Native Tissue Structure Research Articles

Precision Cut Lung Slices as a Preclinical Model for Non-Small Cell Lung Cancer Chemoprevention.

PCLS could be a new model for premalignancy and chemoprevention research, and this work evaluates the model with tissue from prevention-relevant genetic and carcinogen exposed in vivo mouse models, in addition to evaluating chemoprevention agents.

Gelatin-based cell culture device for construction and X-ray irradiation of a three-dimensional oral cancer model.

Bioassays using three-dimensional (3D) tissue models offer several advantages over 2D culture assays because they can reproduce the structure and function of native tissues. In this study, we used our newly designed gelatin device to generate a miniature 3D model of human oral squamous cell carcinoma with stroma and blood vessels. To enable air-liquid interface culture, we conceived a new device structure in which three wells were lined up and separated by a dividing thread; the wells could be connected by removing the dividing thread. Cells were seeded in the center well with the dividing thread to form a multilayer, followed by the supply of media from the side wells after thread removal. Human oral squamous cell carcinoma (HSC-4) cells, human umbilical vein endothelial cells (HUVECs), and normal human dermal fibroblasts (NHDFs) were successfully cocultured, resulting in structures that mimicked 3D-cancer tissues. This 3D-cancer model was subjected to an X-ray sensitivity assay, followed by the evaluation of DNA damage using confocal microscopy and section-scanning electron microscopy.

Cellular Contact Guidance on Liquid Crystalline Networks with Anisotropic Roughness.

Cell contact guidance is widely employed to manipulate cell alignment and differentiation in vitro. The use of nano- or micro-patterned substrates allows efficient control of cell organization, thus opening up to biological models that cannot be reproduced spontaneously on standard culture dishes. In this paper, we explore the concept of cell contact guidance by Liquid Crystalline Networks (LCNs) presenting different surface topographies obtained by self-assembly of the monomeric mixture. The materials are prepared by photopolymerization of a low amount of diacrylate monomer dissolved in a liquid crystalline solvent, not participating in the reaction. The alignment of the liquid crystals, obtained before polymerization, determines the scaffold morphology, characterized by a nanometric structure. Such materials are able to drive the organization of different cell lines, e.g., fibroblasts and myoblasts, allowing for the alignment of single cells or high-density cell cultures. These results demonstrate the capabilities of rough surfaces prepared from the spontaneous assembly of liquid crystals to control biological models without the need of lithographic patterning or complex fabrication procedures. Interestingly, during myoblast differentiation, also myotube structuring in linear arrays is observed along the LCN fiber orientation. The implementation of this technology will open up to the formation of muscular tissue with well-aligned fibers in vitro mimicking the structure of native tissues.

A new generative approach for optical coherence tomography data scarcity: unpaired mutual conversion between scanning presets

In optical coherence tomography (OCT), there is a trade-off between the scanning time and image quality, leading to a scarcity of high quality data. OCT platforms provide different scanning presets, producing visually distinct images, limiting their compatibility. In this work, a fully automatic methodology for the unpaired visual conversion of the two most prevalent scanning presets is proposed. Using contrastive unpaired translation generative adversarial architectures, low quality images acquired with the faster Macular Cube preset can be converted to the visual style of high visibility Seven Lines scans and vice-versa. This modifies the visual appearance of the OCT images generated by each preset while preserving natural tissue structure. The quality of original and synthetic generated images was compared using brisque. The synthetic generated images achieved very similar scores to original images of their target preset. The generative models were validated in automatic and expert separability tests. These models demonstrated they were able to replicate the genuine look of the original images. This methodology has the potential to create multi-preset datasets with which to train robust computer-aided diagnosis systems by exposing them to the visual features of different presets they may encounter in real clinical scenarios without having to obtain additional data.Graphical Unpaired mutual conversion between scanning presets. Two generative adversarial models are trained for the conversion of OCT images into images of another scanning preset, replicating the visual features that characterise said preset.

Three-dimensional biofabrication of nanosecond laser micromachined nanofibre meshes for tissue engineered scaffolds.

There is a high demand for bespoke grafts to replace damaged or malformed bone and cartilage tissue. Three-dimensional (3D) printing offers a method of fabricating complex anatomical features of clinically relevant sizes. However, the construction of a scaffold to replicate the complex hierarchical structure of natural tissues remains challenging. This paper reports a novel biofabrication method that is capable of creating intricately designed structures of anatomically relevant dimensions. The beneficial properties of the electrospun fibre meshes can finally be realised in 3D rather than the current promising breakthroughs in two-dimensional (2D). The 3D model was created from commercially available computer-aided design software packages in order to slice the model down into many layers of slices, which were arrayed. These 2D slices with each layer of a defined pattern were laser cut, and then successfully assembled with varying thicknesses of 100 μm or 200 μm. It is demonstrated in this study that this new biofabrication technique can be used to reproduce very complex computer-aided design models into hierarchical constructs with micro and nano resolutions, where the clinically relevant sizes ranging from a simple cube of 20 mm dimension, to a more complex, 50 mm-tall human ears were created. In-vitro cell-contact studies were also carried out to investigate the biocompatibility of this hierarchal structure. The cell viability on a micromachined electrospun polylactic-co-glycolic acid fibre mesh slice, where a range of hole diameters from 200 μm to 500 μm were laser cut in an array where cell confluence values of at least 85% were found at three weeks. Cells were also seeded onto a simpler stacked construct, albeit made with micromachined poly fibre mesh, where cells can be found to migrate through the stack better with collagen as bioadhesives. This new method for biofabricating hierarchical constructs can be further developed for tissue repair applications such as maxillofacial bone injury or nose/ear cartilage replacement in the future.

In vivo efficacy of a polymer layered decellularized matrix composite as a cell honing cardiovascular tissue substitute.

Cardiovascular surgery involves reconstruction of tissues that are under cyclical mechanical loading, and in constant contact with pulsatile blood flow. Durable biomaterials for such tissue reconstruction are scarce, as they need to be mechanically strong, hemocompatible, and resist structural deterioration from calcification. While homografts are ideal, they are scarce; xenografts are immunogenic and rendered inactive from glutaraldehyde fixation, causing them to calficy and structurally deteriorate over time; decellularized xenografts are devoid of cells, mechanically weak; and synthetic polymeric scaffolds are thrombogenic or too dense to enable host cell infiltration. In this work, we report the in vivo feasibility of a new polymer-decellularized matrix composite material (decellularized bovine pericardium-polycaprolactone: chitosan) fabricated by electrospinning, which is designed to be mechanically strong and achieve programmed host cell honing to integrate into the host. In a rodent and sheep model, this new material was found to be hemocompatible, and enabled host cell infiltration into the polymer and the decellularized matrix core underlying the polymer. Presence of M2 macrophages and several vascular cell types, with matrix remodeling in the vicinity of the cells was observed in the explanted tissues. In summary, the proposed composite material is a novel approach to create in-situ host integrating tissue substitutes, with better non-thrombogenicity, reduced infections and endocarditis, and potentially the ability to grow with the patient and remodeling into a native tissue structure.

The Structural Interactions of Molecular and Fibrillar Collagen Type I with Fibronectin and Its Role in the Regulation of Mesenchymal Stem Cell Morphology and Functional Activity.

The observed differences in the structure of native tissue and tissue formed in vitro cause the loss of functional activity of cells cultured in vitro. The lack of fundamental knowledge about the protein mechanism interactions limits the ability to effectively create in vitro native tissue. Collagen is able to spontaneously assemble into fibrils in vitro, but in vivo, other proteins, for example fibronectin, have a noticeable effect on this process. The molecular or fibrillar structure of collagen plays an equally important role. Therefore, we studied the interaction of the molecular and fibrillar structure of collagen with fibronectin. Atomic force and transmission electron microscopy showed that the presence of fibronectin does not affect the native structure and diameter of collagen fibrils. Confocal microscopy demonstrated that the collagen structure affects the cell morphology. Cells are better spread on molecular collagen compared with cells cultured on fibrillar collagen. Fibronectin promotes the formation of a large number of focal contacts, while in combination with collagen of both forms, its effect is leveled. Thus, understanding the mechanisms of the relationship between the protein structure and composition will effectively manage the creation in vitro of a new tissue with native properties.

Human Lung Organoid Culture in Alginate With and Without Matrigel to Model Development and Disease.

Human lung organoids (HLOs) are enabling the study of human lung development and disease by modeling native organ tissue structure, cellular composition, and cellular organization. In this report, we demonstrate that HLOs derived from human pluripotent stem cells cultured in alginate, a fully defined nonanimal product substrate, exhibit enhanced cellular differentiation compared with HLOs cultured in the commercially available Matrigel. More specifically, we observed an earlier onset and increase in the number of multiciliated cells, along with mucus producing MUC5AC+ goblet-like cells that were not observed in HLOs cultured in Matrigel. The epithelium in alginate-grown HLOs was organized in a pseudostratified epithelium with airway basal cells lining the basal lamina, but with the apical surface of cells on the exterior of the organoid. We further observed that HLOs cultured in Matrigel exhibited mesenchymal overgrowth that was not present in alginate cultures. The containment of the mesenchyme within HLOs in alginate enabled modeling of key features of idiopathic pulmonary fibrosis (IPF) by treatment with transforming growth factor β (TGFβ). TGFβ treatment resulted in morphological changes including an increase in mesenchymal growth, increased expression of IPF markers, and decreased numbers of alveolar-like cells. This culture system provides a model to study the interaction of the mesenchyme with the epithelium during lung development and diseased states such as IPF.

Complex architectural control of ice-templated collagen scaffolds using a predictive model

The architectural and physiomechanical properties of regenerative scaffolds have been shown to improve engineered tissue function at both a cellular and tissue level. The fabrication of regenerative three-dimensional scaffolds that precisely replicate the complex hierarchical structure of native tissue, however, remains a challenge. The aim of this work is therefore two-fold: i) demonstrate an innovative multidirectional freeze-casting system to afford precise architectural control of ice-templated collagen scaffolds; and ii) present a predictive simulation as an experimental design tool for bespoke scaffold architecture. We used embedded heat sources within the freeze-casting mold to manipulate the local thermal environment during solidification of ice-templated collagen scaffolds. The resultant scaffolds comprised complex and spatially varied lamellar orientations that correlated with the imposed thermal environment and could be readily controlled by varying the geometry and power of the heat sources. The complex macro-architecture did not interrupt the hierarchical features characteristic of ice-templated scaffolds, but pore orientation had a significant impact on the stiffness of resultant structures under compression. Furthermore, our finite element model (FEM) accurately predicted the thermal environment and illustrated the freezing front topography within the mold during solidification. The lamellar orientation of freeze-cast scaffolds was also predicted using thermal gradient vector direction immediately prior to phase change. In combination our FEM and bespoke freeze-casting system present an exciting opportunity for tailored architectural design of ice-templated regenerative scaffolds that mimic the complex hierarchical environment of the native extracellular matrix. Statement of significanceBiomimetic scaffold structure improves engineered tissue function, but the fabrication of three-dimensional scaffolds that precisely replicate the complex hierarchical structure of native tissue remains a challenge. Here, we leverage the robust relationship between thermal gradients and lamellar orientation of ice-templated collagen scaffolds to develop a multidirectional freeze-casting system with precise control of the thermal environment and consequently the complex lamellar structure of resultant scaffolds. Demonstrating the diversity of our approach, we identify heat source geometry and power as control parameters for complex lamellar orientations. We simultaneously present a finite element model (FEM) that describes the three-dimensional thermal environment during solidification and accurately predicts lamellar structure of resultant scaffolds. The model serves as a design tool for bespoke regenerative scaffolds.

Electrospun aligned poly(ε-caprolactone) nanofiber yarns guiding 3D organization of tendon stem/progenitor cells in tenogenic differentiation and tendon repair.

Hierarchical anisotropy structure directing 3D cellular orientation plays a crucial role in designing tendon tissue engineering scaffolds. Despite recent development of fabrication technologies for controlling cellular organization and design of scaffolds that mimic the anisotropic structure of native tendon tissue, improvement of tenogenic differentiation remains challenging. Herein, we present 3D aligned poly (ε-caprolactone) nanofiber yarns (NFYs) of varying diameter, fabricated using a dry-wet electrospinning approach, that integrate with nano- and micro-scale structure to mimic the hierarchical structure of collagen fascicles and fibers in native tendon tissue. These aligned NFYs exhibited good in vitro biocompatibility, and their ability to induce 3D cellular alignment and elongation of tendon stem/progenitor cells was demonstrated. Significantly, the aligned NFYs with a diameter of 50 μm were able to promote the tenogenic differentiation of tendon stem/progenitor cells due to the integration of aligned nanofibrous structure and suitable yarn diameter. Rat tendon repair results further showed that bundled NFYs encouraged tendon repair in vivo by inducing neo-collagen organization and orientation. These data suggest that electrospun bundled NFYs formed by aligned nanofibers can mimic the aligned hierarchical structure of native tendon tissue, highlighting their potential as a biomimetic multi-scale scaffold for tendon tissue regeneration.

Physical, Mechanical, and Biological Properties of Fibrin Scaffolds for Cartilage Repair.

Articular cartilage is a highly organized tissue that provides remarkable load-bearing and low friction properties, allowing for smooth movement of diarthrodial joints; however, due to the avascular, aneural, and non-lymphatic characteristics of cartilage, joint cartilage has self-regeneration and repair limitations. Cartilage tissue engineering is a promising alternative for chondral defect repair. It proposes models that mimic natural tissue structure through the use of cells, scaffolds, and signaling factors to repair, replace, maintain, or improve the specific function of the tissue. In chondral tissue engineering, fibrin is a biocompatible biomaterial suitable for cell growth and differentiation with adequate properties to regenerate damaged cartilage. Additionally, its mechanical, biological, and physical properties can be enhanced by combining it with other materials or biological components. This review addresses the biological, physical, and mechanical properties of fibrin as a biomaterial for cartilage tissue engineering and as an element to enhance the regeneration or repair of chondral lesions.

3D bioprinted conductive spinal cord biomimetic scaffolds for promoting neuronal differentiation of neural stem cells and repairing of spinal cord injury

Spinal cord injury (SCI) is a terrible injury of the central nervous system for which there is no ideal treatment in clinic so far. The spinal cord biomimetic scaffolds which mimic the shape and structure of native spinal cord tissue, could effectively promote the repair of SCI. However, current biomimetic scaffolds do not mimic the biological functions, especially the electrical conductivity, of spinal cord tissue, which largely limits the therapeutic effect of SCI. In this study, we have developed novel conductive hydrogels based on gelatin methacrylate (GelMA), hyaluronic acid methacrylate (HAMA) and poly(3,4-ethylenedioxythiophene): sulfonated lignin (PEDOT:LS). The incorporation of PEDOT:LS into GelMA/HAMA hydrogel matrix significantly improved the conductivity of the hydrogels. By precisely regulating the light curing time, the conductive hydrogels showed mechanical properties similar to native spinal cord tissues. Then, the conductive biomimetic scaffolds were fabricated by 3D bioprinting, and the neural stem cells (NSCs) encapsulated in the scaffolds exhibited good survival rate (higher than 90%). Compared to the non-conductive scaffolds reported, the conductive scaffolds remarkably promoted neuronal differentiation of NSCs in vitro. In a rat spinal cord complete transection model, the conductive biomimetic scaffold dramatically promoted the recovery of hindlimb motor function. Immunofluorescence staining results further showed that the conductive biomimetic scaffold effectively promoted the regeneration of neurons at the injury site, reduced the deposition of glial scar, and promoted nerve axon regeneration and myelination. Overall, the 3D bioprinting of the conductive spinal cord biomimetic scaffolds developed in this work represents a promising approach for the stem cell-based treatment of SCI.

A biomimetic multilayered polymeric material designed for heart valve repair and replacement

Materials currently used to repair or replace a heart valve are not durable. Their limited durability related to structural degeneration or thrombus formation is attributed to their inadequate mechanical properties and biocompatibility profiles. Our hypothesis is that a biostable material that mimics the structure, mechanical and biological properties of native tissue will improve the durability of these leaflets substitutes and in fine improve the patient outcome. Here, we report the development, optimization, and testing of a biomimetic, multilayered material (BMM), designed to replicate the native valve leaflets. Polycarbonate urethane and polycaprolactone have been processed as film, foam, and aligned fibers to replicate the leaflet's architecture and anisotropy, through solution casting, lyophilization, and electrospinning. Compared to the commercialized materials, our BMMs exhibited an anisotropic behavior and a closer mechanical performance to the aortic leaflets. The material exhibited superior biostability in an accelerated oxidization environment. It also displayed better resistance to protein adsorption and calcification in vitro and in vivo. These results will pave the way for a new class of advanced synthetic material with long-term durability for surgical valve repair or replacement.

Computer Model-Driven Design in Cardiovascular Regenerative Medicine

Continuing advances in genomics, molecular and cellular mechanobiology and immunobiology, including transcriptomics and proteomics, and biomechanics increasingly reveal the complexity underlying native tissue and organ structure and function. Identifying methods to repair, regenerate, or replace vital tissues and organs remains one of the greatest challenges of modern biomedical engineering, one that deserves our very best effort. Notwithstanding the continuing need for improving standard methods of investigation, including cell, organoid, and tissue culture, biomaterials development and fabrication, animal models, and clinical research, it is increasingly evident that modern computational methods should play increasingly greater roles in advancing the basic science, bioengineering, and clinical application of regenerative medicine. This brief review focuses on the development and application of computational models of tissue and organ mechanobiology and mechanics for purposes of designing tissue engineered constructs and understanding their development in vitro and in situ. Although the basic approaches are general, for illustrative purposes we describe two recent examples from cardiovascular medicine—tissue engineered heart valves (TEHVs) and tissue engineered vascular grafts (TEVGs)—to highlight current methods of approach as well as continuing needs.

Postcare for Repairing Nerve and Tendon Injury Based on Biomimetic Nano-Parallel Material Composite Protein

Natural tendons are composed of ordered parallel arrangement of bundles of type I collagen fibers. It is responsible for transmitting the forces generated by the bones and muscles to move the body. Tendon injuries are common sports injuries, accounting for 50% of sports injuries. In adulthood, factors such as few tendon tissue cells and poor blood supply make it difficult to heal on their own after injury. Therefore, restoring the structure and function of native tendon tissue is still an unsolved problem. The purpose of this study is to study the use of biomimetic nano-parallel material composite proteins to induce the directional differentiation of stem cells to tendon lineages to promote muscle regeneration and repair. This study proposes to prepare composite protein nanomaterials by simulating the parallel arrangement of tendon collagen fibers. This can facilitate the directed differentiation of fibroblasts into tendon lineages. It can use the BP algorithm in the neural network algorithm to simulate the parallel arrangement of tendon collagen fibers, which is more efficient than other schemes. The experimental results in this study show that the rat cells are repaired after about 6 weeks. Tendon repair enters a remodeling phase with reduced cell numbers, collagen, and mucopolysaccharide synthesis. During this period, tendon repair gradually changes from the cellular level to the tissue level, and tenocyte metabolism remains high. Tenocytes and collagen fibers are aligned in the direction of stress. A higher proportion of collagen 1 synthesis occurs during this period. Collagen 1 accounts for 65% to 80% of the dry weight of the tendon, which plays the most important role in the mechanical properties of the tendon.

Application of 3D Bioprinting in Urology.

Tissue engineering is an emerging field to create functional tissue components and whole organs. The structural and functional defects caused by congenital malformation, trauma, inflammation or tumor are still the major clinical challenges facing modern urology, and the current treatment has not achieved the expected results. Recently, 3D bioprinting has gained attention for its ability to create highly specialized tissue models using biological materials, bridging the gap between artificially engineered and natural tissue structures. This paper reviews the research progress, application prospects and current challenges of 3D bioprinting in urology tissue engineering.

3D printed controllable microporous scaffolds support embryonic development in vitro.

Little is known about the complex molecular and cellular events occurring during implantation, which represents a critical step for pregnancy. The conventional 2D culture could not support postimplantation embryos' normal development, and 3D conditions shed light into the “black box”. 3D printing technology has been widely used in recapitulating the structure and function of native tissues in vitro. Here, we 3D printed anisotropic microporous scaffolds to culture embryos by manipulating the advancing angle between printed layers, which affected embryo development. The 30° and 60° scaffolds promote embryo development with moderate embryo‐scaffold attachments. T‐positive cells and FOXA2‐positive cells were observed to appear in the posterior region of the embryo and migrated to the anterior region of the embryo on day 7. These findings demonstrate a 3D printed stand that supports embryonic development in vitro and the critical role of 3D architecture for embryo implantation, in which additive manufacturing is a versatile tool.

Multicompartmental Scaffolds for Coordinated Periodontal Tissue Engineering

Successful periodontal repair and regeneration requires the coordinated responses from soft and hard tissues as well as the soft tissue–to–bone interfaces. Inspired by the hierarchical structure of native periodontal tissues, tissue engineering technology provides unique opportunities to coordinate multiple cell types into scaffolds that mimic the natural periodontal structure in vitro. In this study, we designed and fabricated highly ordered multicompartmental scaffolds by melt electrowriting, an advanced 3-dimensional (3D) printing technique. This strategy attempted to mimic the characteristic periodontal microenvironment through multicompartmental constructs comprising 3 tissue-specific regions: 1) a bone compartment with dense mesh structure, 2) a ligament compartment mimicking the highly aligned periodontal ligaments (PDLs), and 3) a transition region that bridges the bone and ligament, a critical feature that differentiates this system from mono- or bicompartmental alternatives. The multicompartmental constructs successfully achieved coordinated proliferation and differentiation of multiple cell types in vitro within short time, including both ligamentous- and bone-derived cells. Long-term 3D coculture of primary human osteoblasts and PDL fibroblasts led to a mineral gradient from calcified to uncalcified regions with PDL-like insertions within the transition region, an effect that is challenging to achieve with mono- or bicompartmental platforms. This process effectively recapitulates the key feature of interfacial tissues in periodontium. Collectively, this tissue-engineered approach offers a fundament for engineering periodontal tissue constructs with characteristic 3D microenvironments similar to native tissues. This multicompartmental 3D printing approach is also highly compatible with the design of next-generation scaffolds, with both highly adjustable compartmentalization properties and patient-specific shapes, for multitissue engineering in complex periodontal defects.

Fate of silica phytoliths in the industrial crushing of sugarcane stalks

Biomass from grasses such as sugarcane contains bodies of biogenic silica, the phytoliths. In industrial biomass processing, phytoliths can be harmful to equipment causing wear and ash deposits. However, phytoliths can also be beneficial if they are valorized into silica-based products. This work investigates the fate of phytolith in the industrial crushing of sugarcane stalks using biomass fractionation, ashing, and imaging techniques. Phytoliths can be traced by scanning electron microscopy coupled with energy-dispersive X-ray spectroscopy (SEM-EDS) due to their proximity to the outer surface and fingerprint size, shape, and chemistry. In native sugarcane stalks, phytoliths are concentrated in the epidermal region. Industrial crushing promotes tissue ruptures that transform stalks into bagasse, where the epidermal tissues and phytoliths are found with preserved integrity, fractured, or even detached from the native tissue structures. The observations presented in this work suggest novel strategies for the isolation of phytoliths in biorefinery designs integrable to the well-established industrial crushing of sugarcane stalks.

Modulation of 3D Printed Calcium-Deficient Apatite Constructs with Varying Mn Concentrations for Osteochondral Regeneration via Endochondral Differentiation.

Osteochondral regeneration remains a vital problem in clinical situations affecting both bone and cartilage tissues due to the low regeneration ability of cartilage tissue. Additionally, the simultaneous regeneration of bone and cartilage is difficult to attain due to their dissimilar nature. Thus, fabricating a single scaffold for both bone and cartilage regeneration remains challenging. Biomaterials are frequently employed to promote tissue restoration, but they still cannot replicate the structure of native tissue. This study aims to create a single biomaterial that could be used to regenerate both bone and cartilage. This study focuses on synthesizing calcium-deficient apatite (CDA) with the gradual addition of manganese. The phase stability and the effect of heat treatment on manganese-doped CDA were studied using X-ray diffraction (XRD) and Rietveld refinement. The obtained powders were tested for their 3-dimensional (3D) printing ability by fabricating cuboidal 3D structures. The 3D printed scaffolds were examined for external topography using field-emission scanning electron microscopy (FE-SEM) and were subjected to compression testing. In vitro biocompatibility and differentiation studies were performed to access their biocompatibility and differentiation capabilities. Reverse transcription-quantitative PCR (RT-qPCR) analysis was done to determine the gene expression of bone- and cartilage-specific markers. Mn helps in stabilizing the β-TCP phase beyond its sintering temperature without being degraded to α-TCP. Mn addition in CDA improves the compressive strength of the fabricated scaffolds while keeping them biocompatible. The concentrations of Mn in the CDA ceramic were found to influence the differentiation behavior of MSCs in the fabricated scaffolds. Mn-doped CDA is a promising candidate to be used as a substitute material for bone, cartilage, and osteochondral defects to facilitate repair and regeneration via endochondral differentiation. 3D printing can assist in the fabrication of a multifunctional single-unit scaffold with varied Mn concentrations, which might be able to generate the two tissues in situ in an osteochondral defect.