Suture Fiber Reinforcement of a 3D Printed Gelatin Scaffold for Its Potential Application in Soft Tissue Engineering

Gelatin has excellent biological properties, but its poor physical properties are a major obstacle to its use as a biomaterial ink. These disadvantages not only worsen the printability of gelatin biomaterial ink, but also reduce the dimensional stability of its 3D scaffolds and limit its application in the tissue engineering field. Herein, biodegradable suture fibers were added into a gelatin biomaterial ink to improve the printability, mechanical strength, and dimensional stability of the 3D printed scaffolds. The suture fiber reinforced gelatin 3D scaffolds were fabricated using the thermo-responsive properties of gelatin under optimized 3D printing conditions (−10 °C cryogenic plate, 40–80 kPa pneumatic pressure, and 9 mm/s printing speed), and were crosslinked using EDC/NHS to maintain their 3D structures. Scanning electron microscopy images revealed that the morphologies of the 3D printed scaffolds maintained their 3D structure after crosslinking. The addition of 0.5% (w/v) of suture fibers increased the printing accuracy of the 3D printed scaffolds to 97%. The suture fibers also increased the mechanical strength of the 3D printed scaffolds by up to 6-fold, and the degradation rate could be controlled by the suture fiber content. In in vitro cell studies, DNA assay results showed that human dermal fibroblasts’ proliferation rate of a 3D printed scaffold containing 0.5% suture fiber was 10% higher than that of a 3D printed scaffold without suture fibers after 14 days of culture. Interestingly, the supplement of suture fibers into gelatin biomaterial ink was able to minimize the cell-mediated contraction of the cell cultured 3D scaffolds over the cell culture period. These results show that advanced biomaterial inks can be developed by supplementing biodegradable fibers to improve the poor physical properties of natural polymer-based biomaterial inks.     

3D Printing for Tissue Engineering

Tissue engineering aims to fabricate functional tissue for applications in regenerative medicine and drug testing. More recently, 3D printing has shown great promise in tissue fabrication with a structural control from the micro- to the macroscale by using a layer-by-layer approach. Whether through scaffold-based or scaffold-free approaches, the standard for 3D-printed tissue engineering constructs is to provide a biomimetic structural environment that facilitates tissue formation and promotes host tissue integration (e.g., cellular infiltration, vascularization, and active remodeling). This review will cover several approaches that have advanced the field of 3D printing through novel fabrication methods of tissue engineering constructs. It will also discuss the applications of synthetic and natural materials for 3D printing facilitated tissue fabrication.     

Current Advances in 3D Tissue and Organ Reconstruction

Bi-dimensional culture systems have represented the most used method to study cell biology outside the body for over a century. Although they convey useful information, such systems may lose tissue-specific architecture, biomechanical effectors, and biochemical cues deriving from the native extracellular matrix, with significant alterations in several cellular functions and processes. Notably, the introduction of three-dimensional (3D) platforms that are able to re-create in vitro the structures of the native tissue, have overcome some of these issues, since they better mimic the in vivo milieu and reduce the gap between the cell culture ambient and the tissue environment. 3D culture systems are currently used in a broad range of studies, from cancer and stem cell biology, to drug testing and discovery. Here, we describe the mechanisms used by cells to perceive and respond to biomechanical cues and the main signaling pathways involved. We provide an overall perspective of the most recent 3D technologies. Given the breadth of the subject, we concentrate on the use of hydrogels, bioreactors, 3D printing and bioprinting, nanofiber-based scaffolds, and preparation of a decellularized bio-matrix. In addition, we report the possibility to combine the use of 3D cultures with functionalized nanoparticles to obtain highly predictive in vitro models for use in the nanomedicine field.     

Bioprinting of Thermoresponsive Hydrogels for Next Generation Tissue Engineering: A Review

Bioprinting is a breakthrough technology that integrates living cells, biomaterials, and a robotic dispensing system to create complex structures that mimic original tissues and organs. One of the main components of bioprinting is bioink and hydrogel is essential in bioink formulation. In bioprinting, hydrogel should have good biocompatibility, provide good resolution, and have sufficient mechanical strength to support printed structures. Recently, thermoresponsive hydrogels have gained more and more attention due to their unique characteristic of tunable sol‐gel (liquid to solid phase) transition when temperature is changed, and many biomedical applications from drug delivery devices to tissue scaffolds have demonstrated the potentials of bioprinted thermosresponsive constructs. In this review, we discuss bioprintable thermoresponsive hydrogels with a particular focus on their gelation mechanisms, fabrication strategies using bioprinter and applications. The future prospects of the bioprinting‐based use of thermoresponsive hydrogels for next generation tissue engineering have also been discussed.

     

Cryo-3D Printing of Hierarchically Porous Polyhydroxymethylene Scaffolds for Hard Tissue Regeneration

High molecular weight polyhydroxymethylene (PHM) has a repeat unit identical to that of low molecular weight sugar alcohols and exhibits carbohydrate-like properties. Herein, cryogenic extrusion-based 3D printing is combined with a phase separation in water to fabricate hierarchically porous PHM scaffolds containing interconnected macro-, micro-, and nanopores. As PHM is infusible and insoluble in common solvents, its precursor polyvinylene carbonate (PVCA) dissolved in dimethylsulfoxide (DMSO) is used to 3D print hierarchically porous PVCA scaffolds that are converted into PHM by hydrolysis without impairing the pore architectures. Similar to low-temperature deposition manufacturing, the PVCA/DMSO freezes on a build platform at −78 °C. However, instead of removing the frozen solvent by sublimation, the frozen scaffold is immersed in water to recover DMSO and to effect phase separation by precipitation. However, the computer-guided printhead pathway controls macropore formation phase separation of frozen PVCA/DMSO upon contact with water accounts for simultaneous micro- and nanopore formation. Contrary to 3D printing of PVCA/DMSO at ambient temperature, this cryo-3D printing process does not require shear thinning additives and affords significantly improved build precision with macropore sizes variable between 200 and 1500 µm. Cryo-3D-printed PHM scaffolds are biocompatible and promote osteoblast proliferation.     

Fabrication of bone tissue engineering scaffolds with a hierarchical structure using combination of 3D printing/gas foaming techniques

The goal of this research was to study and optimize the structure and geometric features of scaffolds made using a combined method of 3D printing and gas foaming. This endeavor aimed to produce scaffolds with a hierarchical structure that closely resemble bone tissue. The study examined the effects of saturation pressure, foam temperature, and foam time on the scaffolds using response surface methodology (RSM). RSM is statistical technique used for optimizing and analyzing processes by modeling relationship between input variables and output responses. The results of multi-objective optimization showed that highest pressure (55 MPa), the shortest time (40 s), and the temperature of 68°C were the optimal conditions. RSM was also used to develop mathematical models of structural properties, dimensional accuracy, and mechanical strength, focusing on different foam parameters, which could be used to predict desired properties. Subsequently, the designed scaffold underwent MTT assay testing to assess cell toxicity indicating its biocompatibility. The results demonstrate that by using the correct foam parameters in combination with 3D printing, it is possible to achieve polymer scaffolds with proportional dimensions, geometry, and mechanical strength suitable for cell growth to use inside the human body.     

A Review on Stimuli-Actuated 3D Micro/Nanostructures for Tissue Engineering and the Potential of Laser-Direct Writing via Two-Photon Polymerization for Structure Fabrication

In this review, we present the most recent and relevant research that has been done regarding the fabrication of 3D micro/nanostructures for tissue engineering applications. First, we make an overview of 3D micro/nanostructures that act as backbone constructs where the seeded cells can attach, proliferate and differentiate towards the formation of new tissue. Then, we describe the fabrication of 3D micro/nanostructures that are able to control the cellular processes leading to faster tissue regeneration, by actuation using topographical, mechanical, chemical, electric or magnetic stimuli. An in-depth analysis of the actuation of the 3D micro/nanostructures using each of the above-mentioned stimuli for controlling the behavior of the seeded cells is provided. For each type of stimulus, a particular recent application is presented and discussed, such as controlling the cell proliferation and avoiding the formation of a necrotic core (topographic stimulation), controlling the cell adhesion (nanostructuring), supporting the cell differentiation via nuclei deformation (mechanical stimulation), improving the osteogenesis (chemical and magnetic stimulation), controlled drug-delivery systems (electric stimulation) and fastening tissue formation (magnetic stimulation). The existing techniques used for the fabrication of such stimuli-actuated 3D micro/nanostructures, are briefly summarized. Special attention is dedicated to structures’ fabrication using laser-assisted technologies. The performances of stimuli-actuated 3D micro/nanostructures fabricated by laser-direct writing via two-photon polymerization are particularly emphasized.     

3D Printed Poly(𝜀-caprolactone)/Hydroxyapatite Scaffolds for Bone Tissue Engineering: A Comparative Study on a Composite Preparation by Melt Blending or Solvent Casting Techniques and the Influence of Bioceramic Content on Scaffold Properties

Bone tissue engineering has been developed in the past decades, with the engineering of bone substitutes on the vanguard of this regenerative approach. Polycaprolactone-based scaffolds are fairly applied for bone regeneration, and several composites have been incorporated so as to improve the scaffolds’ mechanical properties and tissue in-growth. In this study, hydroxyapatite is incorporated on polycaprolactone-based scaffolds at two different proportions, 80:20 and 60:40. Scaffolds are produced with two different blending methods, solvent casting and melt blending. The prepared composites are 3D printed through an extrusion-based technique and further investigated with regard to their chemical, thermal, morphological, and mechanical characteristics. In vitro cytocompatibility and osteogenic differentiation was also assessed with human dental pulp stem/stromal cells. The results show the melt-blending-derived scaffolds to present more promising mechanical properties, along with the incorporation of hydroxyapatite. The latter is also related to an increase in osteogenic activity and promotion. Overall, this study suggests polycaprolactone/hydroxyapatite scaffolds to be promising candidates for bone tissue engineering, particularly when produced by the MB method.     

Cryogenic 3D Printing of w/o Pickering Emulsions Containing Bifunctional Drugs for Producing Hierarchically Porous Bone Tissue Engineering Scaffolds with Antibacterial Capability

How to fabricate bone tissue engineering scaffolds with excellent antibacterial and bone regeneration ability has attracted increasing attention. Herein, we produced a hierarchical porous β-tricalcium phosphate (β-TCP)/poly(lactic-co-glycolic acid)-polycaprolactone composite bone tissue engineering scaffold containing tetracycline hydrochloride (TCH) through a micro-extrusion-based cryogenic 3D printing of Pickering emulsion inks, in which the hydrophobic silica (h-SiO₂) nanoparticles were used as emulsifiers to stabilize composite Pickering emulsion inks. Hierarchically porous scaffolds with desirable antibacterial properties and bone-forming ability were obtained. Grid scaffolds with a macroscopic pore size of 250.03 ± 75.88 μm and a large number of secondary micropores with a diameter of 24.70 ± 15.56 μm can be fabricated through cryogenic 3D printing, followed by freeze-drying treatment, whereas the grid structure of scaffolds printed or dried at room temperature was discontinuous, and fewer micropores could be observed on the strut surface. Moreover, the impartment of β-TCP in scaffolds changed the shape and density of the micropores but endowed the scaffold with better osteoconductivity. Scaffolds loaded with TCH had excellent antibacterial properties and could effectively promote the adhesion, expansion, proliferation, and osteogenic differentiation of rat bone marrow-derived mesenchymal stem cells afterward. The scaffolds loaded with TCH could realize the strategy to “kill bacteria first, then induce osteogenesis”. Such hierarchically porous scaffolds with abundant micropores, excellent antibacterial property, and improved bone-forming ability display great prospects in treating bone defects with infection.     

Additive Manufacturing of Poly(3-hydroxybutyrate-co-3-hydroxyvalerate)/Poly(D,L-lactide-co-glycolide) Biphasic Scaffolds for Bone Tissue Regeneration

Polyhydroxyalkanoates are biopolyesters whose biocompatibility, biodegradability, environmental sustainability, processing versatility, and mechanical properties make them unique scaffolding polymer candidates for tissue engineering. The development of innovative biomaterials suitable for advanced Additive Manufacturing (AM) offers new opportunities for the fabrication of customizable tissue engineering scaffolds. In particular, the blending of polymers represents a useful strategy to develop AM scaffolding materials tailored to bone tissue engineering. In this study, scaffolds from polymeric blends consisting of poly(3-hydroxybutyrate-co-3-hydroxyvalerate) (PHBV) and poly(D,L-lactide-co-glycolide) (PLGA) were fabricated employing a solution-extrusion AM technique, referred to as Computer-Aided Wet-Spinning (CAWS). The scaffold fibers were constituted by a biphasic system composed of a continuous PHBV matrix and a dispersed PLGA phase which established a microfibrillar morphology. The influence of the blend composition on the scaffold morphological, physicochemical, and biological properties was demonstrated by means of different characterization techniques. In particular, increasing the content of PLGA in the starting solution resulted in an increase in the pore size, the wettability, and the thermal stability of the scaffolds. Overall, in vitro biological experiments indicated the suitability of the scaffolds to support murine preosteoblast cell colonization and differentiation towards an osteoblastic phenotype, highlighting higher proliferation for scaffolds richer in PLGA.     

Fabrication of Gentamicin Sulfate-Loaded 3D-Printed Polyvinyl Alcohol/Sodium Alginate/Gelatin-Methacryloyl Hybrid Scaffolds for Skin Tissue Replacement

3D-printed scaffolds can better mimic the function of human skin, both biologically and mechanically. Within the scope of this study, the effect of the addition of different amounts (10, 15, 20 mg) of gentamicin sulfate (GS) to a 10 mL solution of natural and synthetic polymers is investigated. Sodium alginate (SA), gelatin-methacryloyl (GelMA), and polyvinyl alcohol (PVA) are chosen as bioactive materials. The surface morphology and pore structures are visualized by scanning electron microscopy (SEM). According to the results, it is observed that the pore sizes of all scaffolds are smaller than 270 µm, the lowest value (130 µm) is obtained in the scaffold loaded with 15 mg GS, and it also has the highest tensile strength value (12.5 ± 7.6 MPa). Similarly, it is observed that the tensile strength (9.7 ± 4.5 MPa) is high in scaffold loaded with 20 mg GS. The biocompatibility test is performed with fibroblast cells, and the results show that the scaffolds are biocompatible with cells. The antibacterial test is carried out against the S.aureous and E. coli and the results indicate that all GS-loaded scaffolds demonstrate antibacterial activity.     

基于快速成型技术的组织工程支架制备进展

介绍了组织工程支架的重要性和基本要求,综述了组织工程支架制备中的新技术：三雏打印技术、熔融沉积模型以及选择性激光烧结等三种快速成型技术的基本原理及应用情况,指出了各种技术的特点并对其应用前景进行了展望。     

Comparison of three-dimensional printing for fabricating silk fibroin-blended scaffolds

In this study, two types of scaffolds were fabricated by 3D printing. Morphology, physical properties, biochemical were evaluated. Cell morphology and distribution were observed. It was found that the silk fibroin/collagen (SF/C) scaffold-based material had significantly higher values than the silk fibroin/chitosan (SF/CS) scaffold-based material. Hematoxylin and eosin staining of the scaffolds revealed that the number of cells in the SF/C scaffold was higher. Cells grew well inside the SF/C scaffold as measured by scanning electron microscope. Reverse Transcriotion-Polymerase Chain Reaction (RT-PCR) and Western blot showed that type II collagen and Sox9 can be found in SF/C scaffold. Therefore, the SF/C scaffold exhibited better overall performance compared with the SF/CS scaffold.     

Engineering 3D Aligned Nanofibers for Regulation of Cell Growth Behavior

3D aligned electrospun fibers hold a promising potential in a wide range of biomedical areas, including biosensors, controlled drug release, tissue engineering, etc. Thus, a cost‐effective and easy way to scale‐up fabrication for 3D aligned nanofibers is highly desired. Herein, a novel yet facile preparation process of 3D aligned nanofibers (3D AFs) by an improved electrospinning technique is reported. The obtained 3D AFs show enhanced controllability on morphology and fiber density, and thus facilitate adhesion and growth of human mesenchymal stem cells within their 3D nanofiber microarchitectures, leading to an excellent in vitro biocompatibility. Moreover, the 3D AFs with aligned morphology can enhance the neuron activities and induce directional cell growth along the direction of nanofiber orientation, thereby providing an excellent cue for the anchorage and migration dependent neurons. Combined with controllable morphology and structure, it is anticipated that this finding can lead to great applications of electrospun fibers in nerve tissue engineering, diagnostics, and other biomedical fields.

     

Tissue Engineering Approaches to Uncover Therapeutic Targets for Endothelial Dysfunction in Pathological Microenvironments

Endothelial cell dysfunction plays a central role in many pathologies, rendering it crucial to understand the underlying mechanism for potential therapeutics. Tissue engineering offers opportunities for in vitro studies of endothelial dysfunction in pathological mimicry environments. Here, we begin by analyzing hydrogel biomaterials as a platform for understanding the roles of the extracellular matrix and hypoxia in vascular formation. We next examine how three-dimensional bioprinting has been applied to recapitulate healthy and diseased tissue constructs in a highly controllable and patient-specific manner. Similarly, studies have utilized organs-on-a-chip technology to understand endothelial dysfunction’s contribution to pathologies in tissue-specific cellular components under well-controlled physicochemical cues. Finally, we consider studies using the in vitro construction of multicellular blood vessels, termed tissue-engineered blood vessels, and the spontaneous assembly of microvascular networks in organoids to delineate pathological endothelial dysfunction.     

一辊多色多图立体胶辊印刷技术及产品开发

在高清三维胶辊印刷技术的基础上,通过对胶辊印刷装备进行重大改进和花釉配方组成优化,研究开发一辊多色多图立体胶辊印刷技术及凹凸拼图釉面砖产品。试验表明,采用本工作开发的一辊多色多图立体胶辊印刷技术可成功实现凹凸拼图釉面砖产品生产,大大减少印花装饰过程胶辊数量和明显提高了产品质量稳定性。     

On the Use of Surfactant-Complexed Chitosan for Toughening 3D Printed Polymethacrylate Composites

This work reports a simple approach to prepare toughened 3D-printed polymethacrylate (PMA) composites using surfactant-modified chitosan (SMCS) particles at loadings between 2–10 wt%. Chitosan (CS) is modified with anionic surfactant, sodium dodecyl sulfate, via ionic complexation to facilitate compatibility and dispersion of CS to PMA matrix by non-covalent interactions between the components. The study successfully demonstrates high-accuracy 3D printing of composites with significant improvements in the overall mechanical properties. The composite with the best loading of 8 wt% SMCS shows a tensile modulus of 1.23 ± 0.05 GPa, a tensile strength at 49.8 ± 0.96 MPa, a yield stress at 33.3 ± 1.48 MPa, and a strain-at-failure 10.3 ± 0.61%, which are 45%, 40%, 32%, and 68% higher than neat PMA, respectively. This provides a significant improvement in toughness at 4.92 ± 0.55 MJ m⁻³ for the composite, 184% higher than that of neat PMA. The marked increase in toughness is due to enhanced filler-matrix interactions which improve the ability of the 3D printed composite to absorb energy under tensile load. The results from this work provide new understandings into the strategies for design and preparation of stereolithography 3D printed materials reinforced with toughening fillers from renewable resources.     

Engineering a 3D In Vitro Model of Human Gingival Tissue Equivalent with Genipin/Cytochalasin D

Although three-dimensional (3D) co-culture of gingival keratinocytes and fibroblasts-populated collagen gel can mimic 3D structure of in vivo tissue, the uncontrolled contraction of collagen gel restricts its application in clinical and experimental practices. We here established a stable 3D gingival tissue equivalent (GTE) using hTERT-immortalized gingival fibroblasts (hGFBs)-populated collagen gel directly crosslinked with genipin/cytochalasin D and seeding hTERT-immortalized gingival keratinocytes (TIGKs) on the upper surface for a 2-week air–liquid interface co-culture. MTT assay was used to measure the cell viability of GTEs. GTE size was monitored following culture period, and the contraction was analyzed. Immunohistochemical assay was used to analyze GTE structure. qRT-PCR was conducted to examine the mRNA expression of keratinocyte-specific genes. Fifty µM genipin (G50) or combination (G + C) of G50 and 100 nM cytochalasin D significantly inhibited GTE contraction. Additionally, a higher cell viability appeared in GTEs crosslinked with G50 or G + C. GTEs crosslinked with genipin/cytochalasin D showed a distinct multilayered stratified epithelium that expressed keratinocyte-specific genes similar to native gingiva. Collagen directly crosslinked with G50 or G + C significantly reduced GTE contraction without damaging the epithelium. In summary, the TIGKs and hGFBs can successfully form organotypic multilayered cultures, which can be a valuable tool in the research regarding periodontal disease as well as oral mucosa disease. We conclude that genipin is a promising crosslinker with the ability to reduce collagen contraction while maintaining normal cell function in collagen-based oral tissue engineering.     

3D gel printing of magnetic hydrogel scaffolds assisted by in-situ gelation in a water level-controlled crosslinker bath

Polyvinylalcohol/chitosan (PVA/CS) is an excellent dual-network hydrogel material, but some significant challenges remain in fabricating composites with specific structures. In this study, 3D gel printing (3DGP) combined with a water-level controlled crosslinker bath was proposed for the rapid in-situ prototyping of PVA/CS/Fe₃O₄ magnetic hydrogel scaffolds. Specifically, the PVA/CS/Fe₃O₄ hydrogels were extruded into the crosslinker water to achieve rapid in-situ gelation, improving the printability of hydrogel scaffolds. The effect of the PVA/CS ratio on the rheological and mechanical properties of dual-network magnetic hydrogels was evaluated. The printing parameters were systematically optimized to facilitate the coordination between the crosslinking water bath and printer. The different crosslinking water baths were investigated to improve the printability of PVA/CS/Fe₃O₄ hydrogels. The results showed that the printability of the sodium hydroxide (NaOH) crosslinker was significantly better than that of sodium tripolyphosphate (TPP). The magnetic hydrogels (PVA: CS= 1: 1) crosslinked by NaOH had better compressive strength, swelling rate, and saturation magnetization of 1.17 MPa, 92.43%, and 22.19 emu/g, respectively. The MC3T3-E1 cell culture results showed that the PVA/CS/Fe₃O₄ scaffolds promoted cell adhesion and proliferation, and the scaffolds crosslinked by NaOH had superior cytocompatibility. 3DGP combined with a water-level controlled crosslinker bath offers a promising approach to preparing magnetic hydrogel materials.     

Evaluation of Three-Dimensional Bioprinted Human Cartilage Powder Combined with Micronized Subcutaneous Adipose Tissues for the Repair of Osteochondral Defects in Beagle Dogs

Cartilage lesions are difficult to repair due to low vascular distribution and may progress into osteoarthritis. Despite numerous attempts in the past, there is no proven method to regenerate hyaline cartilage. The purpose of this study was to investigate the ability to use a 3D printed biomatrix to repair a critical size femoral chondral defect using a canine weight-bearing model. The biomatrix was comprised of human costal-derived cartilage powder, micronized adipose tissue, and fibrin glue. Bilateral femoral condyle defects were treated on 12 mature beagles staged 12 weeks apart. Four groups, one control and three experimental, were used. Animals were euthanized at 32 weeks to collect samples. Significant differences between control and experimental groups were found in both regeneration pattern and tissue composition. In results, we observed that the experimental group with the treatment with cartilage powder and adipose tissue alleviated the inflammatory response. Moreover, it was found that the MOCART score was higher, and cartilage repair was more organized than in the other groups, suggesting that a combination of cartilage powder and adipose tissue has the potential to repair cartilage with a similarity to normal cartilage. Microscopically, there was a well-defined cartilage-like structure in which the mid junction below the surface layer was surrounded by a matrix composed of collagen type I, II, and proteoglycans. MRI examination revealed significant reduction of the inflammation level and progression of a cartilage-like growth in the experimental group. This canine study suggests a promising new surgical treatment for cartilage lesions.