimal approach to obtain ring-shaped CS. Cell orientation was observed for the ring-shaped CS and was confirmed by the degree of circularity of their nuclei: cell nuclei in ring-shaped CS were more elongated than those in sheets detached from bulk hydrogels. The 3D printing process appears adequate for the preparation of cell sheets of different shapes for the regeneration of complex tissues.

Electrospun Fibers as a Dressing Material for Drug and Biological Agent Delivery in Wound Healing Applications.

Wound healing is a complex tissue regeneration process that promotes the growth of new tissue to provide the body with the necessary barrier from the outside environment. In the class of non-healing wounds, diabetic wounds, and ulcers, dressing materials that are available clinically (e.g., gels and creams) have demonstrated only a slow improvement with current available technologies. Among all available current technologies, electrospun fibers exhibit several characteristics that may provide novel replacement dressing materials for the above-mentioned wounds. Therefore, in this review, we focus on recent achievements in electrospun drug-eluting fibers for wound healing applications. In particular, we review drug release, including small molecule drugs, proteins and peptides, and gene vectors from electrospun fibers with respect to wound healing. Furthermore, we provide an overview on multifunctional dressing materials based on electrospun fibers, including those that are capable of achieving wound debridement and wound healing simultaneously as well as multi-drugs loading/types suitable for various stages of the healing process. Our review provides important and sufficient information to inform the field in development of fiber-based dressing materials for clinical treatment of non-healing wounds.