Design Of Tissue Engineering Scaffolds Research Articles

Multiphoton microfabrication and micropatternining (MMM)-based screening of multiplex cell niche factors for phenotype maintenance - Bovine nucleus pulposus cell as an example

Upon monolayer cultures on flat and rigid plastic dishes, many cells de-differentiate and lose their native phenotype. Technologies able to identify and reconstitute the cell niche factors that best maintain the physiological cellular phenotype in cultures are critical. We have developed a multiphoton microfabrication and micropatterning (MMM) technology, a robust 3D micro-printing platform capable to fabricate protein microstructures and micropatterns with quantitative, spatial and independent control of the mechanical, topological and extracellular matrix properties. Here, using bovine nucleus pulposus cells (bNPCs) as an example, we aim to reconstitute a spectrum of individual cell niche factors (2 mechanical, 9 topological and 4 matrices) in vitro for multiplex cell niche factor screening, and fabricate the optimal combinations of a series of shortlisted cell niche factors that best maintain the bNPC phenotype. Among all factors screened, two topological (micropillar array; fiber-bead structure) and two matrix (laminin; vitronectin) factors were shortlisted and the combinatory cell niche factors reconstituted from the shortlisted factors were found to synergistically augmented the expression of selected bNPC phenotype markers (Col II, SNAP25 and Keratin 8) and maintained their morphology and phenotype. These optimal cell niches can be micro-printed on culture dishes for physiologically relevant cultures and contribute to biomimetic scaffold design for intervertebral disc tissue engineering.

Biomimetic 3D-printed PCL scaffold containing a high concentration carbonated-nanohydroxyapatite with immobilized-collagen for bone tissue engineering: enhanced bioactivity and physicomechanical characteristics

A challenging approach of three-dimensional (3D)-biomimetic scaffold design for bone tissue engineering is to improve scaffold bioactivity and mechanical properties. We aimed to design and fabricate 3D-polycaprolactone (PCL)-based nanocomposite scaffold containing a high concentration homogeneously distributed carbonated-nanohydroxyapatite (C-nHA)-particles in combination with immobilized-collagen to mimic real bone properties. PCL-scaffolds without/with C-nHA at 30%, 45%, and 60% (wt/wt) were 3D-printed. PCL/C-nHA60%-scaffolds were surface-modified by NaOH-treatment and collagen-immobilization. Physicomechanical and biological properties were investigated experimentally and by finite-element (FE) modeling. Scaffold surface-roughness enhanced by increasing C-nHA (1.7 – 6.1-fold), but decreased by surface-modification (0.6-fold). The contact angle decreased by increasing C-nHA (0.9 – 0.7-fold), and by surface-modification (0.5-fold). The zeta potential decreased by increasing C-nHA (3.2-9.9-fold). Average elastic modulus, compressive strength, and reaction force enhanced by increasing C-nHA and by surface-modification. FE modeling revealed that von Mises stress distribution became less homogeneous by increasing C-nHA, and by surface-modification. Maximal von Mises stress for 2% compression strain in all scaffolds did not exceed yield stress for bulk-material. 3D-printed PCL/C-nHA60% with surface-modification enhanced pre-osteoblast spreading, proliferation, collagen deposition, alkaline phosphatase activity, and mineralization. In conclusion, a novel biomimetic 3D-printed PCL-scaffold containing a high concentration C-nHA with surface-modification was successfully fabricated. It exhibited superior physicomechanical and biological properties, making it a promising biomaterial for bone tissue engineering.

Artificial Intelligence Aided Design of Tissue Engineering Scaffolds Employing Virtual Tomography and 3D Convolutional Neural Networks.

Design requirements for different mechanical metamaterials, porous constructions and lattice structures, employed as tissue engineering scaffolds, lead to multi-objective optimizations, due to the complex mechanical features of the biological tissues and structures they should mimic. In some cases, the use of conventional design and simulation methods for designing such tissue engineering scaffolds cannot be applied because of geometrical complexity, manufacturing defects or large aspect ratios leading to numerical mismatches. Artificial intelligence (AI) in general, and machine learning (ML) methods in particular, are already finding applications in tissue engineering and they can prove transformative resources for supporting designers in the field of regenerative medicine. In this study, the use of 3D convolutional neural networks (3D CNNs), trained using digital tomographies obtained from the CAD models, is validated as a powerful resource for predicting the mechanical properties of innovative scaffolds. The presented AI-aided or ML-aided design strategy is believed as an innovative approach in area of tissue engineering scaffolds, and of mechanical metamaterials in general. This strategy may lead to several applications beyond the tissue engineering field, as we analyze in the discussion and future proposals sections of the research study.

Photoinduced Porcine Gelatin Cross-Linking by Homobi- and Homotrifunctional Tetrazoles.

Gelatin is a costless polypeptide material of natural origin, able to form hydrogels that are potentially useful in biomaterial scaffold design for drug delivery, cell cultures, and tissue engineering. However, gelatin hydrogels are unstable at physiological conditions, losing their features only after a few minutes at 37 °C. Accordingly, treatments to address this issue are of great interest. In the present work, we propose for the first time the use of bi- and trifunctional tetrazoles, most of them unknown to date, for photoinduced gelatin cross-linking towards the production of physiologically stable hydrogels. Indeed, after UV-B irradiation, aryl tetrazoles generate a nitrilimine intermediate that is reactive towards different functionalities, some of them constitutively present in the amino acid side chains of gelatin. The efficacy of the treatment strictly depends on the structure of the cross-linking agent used, and substantial improved stability was observed by switching from bifunctional to trifunctional cross-linkers.

Mechanical Properties of Compact Bone Defined by the Stress-Strain Curve Measured Using Uniaxial Tensile Test: A Concise Review and Practical Guide.

Mechanical properties are crucial parameters for scaffold design for bone tissue engineering; therefore, it is important to understand the definitions of the mechanical properties of bones and relevant analysis methods, such that tissue engineers can use this information to properly design the mechanical properties of scaffolds for bone tissue engineering. The main purpose of this article is to provide a review and practical guide to understand and analyze the mechanical properties of compact bone that can be defined and extracted from the stress–strain curve measured using uniaxial tensile test until failure. The typical stress–strain curve of compact bone measured using uniaxial tensile test until failure is a bilinear, monotonically increasing curve. The associated mechanical properties can be obtained by analyzing this bilinear stress–strain curve. In this article, a computer programming code for analyzing the bilinear stress–strain curve of compact bone for quantifying the associated mechanical properties is provided, such that the readers can use this computer code to perform the analysis directly. In addition to being applied to compact bone, the information provided by this article can also be applied to quantify the mechanical properties of any material having a bilinear stress–strain curve, such as a whole bone, some metals and biomaterials. The information provided by this article can be applied by tissue engineers, such that they can have a reference to properly design the mechanical properties of scaffolds for bone tissue engineering. The information can also be applied by researchers in biomechanics and orthopedics to compare the mechanical properties of bones in different physiological or pathological conditions.

4: Bone Tissue Engineering in the Growing Skull: A Short-term Study Using Dipyridamole-loaded 3D-printed Beta-tricalcium Phosphate Scaffolds to Repair Critically Sized Calvarial Defects in a Translational Animal Model

Purpose:The repair of bony defects remains a challenge to reconstructive surgeons due to limitations of current bone replacement techniques. Previous studies have shown that dipyridamole-loaded 3D-printed bioceramic (DIPY-3DPBC) scaffolds composed of 100% beta-tricalcium phosphate regenerate bone across critically sized defects in multiple in vivo models. However, a critical step to translating this bone tissue engineering construct to bedside is its successful application in a pre-clinical translational model that mimics biologic similarities to humans. Pigs are considered to be an excellent species for bone tissue engineering studies due to their similarities to humans both in bone biology and wound healing. In this short-term study, our group implanted DIPY-3DPBC scaffolds in an effort to repair critically sized bony defects in a translational, skeletally immature, growing craniofacial pig model. The purpose was to evaluate the performance of these 3D-printed constructs in terms of bone growth and scaffold absorption.Methods:Unilateral calvarial defects were created in six-week-old Göttingen minipigs (n=12). Four defects were left empty to serve as negative controls, four defects were filled with a 1000μM DIPY-3DPBC scaffold with a closed scaffold design (contains a solid barrier on the ectocortical side of the scaffold to prevent soft tissue infiltration), and four defects were filled with a 1000μM DIPY-3DPBC scaffold with an open design (no solid barrier on the ectocortical side). Animals were euthanized at 12-weeks post-operatively. Calvaria were subjected to micro-computed tomography (CT) for volumetric (3D) analysis. 3D reconstruction was performed using Amira software to quantitatively assess percent bone volume occupying the defect space (bone volume/total volume x 100%) and percent scaffold volume occupying the defect space (scaffold volume/total volume x 100%). A generalized linear mixed model (GLMM) was used to determine significance between groups. Results are presented using means with corresponding 95% confidence intervals and p-values.Results:Significantly higher mean (± 95% CI) percent bone volume was observed in calvarial defects treated with the scaffolds compared to negative controls (42.6% ± 8.6% vs 4.6% ± 12.1%, p≤0.001). Defects that were filled with closed cap scaffolds had a significantly greater mean percent bone volume compared to defects filled with the open scaffolds (52.9% ± 12.1% vs 32.3% ± 12.1%, p=0.024). No significant difference was detected between percent scaffold volume between closed and open scaffolds (4.4% ± 1.6% vs 3.7% ± 1.6%, p=0.493).Conclusions:The results from this work indicate an effective bone tissue engineering scaffold design that is able to regenerate bone across critically sized calvarial defects in a short-term skeletally immature, growing translational pig model.

4D polycarbonates via stereolithography as scaffolds for soft tissue repair

3D printing has emerged as one of the most promising tools to overcome the processing and morphological limitations of traditional tissue engineering scaffold design. However, there is a need for improved minimally invasive, void-filling materials to provide mechanical support, biocompatibility, and surface erosion characteristics to ensure consistent tissue support during the healing process. Herein, soft, elastomeric aliphatic polycarbonate-based materials were designed to undergo photopolymerization into supportive soft tissue engineering scaffolds. The 4D nature of the printed scaffolds is manifested in their shape memory properties, which allows them to fill model soft tissue voids without deforming the surrounding material. In vivo, adipocyte lobules were found to infiltrate the surface-eroding scaffold within 2 months, and neovascularization was observed over the same time. Notably, reduced collagen capsule thickness indicates that these scaffolds are highly promising for adipose tissue engineering and repair.

Fabrication of Highly Anisotropic and Interconnected Porous Scaffolds to Promote Preosteoblast Proliferation for Bone Tissue Engineering

Mimicking the complex structure of natural bone remains a challenge for bone tissue scaffolds. In this study, a novel processing strategy was developed to prepare the bone-like scaffolds that are featured by highly oriented and fully interconnected pores. This type of biomimetic scaffolds was evolved from solid phase stretching of immiscible polycaprolactone (PCL)/poly(ethylene oxide) (PEO) blends with co-continuous structure and the pore morphology was inherited from selective extraction of water soluble PEO phase. The pore anisotropy was readily tuned by varying the stretching strain without loss of interconnectivity. Significant promotion in preosteoblast proliferation, alkaline phosphatase activity and osteogenic gene expression was observed in the oriented porous scaffolds compared to the isotropic porous counterpart. The oriented architecture provided a topographical cue for aligned growth of preosteoblasts, which activated the Wnt/β-catenin signaling pathway. The proposed strategy enriches the toolbox for the scaffold design and fabrication for bone tissue engineering.

Mechanical Strain Alters the Surface Charge of Collagen Fibrils.

Collagen fibrils act like nanoscale cables in the extracellular matrix of vertebrate tissues and provide a scaffold for cells to attach to. However, beyond this mechanical function, the surface charge of collagen fibrils is also likely to play an important role. Here, we show that native, type I collagen fibrils from a mammal tendon exhibit a particular dependence of surface charge on longitudinal strain. Fibrils first become more positive with strain of up to 10% and then become more negative again with strain between 10 and 17%. The effect correlates with the stiffness of fibrils and can be explained by structural rearrangements, which expose hidden, ionizable residues. Fibrils treated with glutaraldehyde did not show any change in surface charge when strained. The electrical surface potential, which is directly related to the number ratio of exposed amine and carboxy groups on the surface, was determined by Kelvin-probe force microscopy of fibrils attached on an extensible, thin polymer film. By stretching the film, a large number of individual fibrils could be strained simultaneously without resorting to sophisticated nanomechanical devices. It is conceivable that cells react to such changes of the fibril charge and that this effect is an additional contributor, besides mechanics, to a number of physiological processes. It may also need to be considered in the design of tissue-engineering scaffolds.

The effect of highly inhomogeneous biphasic properties on mechanical behaviour of articular cartilage

Background and objectiveInvestigating the biomechanics of cartilage could help to understand the unique load-bearing property of the cartilage and optimize the scaffold design in tissue-engineering. It is important to model the cartilage as a highly inhomogeneous fibril-reinforced biphasic material to represent its complex composition and structure. The depth-dependent and strain-dependent properties of the cartilage would also play an important role in its mechanical behaviour. However, the differences in representing the cartilage as a highly inhomogeneous model or as simplified models still remain unclear. Hence, in this study, a highly inhomogeneous fibril-reinforced biphasic cartilage model considering both the depth-dependent and strain-dependent properties was constructed; the effect of highly inhomogeneous properties on the mechanical behaviour of articular cartilage was investigated. MethodsA finite element model of the cartilage was developed based on a flat-ended indentation test. Compressive forces were applied to four various inhomogeneous layered models through a porous indenter (Model 1: nine layers with strain-dependent permeability; Model 2: three layers with strain-dependent permeability; Model 3: single layer with strain-dependent permeability; Model 4: nine layers with constant permeability). ResultsModels 1 and 2 provided similar results with less than 3% difference in the peak effective stress, contact pressure, fluid pressure as well as fluid support ratio. However, Model 1 to Model 3 differed in stress and strain distribution patterns along depth over prolonged loads, which may provide an important insight into the highly inhomogeneous depth-dependent properties of cartilage. In addition, Model 1 with strain-dependent permeability demonstrated an enhanced capability on fluid pressurisation as compared with Model 4 which had constant permeability. ConclusionsA highly inhomogeneous fibril-reinforced biphasic model considering both depth-dependent and strain-dependent properties was developed in this study, in order to illustrate the effect of highly inhomogeneous properties on the mechanical behaviour of the articular cartilage. The number of layers in the models with depth-dependent properties should be selected according to the research questions and clinical demands. The model with strain-dependent permeability offers an enhanced capability on fluid pressurisation. In future studies, the proposed model could be adopted in cell-models to provide more in-depth information or in tissue-engineering to optimize the depth-dependent scaffold structure.

Macropore design of tissue engineering scaffolds regulates mesenchymal stem cell differentiation fate

Craniosynostosis is a debilitating birth defect characterized by the premature fusion of cranial bones resulting from premature loss of stem cells located in suture tissue between growing bones. Mesenchymal stromal cells in long bone and the cranial suture are known to be multipotent cell sources in the appendicular skeleton and cranium, respectively. We are developing biomaterial constructs to maintain stemness of the cranial suture cell population towards an ultimate goal of diminishing craniosynostosis patient morbidity. Recent evidence suggests that physical features of synthetic tissue engineering scaffolds modulate cell and tissue fate. In this study, macroporous tissue engineering scaffolds with well-controlled spherical pores were fabricated by a sugar porogen template method. Cell-scaffold constructs were implanted subcutaneously in mice for up to eight weeks then assayed for mineralization, vascularization, extracellular matrix composition, and gene expression. Pore size differentially regulates cell fate, where sufficiently large pores provide an osteogenic niche adequate for bone formation, while sufficiently small pores (<125 μm in diameter) maintain stemness and prevent differentiation. Cell-scaffold constructs cultured in vitro followed the same pore size-controlled differentiation fate. We therefore attribute the differential cell and tissue fate to scaffold pore geometry. Scaffold pore size regulates mesenchymal cell fate, providing a novel design motif to control tissue regenerative processes and develop mesenchymal stem cell niches in vivo and in vitro through biophysical features.

Crosstalk between Bone and Nerves within Bone.

For the past two decades, the function of intrabony nerves on bone has been a subject of intense research, while the function of bone on intrabony nerves is still hidden in the corner. In the present review, the possible crosstalk between bone and intrabony peripheral nerves will be comprehensively analyzed. Peripheral nerves participate in bone development and repair via a host of signals generated through the secretion of neurotransmitters, neuropeptides, axon guidance factors and neurotrophins, with additional contribution from nerve‐resident cells. In return, bone contributes to this microenvironmental rendezvous by housing the nerves within its internal milieu to provide mechanical support and a protective shelf. A large ensemble of chemical, mechanical, and electrical cues works in harmony with bone marrow stromal cells in the regulation of intrabony nerves. The crosstalk between bone and nerves is not limited to the physiological state, but also involved in various bone diseases including osteoporosis, osteoarthritis, heterotopic ossification, psychological stress‐related bone abnormalities, and bone related tumors. This crosstalk may be harnessed in the design of tissue engineering scaffolds for repair of bone defects or be targeted for treatment of diseases related to bone and peripheral nerves.

Bacterial cellulose nanofiber distribution on gelatin and silk fibroin scaffolds and the cell behavior

This paper presents a simple strategy to adjust bacterial cellulose (BC) nanofiber distribution on the biomimetic 3D scaffolds by in situ bacteria culture on the protein scaffolds which were prepared by selecting gelatin (Gel) with positive charge and silk fibroin (SF) with negative charge. The protein scaffolds with different structures and properties were obtained by adjusting the proportion of Gel and SF. Due to the electrical attraction and repulsion of bacteria and protein, the density and distribution of bacteria on the protein scaffolds is obviously different. Thus, after in situ culture, protein/BC scaffolds with different nanofibers density and distribution from basically adhering to the pore wall to distributing in the middle pore were obtained, which make different cell shape and distribution. It is can be easily realized by adjusting the properties of scaffold materials to guide the growth of bacteria which provides a simple idea for the design of tissue engineering scaffolds applied in medical implants, cell supports and other tissue regeneration.

Co-electrospun nano-/microfibrous composite scaffolds with structural and chemical gradients for bone tissue engineering

Recent trends in scaffold design for tissue engineering have focused on providing structural, mechanical and chemical cues for guiding cell behaviors. In this study, we presented a structural/compositional gradient nano-/microfibrous mesh by co-electrospinning, using silk fibroin-poly(ε-caprolactone) (SF-PCL) nanofibers and PCL microfibers. The pore size, porosity, and physical property of the gradient meshes were qualified. Cell proliferation of mouse osteoblast-like MC3T3-E1 cells was carried out to estimate the effect of structural and compositional gradients on biocompatibility. Furthermore, the 2-D mesh was rolled up and the compressive property of 3-D cylinder was investigated. The results suggested that the rolled-up gradient cylinder scaffold exhibited higher osteogenic differentiation compared to the pristine nanofibrous cylinder sample. By incorporating Chinese medicine ginsenoside Rg1, sustained release was achieved in composite meshes. Rg1-containing nanofibrous meshes and Rg1 gradient cylinders enhanced the cell proliferation of human umbilical vein endothelial cells (HUVECs). The developed fibrous scaffold may provide structural, compositional, and chemical gradients for bone regeneration. BriefsStructural and chemical gradient fibrous scaffold fabricated by co-electrospinning.

Biodegradable Polymers as the Pivotal Player in the Design of Tissue Engineering Scaffolds

Biodegradable polymers play a pivotal role in in situ tissue engineering. Utilizing various technologies, researchers have been able to fabricate 3D tissue engineering scaffolds using biodegradable polymers. They serve as temporary templates, providing physical and biochemical signals to the cells and determining the successful outcome of tissue remodeling. Furthermore, a biodegradable scaffold also presents the fourth dimension for tissue engineering, namely time. The properties of the biodegradable polymer change over time, presenting continuously changing features during the degradation process. These changes become more complicated when different materials are combined together to fabricate a composite or heterogeneous scaffold. This review undertakes a systematic analysis of the basic characteristics of biodegradable polymers and describe recent advances in making composite biodegradable scaffolds for in situ tissue engineering and regenerative medicine. The interaction between implanted biodegradable biomaterials and the in vivo environment are also discussed, including the properties and functional changes of the degradable scaffold, the local effect of degradation on the contiguous tissue and their evaluation using both in vitro and in vivo models.

Vertical Fibrous Morphology and Structure-Function Relationship in Natural and Biomimetic Suction-Based Adhesion Discs

Summary Recent years have seen rapid development in bio-inspired materials for adhesion. However, it remains challenging to realize robust adhesion on rough aquatic surfaces. An inspiring natural model is the remora fish, which has evolved to retain powerful adhesion to hosts using a dorsal suction disc. We find that the remora suction disc has a unique fibrous architecture of vertically oriented collagen fibers that enable anisotropic mechanical properties and enhanced adhesion performance. In the engineered prototype, vertically oriented nylon fibers are embedded into the soft silicone matrix using electrostatic flocking. The anisotropic mechanical properties are validated in both natural and biomimetic suction discs. Furthermore, the biomimetic suction disc demonstrates an enhanced adhesion function with a maximum 62.5% increase in pull-off force and a 340% increase in attachment time compared with the silicone control. This work can shed light on natural adhesion mechanisms and inspire novel designs for aquatic soft adhesives and actuators.

Ultraviolet Functionalization of Electrospun Scaffolds to Activate Fibrous Runways for Targeting Cell Adhesion.

A critical challenge in scaffold design for tissue engineering is recapitulating the complex biochemical patterns that regulate cell behavior in vivo. In this work, we report the adaptation of a standard sterilization methodology—UV irradiation—for patterning the surfaces of two complementary polymeric electrospun scaffolds with oxygen cues able to efficiently immobilize biomolecules. Independently of the different polymer chain length of poly(ethylene oxide terephthalate)/poly(butylene terephthalate) (PEOT/PBT) copolymers and PEOT/PBT ratio, it was possible to easily functionalize specific regions of the scaffolds by inducing an optimized and spatially controlled adsorption of proteins capable of boosting the adhesion and spreading of cells along the activated fibrous runways. By allowing an efficient design of cell attachment patterns without inducing any noticeable change on cell morphology nor on the integrity of the electrospun fibers, this procedure offers an affordable and resourceful approach to generate complex biochemical patterns that can decisively complement the functionality of the next generation of tissue engineering scaffolds.

Scaffolds with a High Surface Area-to-Volume Ratio and Cultured Under Fast Flow Perfusion Result in Optimal O2 Delivery to the Cells in Artificial Bone Tissues

Tissue engineering has the potential for repairing large bone defects, which impose a heavy financial burden on the public health. However, difficulties with O2 delivery to the cells residing in the interior of tissue engineering scaffolds make it challenging to grow artificial tissues of clinically-relevant sizes. This study uses image-based simulation in order to provide insight into how to better optimize the scaffold manufacturing parameters, and the culturing conditions, in order to resolve the O2 bottleneck. To do this, high resolution 3D X-ray images of two common scaffold types (salt leached foam and non-woven fiber mesh) are fed into Lattice Boltzmann Method fluid dynamics and reactive Lagrangian Scalar Tracking mass transfer solvers. The obtained findings indicate that the scaffolds should have maximal surface area-to-solid volume ratios for higher chances of the molecular collisions with the cells. Furthermore, the cell culture media should be flown through the scaffold pores as fast as practically possible (without detaching or killing the cells). Finally, we have provided a parametric sweep that maps how the molecular transport within the scaffolds is affected by variations in rates of O2 consumption by the cells. Ultimately, the results of this study are expected to benefit the computer-assisted design of tissue engineering scaffolds and culturing experiments.

Scaffold Techniques and Designs in Tissue Engineering Functions and Purposes: A Review

In this review paper, the definition of the tissue engineering (TE) was comprehensively explored towards scaffold fabrication techniques and applications. Scaffold properties and features in TE, biological aspects, scaffold material composition, scaffold structural requirements, and old and current manufacturing technologies were reported and discussed. In almost all the reviewed reports, the TE definition denotes renewal, development, and repairs of damaged tissues caused by various factors such as disease, injury, or congenital disabilities. TE is multidisciplinary that combines biology, biochemistry, clinical medicine, and materials science whose application in cellular systems such as organ transplantation serves as a delivery vehicle for cells and drug. According to the previous literature and this review, the scaffold fabrication techniques can be classified into two main categories: conventional and modern techniques. These TE fabrication techniques are applied in the scaffold building which later on are used in tissue and organ structure. The benefits and drawbacks of each of the fabrication techniques have been described in conjunction with current areas of research devoted to deal with some of the challenges. To figure out, the highlighted aspects aimed to define the advancements and challenges that should be addressed in the scaffold design for tissue engineering. Additionally, this study provides an excellent review of original numerical approaches focused on mechanical characteristics that can be helpful in the scaffold design assessment in the analysis of scaffold parameters in tissue engineering.

The influence of the stiffness of GelMA substrate on the outgrowth of PC12 cells.

Recent studies have shown the importance of cell–substrate interaction on neurone outgrowth, where the Young’s modulus of the matrix plays a crucial role on the neurite length, migration, proliferation, and morphology of neurones. In the present study, PC12 cells were selected as the representative neurone to be cultured on hydrogel substrates with different stiffness to explore the effect of substrate stiffness on the neurone outgrowth. By adjusting the concentration of gelatin methacryloyl (GelMA), the hydrogel substrates with the variation of stiffnesses (indicated by Young’s modulus) from approximately 3–180 KPa were prepared. It is found that the stiffness of GelMA substrates influences neuronal outgrowth, including cell viability, adhesion, spreading, and average neurite length. Our results show a critical range of substrate’s Young’s modulus that support PC12 outgrowth, and modulate the cell characteristics and morphology. The present study provides an insight into the relationship between the stiffness of GelMA hydrogel substrates and PC12 cell outgrowth, and helps the design and optimization of tissue engineering scaffolds for nerve regeneration.