Three-dimensional Tissue Scaffolds Research Articles

Challenges of Periodontal Tissue Engineering: Increasing Biomimicry through 3D Printing and Controlled Dynamic Environment.

In recent years, tissue engineering studies have proposed several approaches to regenerate periodontium based on the use of three-dimensional (3D) tissue scaffolds alone or in association with periodontal ligament stem cells (PDLSCs). The rapid evolution of bioprinting has sped up classic regenerative medicine, making the fabrication of multilayered scaffolds-which are essential in targeting the periodontal ligament (PDL)-conceivable. Physiological mechanical loading is fundamental to generate this complex anatomical structure ex vivo. Indeed, loading induces the correct orientation of the fibers forming the PDL and maintains tissue homeostasis, whereas overloading or a failure to adapt to mechanical load can be at least in part responsible for a wrong tissue regeneration using PDLSCs. This review provides a brief overview of the most recent achievements in periodontal tissue engineering, with a particular focus on the use of PDLSCs, which are the best choice for regenerating PDL as well as alveolar bone and cementum. Different scaffolds associated with various manufacturing methods and data derived from the application of different mechanical loading protocols have been analyzed, demonstrating that periodontal tissue engineering represents a proof of concept with high potential for innovative therapies in the near future.

Preparation of Diatom-Doped Bio-Nanocomposite Materials for Bone Tissue Scaffolds

Naturally sourced materials have an important place in bone tissue engineering due to their biocompatibility and biodegradability. Non-diatom, diatom-doped chitosan/hydroxyapatite (CS/HAp) and collagen/chitosan/hydroxyapatite (Col/CS/HAp) as three-dimensional tissue scaffolds were produced by freeze drying technique. It was determined by SEM analysis that CS/HAp, CS/HAp/Di, Col/CS/HAp, Col/CS/HAp/Di scaffolds have 160 μm, 130 μm, 390 μm and 340 μm pores, respectively. The diatoms in the structure have approximately 9-16 μm in length, 8-20 μm in diameter and nanopore sizes of 260-330 nm. Cell culture studies were performed using the 3T3 cell line to study the non-toxic nature of biocomposite scaffolds that support cell attachment and proliferation. The cells in the scaffolds without diatom proliferate in a reticulated manner, whereas in the scaffolds containing diatom the cells were wrapped around the scaffold like a cover. The suggested scaffolds have the potential to meet the basic requirements in biocompatibility, cytocompatibility and interconnected pore structure.

Tuneable synthetic reduced graphene oxide scaffolds elicit high levels of three-dimensional glioblastoma interconnectivity in vitro.

Three-dimensional tissue scaffolds have utilised nanomaterials to great effect over the last decade. In particular, scaffold design has evolved to consider mechanical structure, morphology, chemistry, electrical properties, and of course biocompatibility - all vital to the performance of the scaffold and how successful they are in developing cell cultures. We have developed an entirely synthetic and tuneable three-dimensional scaffold of reduced graphene oxide (rGO) that shows good biocompatibility, and favourable mechanical properties as well as reasonable electrical conductivity. Importantly, the synthesis is scaleable and suitable for producing scaffolds of any desired geometry and size, and we observe a high level of biocompatibility and cell proliferation for multiple cell lines. In particular, one of the most devastating forms of malignant brain cancer, glioblastoma (GBM), grows especially well on our rGO scaffold in vitro, and without the addition of response-specific growth factors. We have observed that our scaffold elicits spontaneous formation of a high degree of intercellular connections across the GBM culture. This phenomenon is not well documented in vitro and nothing similar has been observed in synthetic scaffolds without the use of response-specific growth factors - which risk obscuring any potential phenotypic behaviour of the cells. The use of scaffolds like ours, which are not subject to the limitations of existing two-dimensional substrate technologies, provide an excellent system for further investigation into the mechanisms behind the rapid proliferation and success of cancers like GBM. These synthetic scaffolds can advance our understanding of these malignancies in the pursuit of improved theranostics against them.

Application of dental stem cells in three-dimensional tissue regeneration.

Dental stem cells can differentiate into different types of cells. Dental pulp stem cells, stem cells from human exfoliated deciduous teeth, periodontal ligament stem cells, stem cells from apical papilla, and dental follicle progenitor cells are five different types of dental stem cells that have been identified during different stages of tooth development. The availability of dental stem cells from discarded or removed teeth makes them promising candidates for tissue engineering. In recent years, three-dimensional (3D) tissue scaffolds have been used to reconstruct and restore different anatomical defects. With rapid advances in 3D tissue engineering, dental stem cells have been used in the regeneration of 3D engineered tissue. This review presents an overview of different types of dental stem cells used in 3D tissue regeneration, which are currently the most common type of stem cells used to treat human tissue conditions.

Printing of Woodpile Scaffold Using Fresnel Lens for Tissue Engineering

Tissue engineering has played a very significant role in the medical field with an ever-growing demand for various tissue donations. One crucial factor is the fabrication of a desirable artificial three-dimensional (3D) tissue scaffold to act as the extracellular matrix (ECM), meeting the complex requirements for specific cell cultures. Existing scaffold fabrication techniques and systems used in constructing extracellular matrix are two-dimensionally limiting, expensive, and time-consuming. For instance, some simple fabrication methods cannot control fabricated structures with morphologies accurately, while others may introduce harmful organic solvents into scaffolds during the fabrication processes. To achieve an optimal scaffold for tissue engineering, we developed a novel 3D printing system capable of printing tissue scaffold structures with improved efficiency. The uniqueness of our system is the transparent diffractive optical elements (DOEs) of linear binary Fresnel lens fabricated to control the luminous intensity distribution. These DOEs of different patterns are arranged in series on a coverslip with each optical element designed to diffact and focus incident light at a particular plane within the device. Coupled with other optical components of the system, 3D woodpile scaffolds were printed in an effective and efficient one-step light exposure process to photo cross-link the polymer solution upon demand. The combination of photo cross-linking and diffractive optical technique incorporated within our system enables the patterning of polymer solutions within seconds, making large-scale fast production not only feasible, but also making printing of complex features simple. With this system, 3D two-layered woodpile structures were successfully fabricated within 3 seconds. While cell toxicity studies showed that the scaffold can be used for tissue engineering.

3D printing of Osage orange extract/Chitosan scaffolds for soft tissue engineering

In this study, three-dimensional tissue scaffolds were fabricated using a 3D printing process. Chitosan (CS) was used as a matrix polymer, and Osage orange extract (OGE) with different amounts was added as a bioactive material. The scaffolds were characterized by SEM, FTIR, tensile testing, antibacterial, and biocompatibility tests. The SEM images revealed that scaffolds had homogeneous morphology, and the size of the pores ranged from 160 µm to 270 µm. FTIR spectrum of the extracted Osage Orange confirmed the successful extraction of the Osage Orange. The OGE addition decreased the swelling capacity of the CS scaffold proportionally. Biocompatibility results proved the biostability properties of the scaffolds. According to the results, the antibacterial activity of the scaffolds against the Staphylococcus Aureus (S. aureus) and Enterococcus Faecalis (E. faecalis) should be provided after detailed optimization.

Effect of Die Clearance on Peak Punching Force During Cryogenic Micropunching of Polycaprolactone

Abstract Microscale holes were punched at cryogenic conditions in polycaprolactone (PCL) membranes to create synthetic three-dimensional (3D) tissue scaffolds through multilayer stacking of two-dimensional (2D) porous membranes. Punching forces were experimentally measured, and finite element modeling of the punching process was validated by comparing punching force results. Holes of nominal diameter of 200 μm were punched in PCL films of two different thicknesses: 40 μm and 70 μm. Die clearances used for holes in 40 μm thick films were 15.0%, 30.0%, and 45.0%. Die clearances used for holes in 70 μm films were 8.6%, 17.1%, and 25.7%. All holes were punched while the PCL film was in thermal equilibrium with a bath of boiling liquid nitrogen. Punching forces were analyzed to study the effect of die clearance and film thickness. A 3D finite element simulation of the punching process was done using deform 3d software. Cryogenic material properties of PCL used in the simulation were determined experimentally. It was concluded that finite element simulation for the cryogenic micropunching process can be used to predict peak punching forces with reasonable accuracy, which is a key factor to be considered while designing the punching dies. The finite element simulations did not predict an optimal die clearance to minimize peak punching force. However, the measured peak punching forces for 70 μm thick film seem to favor the smallest die clearance to minimize peak punching force.

A high-throughput three-dimensional cell culture platform for drug screening

Traditional two-dimensional (2D) cell cultures lack the extracellular matrix (ECM)-like structure or dynamic fluidic microenvironment for cells to maintain in vivo functionality. Three-dimensional (3D) tissue scaffolds, on the other hand, could provide the ECM-like microenvironment for cells to reformulate into tissue or organoids that are highly useful for in vitro drug screening. In this study, a high-throughput two-chamber 3D microscale tissue model platform is developed. Porous scaffolds are selectively foamed on a commercially available compact disk using laser. Perfusion of cell culture medium is achieved with centrifugal force-driven diffusion by disk rotation. Experimental studies were conducted on the fabrication process under various gas saturation and laser power conditions. Cell cultures were performed with two types of human cell lines: M059K and C3A-sub28. It is shown that the structure of microscale porous scaffolds can be controlled with laser foaming parameters and that coating with polydopamine these scaffolds are inducive for cell attachment and aggregation, forming a 3D network. With many such two-chamber models fabricated on a single CD and perfusion driven by the centrifugal force from rotation, the proposed platform provides a simple solution to the high-cost and lengthy drug development process with a high-throughput and physiologically more relevant tissue model system.

Tubular Microcapsules with Polysaccharide Membranes Based on a Co-axial Microfluidic Chip.

The tubularlike three-dimensional tissue scaffold is an important architecture in biomedical engineering, but its construction remains a big challenge for existing techniques. This work reports the polysaccharide-biomaterial-based tubular microcapsule, which was fabricated by integrating a co-axial flow microfluidic chip and a polyelectrolyte complex technique. First, we fabricate the densely packed coiled calcium alginate hydrogel microfibers as the building block by a co-axial microfluid chip. Then, the densely packed coiled microfibers were coated with a multilayer membrane through layer-by-layer adsorption of alginate and chitosan. After that, the microfibers with an alginate-chitosan-alginate membrane were expanded and transformed into a tubular microcapsule structure by liquefaction. The tubular microcapsule exhibits a selectively permeable property of different-molecular-weight FITC-dextran/bovine serum albumin compared with original calcium alginate microfibers. Moreover, the tubular microcapsule with a liquefied lumen and a thin membrane allows the sustainable release of encapsulants under the alkaline environment. Our research paves an alternative way of manufacturing artificial biological tube architectures having potential applications for transporting and delivering drugs.

Engineered Fibrillar Fibronectin Networks as Three-Dimensional Tissue Scaffolds.

Extracellular matrix (ECM) proteins, and most prominently, fibronectin (Fn), are routinely used in the form of adsorbed pre-coatings in an attempt to create a cell-supporting environment in both two- and three-dimensional cell culture systems. However, these protein coatings are typically deposited in a form which is structurally and functionally distinct from the ECM-constituting fibrillar protein networks naturally deposited by cells. Here, the cell-free and scalable synthesis of freely suspended and mechanically robust three-dimensional (3D) networks of fibrillar fibronectin (fFn) supported by tessellated polymer scaffolds is reported. Hydrodynamically induced Fn fibrillogenesis at the three-phase contact line between air, an Fn solution, and a tessellated scaffold microstructure yields extended protein networks. Importantly, engineered fFn networks promote cell invasion and proliferation, enable in vitro expansion of primary cancer cells, and induce an epithelial-to-mesenchymal transition in cancer cells. Engineered fFn networks support the formation of multicellular cancer structures cells from plural effusions of cancer patients. With further work, engineered fFn networks can have a transformative impact on fundamental cell studies, precision medicine, pharmaceutical testing, and pre-clinical diagnostics.

Liquid-phase 3D bioprinting of gelatin alginate hydrogels: influence of printing parameters on hydrogel line width and layer height

Extrusion-based 3D bioprinting is a direct deposition approach used to create three-dimensional (3D) tissue scaffolds typically comprising hydrogels. Hydrogels are hydrated polymer networks that are chemically or physically cross-linked. Often, 3D bioprinting is performed in air, despite the hydrated nature of hydrogels and the potential advantage of using a liquid phase to provide cross-linking and otherwise functionalize the hydrogel. In this work, we print gelatin alginate hydrogels directly into a cross-linking solution of calcium chloride and investigate the influence of nozzle diameter, distance between nozzle and surface, calcium chloride concentration, and extrusion rate on the dimensions of the printed hydrogel. The hydrogel layer height was generally found to increase with increasing extrusion rate and nozzle distance, according to the increased volume extruded and the available space, respectively. In addition, the hydrogel width was generally found to increase with decreasing nozzle distance and cross-linking concentration corresponding to confinement-induced spreading and low cross-linking regimes, respectively. Width/height ratios of ~ 1 were generally achieved when the nozzle diameter and distance were comparable above a certain cross-linking concentration. Using these relationships, biocompatible 3D multilayer structures were successfully printed directly into calcium chloride cross-linking solution.

Gradient platform for combinatorial screening of thermoset polymers for biomedical applications

The goal of this work was to design a device for rapid screening of crosslinked thermoset polymers. This gradient curing platform is capable of yielding a library of polyesters with systematically varying mechanical and physicochemical properties and the resultant cellular response. A library of poly(xylitolsebacate) polyesters was prepared in this device by differential curing to yield a gradient polymer. The resultant polymer exhibits a gradient in the storage modulus (1 to 5 MPa), wettability (70° < water contact angle < 110°), degree of crosslinking, degradation rate (3–25% in 7 days), drug release and biological response (ability to support stem cell proliferation and differentiation) from one end of the polymer to the other. Primary human mesenchymal stem cells were cultured to assess the cellular response in vitro. Maximal stem cell proliferation and osteogenesis was observed on the highly crosslinked polyester segments that provide high stiffness, are hydrophobic and are slow degrading as compared to the lower cured counterparts. Under in vivo conditions, this material showed differential response across the gradient without displaying significant concerns for inflammation or infection. This gradient curing device is capable of ascertaining suitable curing conditions to obtain appropriate polymers for application specific requirements. This gradient platform was further used to identify optimal processing parameters to prepare three-dimensional tissue scaffolds such as electrospun fiber mats and porous foams. Thus, this versatile combinatorial platform is well suited for rapid screening of thermoset polymers for biomedical applications.

Formulation and characterization of tissue scaffolds containing simvastatin loaded nanostructured lipid carriers for treatment of diabetic wounds

The objective of the present investigation was to develop novel three-dimensional (3D) and biodegradable tissue scaffolds (TSs), consisting of biological polymers and simvastatin (SV)-loaded nanostructured lipid carriers (NLCs) for the treatment of diabetic wounds. NLCs were prepared via high-shear homogenization and freeze-drying was used for NLC-incorporated TSs. The resulting NLC suspension contained spherical nanosized (110–158 nm) homogeneous particles (PDI<0.16) with >99% encapsulation efficiency. The NLCs were characterized via differential scanning calorimetry and Fourier transform infrared spectroscopy analysis. TSs have 3D high porous structure (74.38%) and sufficient large pore size (∼100–200 μm), low weight loss (57.7%), high absorption capacity (710.88%) and good mechanical strength. Incorporation of NLCs into TSs delayed the initial SV release and generated a more controlled release profile. Biocompatibility of the TSs was shown using MTT assay. The results suggest that TSs can be considered as suitable candidates for diabetic wound treatment.

Remote Determination of Time-Dependent Stiffness of Surface-Degrading-Polymer Scaffolds Via Synchrotron-Based Imaging.

Surface-degrading polymers have been widely used to fabricate scaffolds with the mechanical properties appropriate for tissue regeneration/repair. During their surface degradation, the material properties of polymers remain approximately unchanged, but the scaffold geometry and thus mechanical properties vary with time. This paper presents a novel method to determine the time-dependent mechanical properties, particularly stiffness, of scaffolds from the geometric changes captured by synchrotron-based imaging, with the help of finite element analysis (FEA). Three-dimensional (3D) tissue scaffolds were fabricated from surface-degrading polymers, and during their degradation, the tissue scaffolds were imaged via the synchrotron-based imaging to characterize their changing geometry. On this basis, the stiffness behavior of scaffolds was estimated from the FEA, and the results obtained were compared to the direct measurements of scaffold stiffness from the load-displacement material testing. The comparison illustrates that the Young's moduli estimated from the FEA and characterized geometry are in agreement with the ones of direct measurements. The developed method of estimating the mechanical behavior was also demonstrated effective with a nondegrading scaffold that displays the nonlinear stress-strain behavior. The in vivo monitoring of Young's modulus by morphology characterization also suggests the feasibility of characterizing experimentally the difference between in vivo and in vitro surface degradation of tissue engineering constructs.

The Role of the Bone Marrow Stromal Compartment in the Hematopoietic Response to Microbial Infections.

Continuous production of blood cells unfolds within a complex three-dimensional tissue scaffold established by highly organized stromal cell networks of mesenchymal, neural, and vascular origin inside bone marrow (BM) cavities. Collectively, stromal cells have been shown to serve two principal roles; first as primary participants of bone remodeling and metabolism and second as master regulators of different stages of blood cell development and production. Indeed, ample evidence demonstrates that stromal cells can sense and integrate systemic signals to shape hematopoietic responses and that these regulatory mechanisms are subverted in multiple pathologic conditions. Microbial infections are stressors that elicit potent inflammatory reactions and induce substantial alterations of hematopoietic output. Whether the cellular components of the BM stromal microenvironment are targeted by infections and participate in infection-induced hematopoiesis has not been investigated in sufficient detail to date. In this manuscript, we provide a succinct updated overview of the different cell populations that are currently known to form BM stroma. We discuss experimental evidence demonstrating that different stromal components are actively damaged or functionally altered by pathogens and/or ensuing inflammatory signals and review how these effects are known to contribute to the hematologic manifestations observed during infections.

Design and Validation of Equiaxial Mechanical Strain Platform, EQUicycler, for 3D Tissue Engineered Constructs.

It is crucial to replicate the micromechanical milieu of native tissues to achieve efficacious tissue engineering and regenerative therapy. In this study, we introduced an innovative loading platform, EQUicycler, that utilizes a simple, yet effective, and well-controlled mechanism to apply physiologically relevant homogenous mechanical equiaxial strain on three-dimensional cell-embedded tissue scaffolds. The design of EQUicycler ensured elimination of gripping effects through the use of biologically compatible silicone posts for direct transfer of the mechanical load to the scaffolds. Finite Element Modeling (FEM) was created to understand and to quantify how much applied global strain was transferred from the loading mechanism to the tissue constructs. In vitro studies were conducted on various cell lines associated with tissues exposed to equiaxial mechanical loading in their native environment. In vitro results demonstrated that EQUicycler was effective in maintaining and promoting the viability of different musculoskeletal cell lines and upregulating early differentiation of osteoprogenitor cells. By utilizing EQUicycler, collagen fibers of the constructs were actively remodeled. Residing cells within the collagen construct elongated and aligned with strain direction upon mechanical loading. EQUicycler can provide an efficient and cost-effective tool to conduct mechanistic studies for tissue engineered constructs designed for tissue systems under mechanical loading in vivo.

Modeling and Additive Manufacturing of Biomimetic Heterogeneous Scaffold

In this paper, a feature-based bio-CAD modeling of three-dimensional tissue scaffolds by considering spatial distribution of biologically active materials is presented for biomanufacturing and tissue engineering applications. Proposed model is based on uniform distribution of bio-active particles in different regions of scaffold, which is constrained by geometrical and biological features. The proposed method was integrated with a recently developed method of multi-material additive manufacturing of hydrogel structures, for bio-additive manufacturing of the heterogeneous scaffolds. 3D bioprinted heterogeneous scaffolds were provided as an example for physical implementation of developed algorithm to validate the model.

Adhesion and growth of human bone marrow mesenchymal stem cells on precise-geometry 3D organic-inorganic composite scaffolds for bone repair.

Engineering biomaterial scaffolds that promote attachment and growth of mesenchymal stem cells in three dimensions is a crucial parameter for successful bone tissue engineering. Towards this direction, a lot of research effort has focused recently into the development of three-dimensional porous scaffolds, aiming to elicit positive cellular behavior. However, the fabrication of three-dimensional tissue scaffolds with a precise geometry and complex micro- and nano-features, supporting cell in-growth remains a challenge. In this study we report on a positive cellular response of human bone marrow-derived (BM) mesenchymal stem cells (MSCs) onto hybrid material scaffolds consisting of methacryloxypropyl trimethoxysilane, zirconium propoxide, and 2-(dimethylamino)ethyl methacrylate (DMAEMA). First, we use Direct fs Laser Writing, a 3D scaffolding technology to fabricate the complex structures. Subsequently, we investigate the morphology, viability and proliferation of BM-MSCs onto the hybrid scaffolds and examine the cellular response from different donors. Finally, we explore the effect of the materials' chemical composition on cell proliferation, employing three different material surfaces: (i) a hybrid consisting of methacryloxypropyl trimethoxysilane, zirconium propoxide and 50mol% DMAEMA, (ii) a hybrid material comprising methacryloxypropyl trimethoxysilane and zirconium propoxide, and (iii) a purely organic polyDMAEMA. Our results show a strong adhesion of BM-MSCs onto the hybrid material containing 50% DMAEMA from the first 2h after seeding, and up to several days, and a proliferation increase after 14 and 21days, similar to the polystyrene control, independent of cell donor. These findings support the potential use of our proposed cell-material combination in bone tissue engineering.

Bioplotting Alginate/Hyaluronic Acid Hydrogel Scaffolds with Structural Integrity and Preserved Schwann Cell Viability

Bioplotting is an emerging freeform scaffold fabrication technique useful for creating artificial tissue scaffolds containing living cells. Simultaneous maintenance of scaffold structural integrity and cell viability is a challenging task. In this article, we present strategies developed to bioplot alginate-based three-dimensional tissue scaffolds containing hyaluronic acid and living Schwann cells for potential use in peripheral nerve tissue engineering. The fabrication platform, upon which the scaffold is created, was coated with the polycation polyethylenimine to immobilize the first layer of the scaffold on the platform. Each layer was then dispensed into a bath containing calcium chloride to cross-link the alginate, polyvinyl alcohol, and low concentrations of polyethylenimine to improve the structural integrity of the resulting scaffolds while retaining biocompatibility. The present study illustrated that with these strategies, porous alginate/hyaluronic acid scaffolds can be fabricated with good structural integrity and long-term cell viability. Plotting method and speed were also important factors determining the printability of single-layered scaffolds. The methods developed and the results obtained from this study form the basis for a general bioplotting method by which more sophisticated alginate/hyaluronic acid scaffolds can be fabricated with the inclusion of living cells into desired structures.

Advanced projection image generation algorithm for fabrication of a tissue scaffold using volumetric distance field

Advanced additive manufacture (AM) techniques have been developed to generate three-dimensional (3D) tissue scaffolds with complex topography and controlled internal pore architecture. Among the various AM methods, projection stereolithography (PSL) can be used to fabricate intricate 3D tissue scaffolds that can be engineered to mimic the microarchitecture of tissues. PSL system offers the advantages of enhanced fabrication speed and accuracy compared with conventional stereolithography system. To design and fabricate a complex 3D scaffold, we propose a new projection image generation algorithm that automatically and robustly generates 2D projection image data. The method uses the volumetric distance field (VDF) and triply periodic minimal surface (TPMS) pore morphology. By the creative combination of VDF and TPMS-based pore architecture, we can easily and rapidly generate projection image data for PSL system without using complicated 3D scaffold models. An effective Boolean operation based on VDF was utilized to improve the efficiency of geometrical manipulations required in the scaffold projection image generation. The design results demonstrated that the proposed algorithm can completely alleviate all the limitations and problems of the previous approaches mostly based on time-consuming and error-prone slicing process.