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1. Fabricating spatially functionalized 3D-printed scaffolds for osteochondral tissue engineering

   https://doi.org/10.14440/jbm.2021.353  

   Camacho, P; Fainor, M; Seims, KB; Tolbert, JW; Chow, LW (January 2021, Journal of biological methods)

   null (Ed.)

   Three-dimensional (3D) printing of biodegradable polymers has rapidly become a popular approach to create scaffolds for tissue engineering. This technique enables fabrication of complex architectures and layer-by-layer spatial control of multiple components with high resolution. The resulting scaffolds can also present distinct chemical groups or bioactive cues on the surface to guide cell behavior. However, surface functionalization often includes one or more post-fabrication processing steps, which typically produce biomaterials with homogeneously distributed chemistries that fail to mimic the biochemical organization found in native tissues. As an alternative, our laboratory developed a novel method that combines solvent-cast 3D printing with peptide-polymer conjugates to spatially present multiple biochemical cues in a single scaffold without requiring post-fabrication modification. Here, we describe a detailed, stepwise protocol to fabricate peptide-functionalized scaffolds and characterize their physical architecture and biochemical spatial organization. We used these 3D-printed scaffolds to direct human mesenchymal stem cell differentiation and osteochondral tissue formation by controlling the spatial presentation of cartilage-promoting and bone-promoting peptides. This protocol also describes how to seed scaffolds and evaluate matrix deposition driven by peptide organization. 

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2. Spatial organization of biochemical cues in 3D-printed scaffolds to guide osteochondral tissue engineering

   https://doi.org/10.1039/D1BM00859E  

   Camacho, Paula; Behre, Anne; Fainor, Matthew; Seims, Kelly B.; Chow, Lesley W. (October 2021, Biomaterials Science)

   Functional repair of osteochondral (OC) tissue remains challenging because the transition from bone to cartilage presents gradients in biochemical and physical properties necessary for joint function. Osteochondral regeneration requires strategies that restore the spatial composition and organization found in the native tissue. Several biomaterial approaches have been developed to guide chondrogenic and osteogenic differentiation of human mesenchymal stem cells (hMSCs). These strategies can be combined with 3D printing, which has emerged as a useful tool to produce tunable, continuous scaffolds functionalized with bioactive cues. However, functionalization often includes one or more post-fabrication processing steps, which can lead to unwanted side effects and often produce biomaterials with homogeneously distributed chemistries. To address these challenges, surface functionalization can be achieved in a single step by solvent-cast 3D printing peptide-functionalized polymers. Peptide-poly(caprolactone) (PCL) conjugates were synthesized bearing hyaluronic acid (HA)-binding (HAbind–PCL) or mineralizing (E3–PCL) peptides, which have been shown to promote hMSC chondrogenesis or osteogenesis, respectively. This 3D printing strategy enables unprecedented control of surface peptide presentation and spatial organization within a continuous construct. Scaffolds presenting both cartilage-promoting and bone-promoting peptides had a synergistic effect that enhanced hMSC chondrogenic and osteogenic differentiation in the absence of differentiation factors compared to scaffolds without peptides or only one peptide. Furthermore, multi-peptide organization significantly influenced hMSC response. Scaffolds presenting HAbind and E3 peptides in discrete opposing zones promoted hMSC osteogenic behavior. In contrast, presenting both peptides homogeneously throughout the scaffolds drove hMSC differentiation towards a mixed population of articular and hypertrophic chondrocytes. These significant results indicated that hMSC behavior was driven by dual-peptide presentation and organization. The downstream potential of this platform is the ability to fabricate biomaterials with spatially controlled biochemical cues to guide functional tissue regeneration without the need for differentiation factors. 

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3. Gradient scaffolds for osteochondral tissue engineering and regeneration

   https://doi.org/10.1039/D0TB00688B  

   Zhang, Bin; Huang, Jie; Narayan, Roger J. (September 2020, Journal of Materials Chemistry B)

   null (Ed.)

   The tissue engineering approach for repairing osteochondral (OC) defects involves the fabrication of a biological tissue scaffold that mimics the physiological properties of natural OC tissue ( e.g. , the gradient transition between the cartilage surface and the subchondral bone). The OC tissue scaffolds described in many research studies exhibit a discrete gradient ( e.g. , a biphasic or tri/multiphasic structure) or a continuous gradient to mimic OC tissue attributes such as biochemical composition, structure, and mechanical properties. One advantage of a continuous gradient scaffold over biphasic or tri/multiphasic tissue scaffolds is that it more closely mimics natural OC tissue since there is no distinct interface between each layer. Although research studies to this point have yielded good results related to OC regeneration with tissue scaffolds, differences between engineered scaffolds and natural OC tissue remain; due to these differences, current clinical therapies to repair OC defects with engineered scaffolds have not been successful. This paper provides an overview of both discrete and continuous gradient OC tissue scaffolds in terms of cell type, scaffold material, microscale structure, mechanical properties, fabrication methods, and scaffold stimuli. Fabrication of gradient scaffolds with three-dimensional (3D) printing is given special emphasis due to its ability to accurately control scaffold pore geometry. Moreover, the application of computational modeling in OC tissue engineering is considered; for example, efforts to optimize the scaffold structure, mechanical properties, and physical stimuli generated within the scaffold–bioreactor system to predict tissue regeneration are considered. Finally, challenges associated with the repair of OC defects and recommendations for future directions in OC tissue regeneration are proposed. 

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4. 4D anisotropic skeletal muscle tissue constructs fabricated by staircase effect strategy

   https://doi.org/10.1088/1758-5090/ab1d07  

   Miao, Shida; Nowicki, Margaret; Cui, Haitao; Lee, Se-Jun; Zhou, Xuan; Mills, David K; Zhang, Lijie Grace (January 2019, Biofabrication)

   Like the morphology of native tissue fiber arrangement (such as skeletal muscle), unidirectional anisotropic scaffolds are highly desired as a means to guide cell behavior in anisotropic tissue engineering. In contrast, contour-like staircases exhibit directional topographical cues and are judged as an inevitable defect of fused deposition modeling (FDM). In this study, we will translate this staircase defect into an effective bioengineering strategy by integrating FDM with surface coating technique (FCT) to investigate the effect of topographical cues on regulating behaviors of human mesenchymal stem cells (hMSCs) toward skeletal muscle tissues. This integrated approach serves to fabricate shape-specific, multiple dimensional, anisotropic scaffolds using different biomaterials. 2D anisotropic scaffolds, first demonstrated with different polycaprolactone concentrations herein, efficiently direct hMSC alignment, especially when the scaffold is immobilized on a support ring. By surface coating the polymer solution inside FDM-printed sacrificial structures, 3D anisotropic scaffolds with thin wall features are developed and used to regulate seeded hMSCs through a self-established rotating bioreactor. Using layer-by-layer coating, along with a shape memory polymer, smart constructs exhibiting shape fix and recovery processes are prepared, bringing this study into the realm of 4D printing. Immunofluorescence staining and real-time quantitative polymerase chain reaction analysis confirm that the topographical cues created via FCT significantly enhance the expression of myogenic genes, including myoblast differentiation protein-1, desmin, and myosin heavy chain-2. We conclude that there are broad application potentials for this FCT strategy in tissue engineering as many tissues and organs, including skeletal muscle, possess highly organized and anisotropic extracellular matrix components. 

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5. Nanoscale 3D printing of hydrogels for cellular tissue engineering

   https://doi.org/10.1039/C8TB00301G  

   You, Shangting; Li, Jiawen; Zhu, Wei; Yu, Claire; Mei, Deqing; Chen, Shaochen (January 2018, Journal of Materials Chemistry B)

   Hydrogel scaffolds that mimic the native extracellular matrix (ECM) environment play a crucial role in tissue engineering. It has been demonstrated that cell behaviors can be affected by not only the hydrogel's physical and chemical properties, but also its three dimensional (3D) geometrical structures. In order to study the influence of 3D geometrical cues on cell behaviors as well as the maturation and function of engineered tissues, it is imperative to develop 3D fabrication techniques for creating micro and nanoscale hydrogel constructs. Among existing techniques that can effectively pattern hydrogels, two-photon polymerization (2PP)-based femtosecond laser 3D printing technology allows one to produce hydrogel structures with a resolution of 100 nm. This article reviews the basics of this technique and some of its applications in tissue engineering. 

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