Tissue Engineering Research Articles

Biomimetic grafts from ultrafine fibers for collagenous tissues.

The ligament is the soft tissue that connects bone to bone and, in case of severe injury or rupture, it cannot heal itself mainly because of its poor vascularity and dynamic nature. Tissue engineering carries the potential to restore the injured tissue functions by utilization of scaffolds mimicking the structure of native ligament. Collagen fibrils in the anterior cruciate ligament (ACL) have a diameter ranging from 20 to 300nm, which defines the physical and mechanical properties of the tissue. Also, the ACL tissue exhibited a bimodal distribution of collagen fibrils. Currently, the ability to fabricate scaffolds replicating this structure is a significant challenge. This work aims at i) measuring the diameter of collagens of bovine ACL tissue, ii) investigating the fabrication of sub-100nm fibers, and iii) fabricating aligned scaffolds with bimodal diameter distribution (with two peaks) resembling the healthy ACL structure. It is hypothesized that such scaffolds can be produced by electrospinning polycaprolactone (PCL) solutions. To test the hypothesis, various PCL solutions were formulated in acetone and formic acid in combination with pyridine, and electrospun to generate sub-100nm fibers. Next, this formulation was adjusted to produce nanofibers with a diameter between 100nm and 200nm. Finally, these solutions were combined in the co-electrospinning process, i.e., two-spinneret electrospinning, to fabricate biomimetic scaffolds with a bimodal distribution. Electrospinning of 8% and 15% PCL solutions, respectively, resulted in the production of fibers with diameters below and above 100nm. The combined scaffold exhibited a bimodal distribution of aligned fibers with peaks around 80 and 180nm, thus mimicking the collagen fibrils of healthy ACL tissue. This research is expected to have a society-wide impact because it aims to enhance the health condition and life quality of a wide range of patients.

ATPSpin: A Single Microfluidic Platform that Produces Diversified ATPS-Alginate Microfibers.

Microfluidic spinning is emerging as a useful technique in the fabrication of alginate fibers, enabling applications in drug screening, disease modeling, and disease diagnostics. In this paper, by capitalizing on the benefits of aqueous two-phase systems (ATPS) to produce diverse alginate fiber forms, we introduce an ATPS-Spinning platform (ATPSpin). This ATPS-enabled method efficiently circumvents the rapid clogging challenges inherent to traditional fiber production techniques by regulating the interaction between alginate and cross-linking agents like Ba2+ ions. By varying system parameters under the guidance of a regime map, our system produces several fiber forms─solid, hollow, and droplet-filled─consistently and reproducibly from a single device. We demonstrate that the resulting alginate fibers possess distinct features, including biocompatibility. We also encapsulate HEK293 cells in the microfibers as a proof-of-concept that this versatile microfluidic fiber generation platform may have utility in tissue engineering and regenerative medicine applications.

Understanding the Role of Loss Modulus of Viscoelastic Substrates in the Evaporation Dynamics of Sessile Drops.

Viscoelastic properties of soft substrates play a crucial role in the evaporation dynamics of sessile drops. Recent studies have revealed that the modification of the viscoelastic properties of substrates changes the dynamics of the three-phase contact line, consequently affecting the evaporation behavior of sessile drops. Notably, these modifications occur without any noticeable changes to the substrate's wetting characteristics or surface topography. However, the individual role of storage (G') and loss (G″) moduli of substrates on drop evaporation dynamics remains unexplored. In this study, we investigate the evaporation dynamics of water drops on two groups of poly(dimethylsiloxane)-based viscoelastic substrates possessing either identical G' with varying G″ or identical G″ with varying G'. Our study reveals that on a substrate with constant shear modulus (G'), a reduction of an order of magnitude in loss modulus shifts the evaporation process from the constant contact radius mode to the constant contact angle mode. We hypothesize that this observed shift in behavior stems from the varying viscoelastic dissipation influenced by the plateau modulus and characteristic relaxation time of polymer gels. Our hypothesis is further supported from the observation that the evaporation process persists on the substrate with constant loss modulus (G″). Our study advances the current understanding of drop evaporation on soft substrates that may find potential applications involving soft composites, biological entities, tissue engineering, and wearable electronics.

Ultrasound-Responsive Aligned Piezoelectric Nanofibers Derived Hydrogel Conduits for Peripheral Nerve Regeneration.

Nerve guidance conduits (NGCs) have been considered as promising treatment strategy and frontier trend for peripheral nerve regeneration; while their therapeutic outcomes are limited by the lack of controllable drug delivery and available physicochemical cues. Herein, we propose novel aligned piezoelectric nanofibers derived hydrogel NGCs with ultrasound (US)-triggered electrical stimulation (ES) and controllable drug release for repairing peripheral nerve injury. The inner layer of the NGCs was the barium titanate piezoelectric nanoparticles (BTNPs)-doped polyvinylidene fluoride-trifluoroethylene [BTNPs/P(VDF-TrFE)] electrospinning nanofibers with improved piezoelectricity and aligned orientation. The outer side of the NGCs was the thermoresponsive poly(N-isopropylacrylamide) (pNIPAM) hybrid hydrogel with bioactive drug encapsulation. Such NGCs could not only induce neuronal oriented extension and promote neurite outgrowth with US-triggered wireless ES, but also realize the controllable nerve growth factor (NGF) release with the hydrogel shrinkage under US-triggered heating. Thus, the NGC could positively accelerate the functional recovery and nerve axonal regeneration of rat models with long sciatic nerve defects. We believe that the proposed US-responsive aligned piezoelectric nanofibers derived hydrogel NGCs will find important applications in clinic neural tissue engineering. This article is protected by copyright. All rights reserved.

A Biomimetic Fibrous Composite Scaffold with Nanotopography-Regulated Mineralization for Bone Defect Repair.

The effective regeneration of large bone defects via bone tissue engineering is challenging due to the difficulty in creating an osteogenic microenvironment. Inspired by the fibrillar architecture of the natural extracellular matrix, we developed a nanoscale bioengineering strategy to produce bone fibril-like composite scaffolds with enhanced osteogenic capability. To activate the surface for biofunctionalization, self-adaptive ridge-like nanolamellae were constructed on poly(ε-caprolactone) (PCL) electrospinning scaffolds via surface-directed epitaxial crystallization. This unique nanotopography with a markedly increased specific surface area offered abundant nucleation sites for Ca2+ recruitment, leading to a 5-fold greater deposition weight of hydroxyapatite than that of the pristine PCL scaffold under stimulated physiological conditions. Bone marrow mesenchymal stem cells (BMSCs) cultured on bone fibril-like scaffolds exhibited enhanced adhesion, proliferation, and osteogenic differentiation in vitro. In a rat calvarial defect model, the bone fibril-like scaffold significantly accelerated bone regeneration, as evidenced by micro-CT, histological histological and immunofluorescence staining. This work provides the way for recapitulating the osteogenic microenvironment in tissue-engineered scaffolds for bone repair.

Evaluating osteogenic potential of a 3D-printed bioceramic-based scaffold for critical-sized defect treatment: an in vivo and in vitro investigation.

The integration of precision medicine principles into bone tissue engineering has ignited a wave of research focused on customizing intricate scaffolds through advanced 3D printing techniques. Bioceramics, known for their exceptional biocompatibility and osteoconductivity, have emerged as a promising material in this field. This article aims to evaluate the regenerative capabilities of a composite scaffold composed of 3D-printed gelatin combined with hydroxyapatite/tricalcium phosphate bioceramics (G/HA/TCP), incorporating human dental pulp-derived stem cells (hDPSCs). Using 3D powder printing, we created cross-shaped biphasic calcium phosphate scaffolds with a gelatin layer. The bone-regenerating potential of these scaffolds, along with hDPSCs, was assessed through in vitro analyses and in vivo studies with 60 rats and critical-sized calvarial defects. The assessment included analyzing cellular proliferation, differentiation, and alkaline phosphatase activity (ALP), and concluded with a detailed histological evaluation of bone regeneration. Our study revealed a highly favorable scenario, displaying not only desirable cellular attachment and proliferation on the scaffolds but also a notable enhancement in the ALP activity of hDPSCs, underscoring their pivotal role in bone regeneration. However, the histological examination of calvarial defects at the 12-wk mark yielded a rather modest level of bone regeneration across all experimental groups. The test and cell group exhibited significant bone formation compared to all other groups except the control and cell group. This underscores the complexity of the regenerative process and paves the way for further in-depth investigations aimed at improving the potential of the composite scaffolds.

Unlocking the Future of Periodontal Regeneration: An Interdisciplinary Approach to Tissue Engineering and Advanced Therapeutics

Unlocking the Future of Periodontal Regeneration: An Interdisciplinary Approach to Tissue Engineering and Advanced Therapeutics

Egg White Photocrosslinkable Hydrogels as Versatile Bioinks for Advanced Tissue Engineering Applications

AbstractThree dimensional (3D) bioprinting using photocrosslinkable hydrogels has gained considerable attention due to its versatility in various applications, including tissue engineering and drug delivery. Egg White (EW) is an organic biomaterial with excellent potential in tissue engineering. It provides abundant proteins, along with biocompatibility, bioactivity, adjustable mechanical properties, and intrinsic antiviral and antibacterial features. Here, a photocrosslinkable hydrogel derived from EW is developed through methacryloyl modification, resulting in Egg White methacryloyl (EWMA). Upon exposure to UV light, synthesized EWMA becomes crosslinked, creating hydrogels with remarkable bioactivity. These hydrogels offer adjustable mechanical and physical properties compatible with most current bioprinters. The EWMA hydrogels closely resemble the native extracellular matrix (ECM) due to cell‐binding and matrix metalloproteinase‐responsive motifs inherent in EW. In addition, EWMA promotes cell growth and proliferation in 3D cultures. It facilitates endothelialization when investigated with human umbilical vein endothelial cells (HUVECs), making it an attractive replacement for engineering hemocompatible vascular grafts and biomedical implants. In summary, the EWMA matrix enables the biofabrication of various living constructs. This breakthrough enhances the development of physiologically relevant 3D in vitro models and opens many opportunities in regenerative medicine.

Biofabrication of 3D adipose tissue via assembly of composite stem cell spheroids containing adipo-inductive dual-signal delivery nanofibers.

Reconstruction of large 3D tissues based on assembly of micro-sized multi-cellular spheroids has gained attention in tissue engineering. However, formation of 3D adipose tissue from spheroids has been challenging due to the limited adhesion capability and restricted cell mobility of adipocytes in culture media. In this study, we addressed this problem by developing adipo-inductive nanofibers enabling dual delivery of indomethacin and insulin. These nanofibers were introduced into composite spheroids comprising human adipose-derived stem cells (hADSCs). This approach led to a significant enhancement in the formation of uniform lipid droplets, as evidenced by the significantly increased Oil red O-stained area in spheroids incorporating indomethacin and insulin dual delivery nanofibers (56.9 ± 4.6%) compared to the control (15.6 ± 3.5%) with significantly greater gene expression associated with adipogenesis (C/EBPA, PPARG, FABP4, and adiponectin) of hADSCs. Furthermore, we investigated the influence of culture media on the migration and merging of spheroids and observed significant decrease in migration and merging of spheroids in adipogenic differentiation media. Conversely, the presence of adipo-inductive nanofibers promoted spheroid fusion, allowing the formation of macroscopic 3D adipose tissue in the absence of adipogenic supplements while facilitating homogeneous adipogenesis of hADSCs. The approach described here holds promise for the generation of 3D adipose tissue constructs by scaffold-free assembly of stem cell spheroids with potential applications in clinical and organ models.

Synthesis, modifications, and applications of iron-based nanoparticles.

Magnetic nanoparticles (MNPs) are appealing materials as assistant to resolve environmental pollution issues and as recyclable catalysts for the oxidative degradation of resistant contaminants. Moreover, they can significantly influence the advancement of medical applications for imaging, diagnostics, medication administration, and biosensing. On the other hand, due to unique features, excellent biocompatibility, high curie temperatures and low cytotoxicity of the Iron-based nanoparticles, they have received increasing attention in recent years. Using an external magnetic field, in which the ferrite magnetic nanoparticles (FMNPs) in the reaction mixtures can be easily removed, make them more efficient approach than the conventional method for separating the catalyst particles by centrifugation or filtration. Ferrite magnetic nanoparticles (FMNPs) provide various advantages in food processing, environmental issues, pharmaceutical industry, sample preparation, wastewater management, water purification, illness therapy, identification of disease, tissue engineering, and biosensor creation for healthcare monitoring. Modification of FMNPs with the proper functional groups and surface modification techniques play a significant role in boosting their capability. Due to flexibility of FMNPs in functionalization and synthesis, it is possible to make customized FMNPs that can be utilized in variety of applications. This review focuses on synthesis, modifications, and applications of Iron-based nanoparticles.

Fibrillogenesis in collagen hydrogels accelerated by carboxylated microbeads

Collagen type I is a material widely used for 3D cell culture and tissue engineering. Different architectures, such as gels, sponges, membranes, and nanofibers, can be fabricated with it. In collagen hydrogels, the formation of fibrils and fibers depends on various parameters, such as the source of collagen, pH, temperature, concentration, age, etc. In this work, we study the fibrillogenesis process in collagen type I hydrogels with different types of microbeads embedded, using optical techniques such as turbidity assay and confocal reflectance microscopy. We observe that microbeads embedded in the collagen matrix hydrogels modify the fibrillogenesis. Our results show that carboxylated fluorescent microbeads accelerate 3.6 times the gelation, while silica microbeads slow down the formation of collagen fibrils by a factor of 1.9, both compared to pure collagen hydrogels. Our observations suggest that carboxylate microbeads act as nucleation sites and the early collagen fibrils bind to the microbeads.

Publisher Correction: An Injectable In Situ Forming Collagen/Alginate/CaSO4 Composite Hydrogel for Tissue Engineering Applications: Optimization, Characterization and In Vitro Assessments

Publisher Correction: An Injectable In Situ Forming Collagen/Alginate/CaSO4 Composite Hydrogel for Tissue Engineering Applications: Optimization, Characterization and In Vitro Assessments

Stiffness-tunable biomaterials provide a good extracellular matrix environment for axon growth and regeneration

Abstract Neuronal growth, extension, branching, and formation of neural networks are markedly influenced by the extracellular matrix–a complex network composed of proteins and carbohydrates secreted by cells. In addition to providing physical support for cells, the extracellular matrix also conveys critical mechanical stiffness cues. During the development of the nervous system, extracellular matrix stiffness plays a central role in guiding neuronal growth, particularly in the context of axonal extension, which is crucial for the formation of neural networks. In neural tissue engineering, manipulation of biomaterial stiffness is a promising strategy to provide a permissive environment for the repair and regeneration of injured nervous tissue. Recent research has fine-tuned synthetic biomaterials to fabricate scaffolds that closely replicate the stiffness profiles observed in the nervous system. In this review, we highlight the molecular mechanisms by which extracellular matrix stiffness regulates axonal growth and regeneration. We highlight the progress made in the development of stiffness-tunable biomaterials to emulate in vivo extracellular matrix environments, with an emphasis on their application in neural repair and regeneration, along with a discussion of the current limitations and future prospects. The exploration and optimization of the stiffness-tunable biomaterials has the potential to markedly advance the development of neural tissue engineering.

3D-Printed Polycaprolactone-Based Containing Calcium Zirconium Silicate: Bioactive Scaffold for Accelerating Bone Regeneration

There is an essential clinical need to develop rapid process scaffolds to repair bone defects. The current research presented the development of calcium zirconium silicate/polycaprolactone for bone tissue engineering utilising melt extrusion-based 3D printing. Calcium zirconium silicate (CZS) nanoparticles were added to polycaprolactone (PCL) porous scaffolds to enhance their biological and mechanical properties, while the resulting properties were studied extensively. No significant difference was found in the melting point of the samples, while the crystallisation temperature points of the samples containing bioceramic increased from 36.1 to 40.2 °C. Thermal degradation commenced around 350 °C for all materials. According to our results, increasing the CZS content from 0 to 40 wt.% (PC40) in porous scaffolds (porosity about 55–62%) improved the compressive strength from 2.8 to 10.9 MPa. Furthermore, apatite formation ability in SBF solution increased significantly by enhancing the CZS percentage. According to MTT test results, the viability of MG63 cells improved remarkably (~29%) in PC40 compared to pure PCL. These findings suggest that a 3D-printed PCL/CZS composite scaffold can be fabricated successfully and shows great potential as an implantable material for bone tissue engineering applications.

Recent advancements in cartilage tissue engineering innovation and translation.

Articular cartilage was expected to be one of the first successfully engineered tissues, but today, cartilage repair products are few and they exhibit considerable limitations. For example, of the cell-based products that are available globally, only one is marketed for non-knee indications, none are indicated for severe osteoarthritis or rheumatoid arthritis, and only one is approved for marketing in the USA. However, advances in cartilage tissue engineering might now finally lead to the development of new cartilage repair products. To understand the potential in this field, it helps to consider the current landscape of tissue-engineered products for articular cartilage repair and particularly cell-based therapies. Advances relating to cell sources, bioactive stimuli and scaffold or scaffold-free approaches should now contribute to progress in therapeutic development. Engineering for an inflammatory environment is required because of the need for implants to withstand immune challenge within joints affected by osteoarthritis or rheumatoid arthritis. Bringing additional cartilage repair products to the market willrequire an understanding of the translational vector for their commercialization. Advances thus far can facilitate the future translation of engineered cartilage products to benefit the millions of patients who suffer from cartilage injuries and arthritides.

Effect of 3D-printed polycaprolactone/osteolectin scaffolds on the odontogenic differentiation of human dental pulp cells.

Cell-based tissue engineering often requires the use of scaffolds to provide a 3-dimensional (3D) framework for cell proliferation and tissue formation. Polycaprolactone (PCL), a type of polymer, has good printability, favorable surface modifiability, adaptability, and biodegradability. However, its large-scale applicability is hindered by its hydrophobic nature, which affects biological properties. Composite materials can be created by adding bioactive materials to the polymer to improve the properties of PCL scaffolds (PSs). Osteolectin is an odontogenic factor that promotes the maintenance of the adult skeleton by promoting the differentiation of LepR+ cells into osteoblasts. Therefore, the aim of this study was to evaluate whether 3D-printed PCL/osteolectin scaffolds supply a suitable microenvironment for the odontogenic differentiation of human dental pulp cells (hDPCs). The hDPCs were cultured on 3D-printed PSs with or without pores. Cell attachment and cell proliferation were evaluated using EZ-Cytox. The odontogenic differentiation of hDPCs was evaluated by alizarin red S staining and alkaline phosphatase assays. Western blotting was used to evaluate the expression of the proteins DSPP and DMP-Results: The attachment of hDPCs to PSs with pores was significantly higher than to PSs without pores. The odontogenic differentiation of hDPCs was induced more in PCL/osteolectin scaffolds than in PSs, but there was no statistically significant difference. 3D-printed PSs with pores are suitable for the growth of hDPCs, and the PCL/osteolectin scaffolds can provide a more favorable microenvironment for the odontogenic differentiation of hDPCs.&#xD.

Development of Improved Confined Compression Testing Setups for Use in Stress Relaxation Testing of Viscoelastic Biomaterials

The development of cell-based biomaterial alternatives holds significant promise in tissue engineering applications, but it requires accurate mechanical assessment. Herein, we present the development of a novel 3D-printed confined compression apparatus, fabricated using clear resin, designed to cater to the unique demands of biomaterial developers. Our objective was to enhance the precision of force measurements and improve sample visibility during compression testing. We compared the performance of our innovative 3D-printed confined compression setup to a conventional setup by performing stress relaxation testing on hydrogels with variable degrees of crosslinking. We assessed equilibrium force, aggregate modulus, and peak force. This study demonstrates that our revised setup can capture a larger range of force values while simultaneously improving accuracy. We were able to detect significant differences in force and aggregate modulus measurements of hydrogels with variable degrees of crosslinking using our revised setup, whereas these were indistinguishable with the convectional apparatus. Further, by incorporating a clear resin in the fabrication of the compression chamber, we improved sample visibility, thus enabling real-time monitoring and informed assessment of biomaterial behavior under compressive testing.

3D-printed scaffolds for tissue engineering applications using thermosensitive hydrogels based on biopolymer blends

AbstractThree-dimensional (3D) printing is an emerging technology for the construction of complex 3D constructs used for tissue engineering applications. In this study, we are proposing the preparation of 3D printing hydrogel inks consisting of the synthetic polymers poly(caprolactone) and poly(lactic acid), the biopolymer chitosan, and naturally derived gelatin. In addition, pluronic F-127 was used to improve the miscibility between the hydrophobic and hydrophilic components due to its amphiphilic character, as well as for its good 3D printability. The printability of the hydrogel inks was optimized by varying the composition, the extrusion nozzle, and the temperature, while the integrity of the 3D scaffolds was secured via sol–gel transition. The produced hydrogels with PCL-pluronic-chitosan-gelatin/15-20-4-2 wt% (PC3.75-Pl5-CG) and PLA-pluronic-chitosan-gelatin/10-20-4-2 wt% (PL2.5-Pl5-CG) presented the best printability, producing smooth and uniform porous scaffolds. The prepared hydrogels were formed via the interactions between the polymers through hydrogen bonding. Additionally, the produced hydrogels exhibited temperature-dependent swelling behavior, and the scaffolds with PCL presented lower swelling capacity than the scaffolds with PLA. The produced scaffolds presented slower hydrolyzation rate in simulated body fluid (SBF) at 25 °C compared to 37 °C. Biological studies proved that the 3D-printed porous scaffolds were non-cytotoxic and promoted human adipose-derived mesenchymal stem cell adhesion. Graphical abstract

Enhancing Osteogenic Potential in Bone Tissue Engineering: Optimizing Pore Size in Alginate‐Gelatin Composite Hydrogels

Bone tissue engineering is a complex process where the scaffold plays a significant role as it contributes to phenotypically stable tissue formation and stem cell differentiation. These properties can be reached with a composite scaffold of alginate‐gelatin three‐dimensional network structure. The primary purpose of this study was to design a three‐dimensional and porous alginate‐gelatin hydrogel and evaluate the effects of pore size on cell morphology, cell adhesion, and proliferation under different osteogenic microenvironments. For this purpose, hydrogels with various concentrations of alginate and gelatin were prepared by mixing 4% (w/v) alginate and 6% gelatin (4A6G), or 3% (w/v) alginate and 5% (w/v) gelatin (3A5G) and cross‐linked with 2% (w/v) CaCl2. The pore size of these hydrogels was optimized using a very simple method based on different freezing and thawing cycles. The scanning electron microscopy (SEM) analysis showed porous structures with different pore sizes, ranging from 340 µm ± 30 µm with the 4A6G hydrogel to 635 µm ± 25 µm with the 3A5G hydrogel. The stiffness measurement showed a significant difference, with approximately 26.3 kPa ± 0.6 KPa for the 4A6G hydrogel and 21.6 kPa ± 0.2 KPa for the 3A5G hydrogel. Cell interaction with both types of hydrogel showed higher cell adhesion and proliferation rates in the hydrogel with larger pore size. Bone tissue formation, as evaluated by RT‐PCR, ALP (alkaline phosphatase) activity, and ARS (Alizarin Red S) staining, demonstrated strong osteogenic potential of the 3A5G hydrogel compared to the 4A6G. In summary, the 3A5G hydrogel (3% (w/v) alginate and 5% (w/v) gelatin) can be considered a potential material for bone tissue regeneration construction due to its biodegradability and favorable bone‐forming properties.This article is protected by copyright. All rights reserved.

Engineering Cell Instructive Microenvironments for In Vitro Replication of Functional Barrier Organs.

Multicellular organisms exhibit synergistic effects among their components, giving rise to emergent properties crucial for their genesis and overall functionality and survival. Morphogenesis involves and relies upon intricate and biunivocal interactions among cells and their environment, that is, the extracellular matrix (ECM). Cells secrete their own ECM, which in turn, regulates their morphogenetic program by controlling time and space presentation of matricellular signals. The ECM, once considered passive, is now recognized as an informative space where both biochemical and biophysical signals are tightly orchestrated. Replicating this sophisticated and highly interconnected informative media in a synthetic scaffold for tissue engineering is unattainable with current technology and this limits the capability to engineer functional human organs in vitro and in vivo. This review explores current limitations to in vitro organ morphogenesis, emphasizing the interplay of gene regulatory networks, mechanical factors, and tissue microenvironment cues. In vitro efforts to replicate biological processes for barrier organs such as the lung and intestine, are examined. The importance of maintaining cells within their native microenvironmental context is highlighted to accurately replicate organ-specific properties. The review underscores the necessity for microphysiological systems that faithfully reproduce cell-native interactions, for advancing the understanding of developmental disorders and disease progression.