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In this study, zinc silicate hybrid scaffolds with hierarchical meso/macroporous structures were synthesized using evaporation-induced self-assembly and sacrificial foamy templates. F127 triblock copolymer and polyurethane (PU) foam served as templates for mesoporosity and macroporosity, respectively. The scaffolds were calcined at 550, 650, and 750 °C for 2 h to remove the templates and form the crystalline phase. Analytical techniques, including BET, XRD, SEM, FTIR, and STA, were used to study the impact of calcination temperature on the scaffolds' meso-texture and crystalline phase. Results showed that increasing the calcination temperature to 750 °C significantly enhanced the overall crystallinity. The crystallized ZnO content increased from 10.3 wt% to 32 wt%, and the willemite (Zn 2 SiO 4) crystalline phase formed. Willemite content was approximately 3 wt% at 650 °C and 67 wt% at 750 °C, as determined by quantitative XRD analyses via Rietveld refinement. The emergence of the willemite phase adversely impacted the specific surface area, leading to a reduction from 123.18 m2/g to 2.18 m2/g, and compromised meso-texture-related characteristics, indicating a substantial disruption in mesoporosity. The pure willemite sample was also obtained by increasing the calcination temperature up to 1000 °C. Although the sample contained no secondary phase, the specific surface area and total mesopore volume were drastically reduced, indicating the negative effect of high calcination temperature on mesoporosity. In contrast to the mesostructure, the macrostructure of the scaffolds exhibited negligible sensitivity to calcination temperature, maintaining a mean macropore diameter of around 200 μm. Furthermore, the in vitro performance of the scaffolds was evaluated by assessing apatite formation ability, degradability, and cytocompatibility. It was demonstrated that the scaffolds calcined at 750 °C exhibited superior performance in terms of apatite formation and cytocompatibility when cultivated with MG-63 human osteosarcoma cells. [ABSTRACT FROM AUTHOR]

Osteochondral regeneration involves the highly challenging and complex reconstruction of cartilage and subchondral bone. Silicon (Si) ions play a crucial role in bone development. Current research on Si ions mainly focuses on bone repair, by using silicate bioceramics with complex ion compositions. However, it is unclear whether the Si ions have important effect on cartilage regeneration. Developing a scaffold that solely releases Si ions to simultaneously promote subchondral bone repair and stimulate cartilage regeneration is critically important. Diatomite (DE) is a natural diatomaceous sediment that can stably release Si ions, known for its abundant availability, low cost, and environmental friendliness. Herein, a hierarchical osteochondral repair scaffold is uniquely designed by incorporating gradient DE into GelMA hydrogel. The adding DE microparticles provides a specific Si source for controlled Si ions release, which not only promotes osteogenic differentiation of rBMSCs (rabbit bone marrow mesenchymal stem cells) but also enhances proliferation and maturation of chondrocytes. Moreover, DE-incorporated hierarchical scaffolds significantly promoted the regeneration of cartilage and subchondral bone. The study suggests the significant role of Si ions in promoting cartilage regeneration and solidifies their foundational role in enhancing bone repair. Furthermore, it offers an economic and eco-friendly strategy for developing high value-added osteochondral regenerative bioscaffolds from low-value ocean natural materials.

The periodontium is a complex hierarchical structure composed of alveolar bone, periodontal ligament, cementum, and gingiva. Periodontitis is an inflammatory disease that damages and destroys the periodontal tissues supporting the tooth. Periodontal therapies aim to regenerate the lost tissues, yet current treatments lack the integration of multiple structural/biochemical instructive cues to induce a coordinated regeneration, which leads to limited clinical outcomes. Hierarchical biomaterial scaffolds offer the opportunity to recreate the organization and architecture of the periodontium with distinct compartments, providing structural biomimicry that facilitates periodontal regeneration. Various scaffolds have been fabricated and tested preclinically, showing positive regenerative results. This review provides an overview of the recent research on hierarchical scaffolds for periodontal tissue engineering (TE). First, the hierarchical structure of the periodontium is described, covering the limitations of the current treatments used for periodontal regeneration and presenting alternative therapeutic strategies, including scaffolds and biochemical factors. Recent research regarding hierarchical scaffolds is highlighted and discussed, in particular, the scaffold composition, fabrication methods, and results from in vitro/in vivo studies are summarized. Finally, current challenges associated with the application of hierarchical scaffolds for periodontal TE are debated and future research directions are proposed. [ABSTRACT FROM AUTHOR]

Tendon mimetic scaffolds that recreate the tendon hierarchical structure and niche have increasing potential to fully restore tendon functionality. However, most scaffolds lack biofunctionality to boost the tenogenic differentiation of stem cells. In this study, we assessed the role of platelet-derived extracellular vesicles (EVs) in stem cells' tenogenic commitment using a 3D bioengineered in vitro tendon model. First, we relied on fibrous scaffolds coated with collagen hydrogels encapsulating human adipose-derived stem cells (hASCs) to bioengineer our composite living fibers. We found that the hASCs in our fibers showed high elongation and cytoskeleton anisotropic organization, typical of tenocytes. Moreover, acting as biological cues, platelet-derived EVs boosted the hASCs' tenogenic commitment, prevented phenotypic drift, enhanced the deposition of the tendon-like extracellular matrix, and induced lower collagen matrix contraction. In conclusion, our living fibers provided an in vitro system for tendon tissue engineering, allowing us to study not only the tendon microenvironment but also the influence of biochemical cues on stem cell behavior. More importantly, we showed that platelet-derived EVs are a promising biochemical tool for tissue engineering and regenerative medicine applications that are worthy of further exploration, as paracrine signaling might potentiate tendon repair and regeneration. [ABSTRACT FROM AUTHOR]

Porous metals have been widely investigated as bioimplants to repair cancellous bone defects. Nevertheless, how to endure extreme stress and strike a balance between load-bearing performance, elastic modulus, and permeability are important issues. Freeze-casting is a novel technique for manufacturing micro-scale open-cellular lamellar porous materials. However, the pore size is relatively small because of the inherent constraints of the ice template. On the other hand, the scaffold with gyroid pore structures has been proven to have higher permeability due to its larger and smoother channels. To combine the advantages of both porous structures, the present study aims to develop dual-scale 316L stainless steel (316L SS) porous structures by incorporating the 3D-printed gyroid templates into the freeze-casting to further improve the lightweight, specific strength, energy absorption, and permeability of the porous structures. The results showed that macro-scale gyroid channels were successfully built into the micro-scale open-cellular lamellar porous scaffolds. The porosity of dual-scale scaffolds was 70.1 %–74.4 %, which was higher than that of 53.1 %–66.5 % in the single-scale scaffolds. The elastic moduli of the single-scale scaffolds were higher than that of the dual-scale scaffolds; both were within the range of human cancellous bone, demonstrating their mechanical compatibility. Furthermore, the dual-scale scaffolds presented a prominent rise in permeability compared to the single-scale scaffolds. Notably, the permeability of the 3G series scaffolds was comparable to human cancellous bone. In summary, the hierarchical 316L SS scaffolds with controllable macro-scale gyroid channels exhibit great potential for biomedical applications. [ABSTRACT FROM AUTHOR]

The broad applications of graphene-based scaffolds demand fabrication methods that are simple and yet can create complex architectures. This is particularly true for bone tissue engineering, where interconnected, micro and macro porosity is required for cell penetration and nutrient exchange. Here, a dual-templating method is developed to produce graphene oxide (GO) scaffolds with interconnected hierarchical porosity: upon freezing and drying high internal phase emulsions exclusively stabilized by GO sheets, large pores are templated by the oil droplets, and small pores by ice crystals formed in the water phase, whose size can be controlled by freezing temperature. The resulting scaffolds are excellent substrates for mesenchymal stem cell penetration and growth. This method also enables the synthesis of complex hierarchical architectures in reduced graphene oxide (rGO) and GO composite scaffolds, through a single, versatile strategy. [Display omitted] [ABSTRACT FROM AUTHOR]

This study focuses on developing hybrid scaffolds incorporating phytotherapeutic agents via a combination of three-dimensional (3D) printing and electrospinning to enhance mechanical properties and provide antibacterial activity, in order to address the limitations of traditional antibiotics. In this regard, 3D-printed polycaprolactone (PCL) struts are first fabricated using fused deposition modeling (FDM). Then, alkaline surface treatment is applied to improve the adhesion of electrospun nanofibers. Finally, peppermint oil (PEP) or clove oil (CLV)-incorporated PCL-gelatin (GEL) electrospun nanofibers are collected on top of the 3D-printed PCL scaffolds by electrospinning. Incorporating PEP or CLV into PCL-GEL electrospun nanofibers enhances the scaffold's layer detachment and adhesion force. In addition, the DPPH free radical scavenging activity assay indicates that incorporating PEP or CLV improves the antioxidant properties of the scaffolds. Further, antibacterial activity results reveal that PEP or CLV incorporated scaffolds exhibit inhibition against Staphylococcus aureus and Escherichia coli bacteria. Moreover, anti-inflammatory assays show that scaffolds reduce the concentration of nitric oxide (NO) released from Raw 264.7 macrophage-like cells. On the other hand, the phytotherapeutic hierarchical scaffolds have no toxic effect on normal human dermal fibroblast (NHDF) cells, and PEP or CLV enhance cell attachment and proliferation. Overall, incorporating natural phytotherapeutic agents into hierarchical scaffolds shows promise for advancing wound healing applications., (© 2024 The Author(s). Macromolecular Bioscience published by Wiley‐VCH GmbH.)

The biomaterials requirements for osteochondral (OC) defects restoration simultaneously include adequate mechanical behavior, and the prevention of bacterial adherence and biofilm formation, without impairing local tissue integration. Bilayered and hierarchical scaffolds combining a cartilage-like layer interconnected to an underlying subchondral bone-like layer appeared as innovative technological solutions able to mimic the native OC tissue hierarchical architecture. This study is focused on the assessment of the combined compression-shear stresses and possible bacterial biofilm formation of hierarchical scaffolds prepared from a horseradish peroxidase-crosslinking reaction of silk fibroin (SF) combined with zinc (Zn) and strontium (Sr)-doped β-tricalcium phosphate (β-TCP) for OC tissue regeneration. Scaffolds with undoped-β-TCP incorporation were used as control. Results showed that the bilayered scaffolds presented suitable aptitude to support compression and shear loading for OC tissue, with better mechanical properties for the ZnSr-containing structures. Young and shear moduli presented values close to 0.01 MPa in the region 10–20% strain. The investigation of biomaterials surface ability to prevent biofilm formation showed reduced bacterial adhesion of Escherichia coli (E. coli, gram-negative) and Staphylococcus aureus (S. aureus, gram-positive) on both scaffolds, thus suggesting that the proposed hierarchical scaffolds have a positive effect in preventing gram-positive and gram-negative bacteria proliferation.

Summary: The regeneration of injured tendons and ligaments is challenging because the scaffolds needs proper mechanical properties and a biomimetic morphology. In particular, the morphological arrangement of scaffolds is a key point to drive the cells growth to properly regenerate the collagen extracellular matrix. Electrospinning is a promising technique to produce hierarchically structured nanofibrous scaffolds able to guide cells in the regeneration of the injured tissue. Moreover, the dynamic stretching in bioreactors of electrospun scaffolds had demonstrated to speed up cell shape modifications in vitro. The aim of the present study was to combine different imaging techniques such as high‐resolution X‐ray tomography (XCT), scanning electron microscopy (SEM), fluorescence microscopy and histology to investigate if hierarchically structured poly (L‐lactic acid) and collagen electrospun scaffolds can induce morphological modifications in human fibroblasts, while cultured in static and dynamic conditions. After 7 days of parallel cultures, the results assessed that fibroblasts had proliferated on the external nanofibrous sheath of the static scaffolds, elongating themselves circumferentially. The dynamic cultures revealed a preferential axial orientation of fibroblasts growth on the external sheath. The aligned nanofibre bundles inside the hierarchical scaffolds instead, allowed a physiological distribution of the fibroblasts along the nanofibre direction. Inside the dynamic scaffolds, cells appeared thinner compared with the static counterpart. This study had demonstrated that hierarchically structured electrospun scaffolds can induce different fibroblasts morphological modifications during static and dynamic conditions, modifying their shape in the direction of the applied loads. Lay Description: To enhance the regeneration of injured tendons and ligaments cells need to growth on dedicated structures (scaffolds) with mechanical properties and a fibrous morphology similar to the natural tissue. In particular, the morphological organisation of scaffolds is fundamental in leading cells to colonise them, regenerating the collagen extracellular matrix. Electrospinning is a promising technique to produce fibres with a similar to the human collagen fibres, suitable to design complex scaffolds able to guide cells in the reconstruction of the natural tissue. Moreover, it is well established that the cyclic stretching of these scaffolds inside dedicated systems called bioreactors, can speed up cells growth and their shape modification. The aim of the present study was to investigate how hierarchically structured electrospun scaffolds, made of resorbable material such as poly(L‐lactic acid) and collagen, could induce morphological changes in human fibroblasts, while cultured during static and dynamic conditions. These scaffolds were composed by an external electrospun membrane that grouped inside it a ring‐shaped bundle, made of axially aligned nanofibres, resembling the morphological arrangement of tendon and ligament tissue. After 7 days of parallel cultures, the scaffolds were investigated using the following imaging techniques: (i) high‐resolution X‐ray tomography (XCT); (ii) scanning electron microscopy (SEM); (iii) fluorescence microscopy and (iv) histology. The results showed that fibroblasts were able to grow on the external nanofibrous sheath of the static scaffolds, by elongating themselves along their circumference. The dynamic cultures revealed instead a preferential axial orientation of fibroblasts grown on the external sheath. The aligned nanofibre bundles inside the hierarchical scaffolds allowed an axial distribution of the fibroblasts along the nanofibres direction. This study has demonstrated that the electrospun hierarchically structured scaffolds investigated can modify the fibroblasts morphology both in static and dynamic conditions, in relation with the direction of the applied loads. [ABSTRACT FROM AUTHOR]

Abstract This paper reports the synthesis of hierarchical meso /macroporous TiO 2 scaffolds by mimicking bone structure. Evaporation-induced self-assembly (EISA) method along with foamy method was utilized to produce TiO 2 scaffolds in situ. Simultaneous thermal analysis (STA), small angle X-ray diffraction (SAXD), wide-angle X-ray diffraction (WAXD), nitrogen adsorption–desorption isotherm analysis, scanning electron microscopy (SEM) and transmission electron microscopy (TEM) techniques were applied to characterize the scaffolds. The results showed that TiO 2 scaffolds can be produced after sintering the specimens at 550 °C for 2 h. The obtained scaffolds had a pore size in the range of 150–300 μm and meso pores in the range of 6–12 nm. The specific surface area was obtained from the Brunauer–Emmett–Teller theory, the total volume and mean diameter of pores were 67.89 m3 g−1, 0.1245 cm3 g−1 and 7.3 nm, respectively. Highlights • Hierarchical meso/macroporous TiO 2 scaffolds were produced. • TiO 2 formation mechanism was investigated. • The scaffolds had a pore size of 150–300 μm and meso pores of 6–12 nm. • The specific surface area obtained from BET theory was 67.89 m3 g−1. [ABSTRACT FROM AUTHOR]

Collagen is the most abundant protein found in mammals and it exhibits a low immunogenicity, high biocompatibility and biodegradability when compared with others natural polymers. For this reason, it has been explored for the development of biologically instructive biomaterials with applications for tissue substitution and regeneration. Marine origin collagen has been pursued as an alternative to the more common bovine and porcine origins. This study focused on squid (Teuthoidea: Cephalopoda), particularly the Antarctic squid Kondakovia longimana and the Sub-Antarctic squid Illex argentinus as potential collagen sources. In this study, collagen has been isolated from the skins of the squids using acid-based and pepsin-based protocols, with the higher yield being obtained from I. argentinus in the presence of pepsin. The produced collagen has been characterized in terms of physicochemical properties, evidencing an amino acid profile similar to the one of calf collagen, but exhibiting a less preserved structure, with hydrolyzed portions and a lower melting temperature. Pepsin-soluble collagen isolated from I. argentinus was selected for further evaluation of biomedical potential, exploring its incorporation on poly-ε-caprolactone (PCL) 3D printed scaffolds for the development of hybrid scaffolds for tissue engineering, exhibiting hierarchical features. [ABSTRACT FROM AUTHOR]

Hierarchical scaffolds have been extensively used in various tissue engineering applications as they can provide unique biofunctional properties, including biophysical cues, to successfully accomplish preferred cell attachment, growth, and differentiation depending on the target tissues. In this study, we propose a new strategy for fabricating hierarchical structures consisting of microfibrous collagen/poly(ε-caprolactone) (PCL) bundles obtained using a 3D printing process supplemented with a unique bioink, plasma treatment, and collagen coating process. To obtain the hierarchical fibrous PCL structure, we used an alginate matrix hydrogel to form a microfibrous bundle of dispersed PCL components using elongational and shear stresses within a printing nozzle. By controlling various mixture ratios of alginate and PCL, processing temperature, pneumatic pressure, and nozzle moving speeds, a stable hierarchical structure consisting of PCL microfibers was obtained. To demonstrate the feasibility of hierarchical PCL-based structures coated with type I collagen, we investigated whether the construct affects the alignment and differentiation of myoblasts. The hierarchical fibrous biocomposite constructs presented a higher degree of myogenic activity than the control, which was fabricated using normal printing/collagen coating processes. Furthermore, the hierarchical PCL structures were further elaborated using 10 × simulated body fluid (SBF) resulting in a scaffold consisting of a hydroxyapatite (HA) layer. The hierarchical collagen/PCL/HA structures were evaluated with the aim of obtaining a functional scaffold that could efficiently induce osteogenesis to the human adipose stem cells (hASCs). These in vitro results suggest that the fibrous composite structure can provide enhanced osteogenic activity compared to the normally printed control (collagen/PCL/HA). [Display omitted] • We introduced hierarchical fibrous collagen/PCL biocomposite structures. • 3D-printing, alginate-leaching, and coating process were used to fabricate the hierarchical scaffold. • The hierarchical scaffold cultured with myoblasts induced significant myogenic activities. • The fibrous biomineralized collagen/PCL composites were used for bone tissue engineering. [ABSTRACT FROM AUTHOR]

In the past few years, researchers have focused on the development of three-dimensional (3D) advanced scaffolds and multifunctional hydrogel-based materials. As reported in literature, 3D polymer-based composite scaffolds for tissue engineering have been manufactured through conventional and advanced manufacturing techniques, and different injectable materials and hydrogel-based systems have been proposed and studied. The aim of the current research was to define an approach in the development of multifunctional tools spanning from 3D hierarchical scaffolds for soft tissue engineering to advanced hydrogel-based devices for in situ cell or drug release. The mechanical/rheological behaviour as well as the structural/functional features of the designed devices were discussed and analyzed. [ABSTRACT FROM AUTHOR]

A biomimetic collagen-apatite (Col-Ap) scaffold resembling the composition and structure of natural bone from the nanoscale to the macroscale has been successfully prepared for bone tissue engineering. We have developed a bottom-up approach to fabricate hierarchically biomimetic Col-Ap scaffolds with both intrafibrillar and extrafibrillar mineralization. To achieve intrafibrillar mineralization, polyacrylic acid (PAA) was used as a sequestrating analog of noncollagenous proteins (NCPs) to form a fluidic amorphous calcium phosphate (ACP) nanoprecursor through attraction of calcium and phosphate ions. Sodium tripolyphosphate was used as a templating analog to regulate orderly deposition of apatite within collagen fibrils. Both X-ray diffraction and Fourier transform infrared spectroscopy suggest that the mineral phase was apatite. Field emission scanning electron microscopy, transmission electron microscopy, and selected area electron diffraction confirmed that hierarchical collagen-Ap scaffolds were produced with both intrafibrillar and extrafibrillar mineralization. Biomimetic Col-Ap scaffolds with both intrafibrillar and extrafibrillar mineralization (IE-Col-Ap) were compared with Col-Ap scaffolds with extrafibrillar mineralization only (E-Col-Ap) as well as pure collagen scaffolds in vitro for cellular proliferation using MC3T3-E1 cells. Pure collagen scaffolds had the highest rate of proliferation, while there was no statistically significant difference between IE-Col-Ap and E-Col-Ap scaffolds. Thus, the bottom-up biomimetic fabrication approach has rendered a group of promising Col-Ap scaffolds, which bear high resemblance to natural bone in terms of composition and structure. © 2016 Wiley Periodicals, Inc. J Biomed Mater Res Part A: 104A: 1153-1161, 2016. [ABSTRACT FROM AUTHOR]

Low-temperature deposition modeling (LDM), otherwise termed freeze-form extrusion fabrication or rapid freeze prototyping, involves dispensing an aqueous-based ceramic paste or polymeric hydrogel along predefined paths in subzero ambient temperatures, followed by freeze-drying. The solidification of the material after the deposition of each layer enables large parts to be built without the need for organic binders, which can often have cytotoxic effects. Freeze-dried parts obtained from LDM typically exhibit pores with openings that range in average between 1 and 40 μm. The technique offers the ability to control their size distribution and orientation through varying a number of processing and material parameters. Herein, we describe the construction of an LDM system from readily available electromechanical components, as well as the preparation of a β-ΤCP paste formulation with the appropriate flow characteristics for fabricating hierarchical scaffolds with tailorable bimodal porosity for applications in bone tissue engineering.