Effects of Three-Dimensional Platform Stiffness and Layer Dimensions on Separation of Carcinoma Cells

Cancer cell separation is highly desirable for cancer diagnosis and therapy. Besides biochemical methods, engineered platforms are effective alternatives for sorting carcinoma cells from normal cells based on their unique properties in responding to the physical changes of the surrounding microenvironment. In this work, three-dimensional (3D) biomimetic scaffold platforms were developed to separate nasopharyngeal carcinoma 43 (NPC43) cells from immortalized nasopharyngeal epithelial 460 (NP460) cells based on precisely controlled design parameters including stiffness, number of layers, and structural layout. The migration characteristics of NPC43 and NP460 cells on the scaffold platforms revealed that NPC43 cells could squeeze into 10 μm wide, 15 μm deep trenches while NP460 cells could not. The different migration behavior was mainly due to cells having different interactions with the surrounding microenvironment. NPC43 cells had filopodia-like protrusions, while NP460 cells exhibited a sheet-like morphology. Using these 3D biomimetic platforms, 89% separation efficiency of NPC43 cells from NP460 cells was achieved on stiffer two-layer scaffold platforms with a 40/10 μm ridge/trench (R/T) grating on the top layer and a 20/10 μm R/T grid on the bottom layer. Moreover, the separation efficiency was further increased to 93% by adding an active conditioned medium (ACM) that caused the cells to have higher motility and deformability. These results demonstrate the capability to apply biomimetic engineered platforms with appropriate designs to separate cancer cells from normal cells for potential cancer diagnosis and treatment.     

Improved angiogenic cell penetration in vitro and in vivo in collagen scaffolds with internal channels

Porous scaffolds are limited in volume due to diffusion constraint and delay of vascular network formation. Channels have the potential to speed up cellular penetration. Their effectiveness in improving angiogenic cell penetration was assessed in vitro and in vivo in 3-D collagen scaffolds. In vitro, channelled and non-channelled scaffolds were seeded with vascular smooth muscle cells. Results demonstrated that the scaffolds supported angiogenic cell ingrowth in culture and the channels improved the depth of cell penetration into the scaffold (P < 0.05). The cells reside mainly around and migrate along the channels. In vivo, channels increased cell migration into the scaffolds (P < 0.05) particularly angiogenic cells (P < 0.05) resulting in a clear branched vascular network of microvessels after 2 weeks in the channelled samples which was not apparent in the non-channelled samples. Channels could aid production of tissue engineered constructs by offering the possibility of rapid blood vessel infiltration into collagen scaffolds.     

Mechanistic adaptability of cancer cells strongly affects anti-migratory drug efficacy

Cancer metastasis involves the dissemination of cancer cells from the primary tumour site and is responsible for the majority of solid tumour-related mortality. Screening of anti-metastasis drugs often includes functional assays that examine cancer cell invasion inside a three-dimensional hydrogel that mimics the extracellular matrix (ECM). Here, we built a mechanically tuneable collagen hydrogel model to recapitulate cancer spreading into heterogeneous tumour stroma and monitored the three-dimensional invasion of highly malignant breast cancer cells, MDA-MB-231. Migration assays were carried out in the presence and the absence of drugs affecting four typical molecular mechanisms involved in cell migration, as well as under five ECMs with different biophysical properties. Strikingly, the effects of the drugs were observed to vary strongly with matrix mechanics and microarchitecture, despite the little dependence of the inherent cancer cell migration on the ECM condition. Specifically, cytoskeletal contractility-targeting drugs reduced migration speed in sparse gels, whereas migration in dense gels was retarded effectively by inhibiting proteolysis. The results corroborate the ability of cancer cells to switch their multiple invasion mechanisms depending on ECM condition, thus suggesting the importance of factoring in the biophysical properties of the ECM in anti-metastasis drug screenings.     

Reverse Reconstruction and Bioprinting of Bacterial Cellulose‐Based Functional Total Intervertebral Disc for Therapeutic Implantation

The degradation of intervertebral discs (IVD), a typical hierarchical structured tissue, causes serious neck and back pain. The current methods cannot fully reconstitute the unique structure and function of native IVD. In this study, by reverse reconstruction of the structure of native IVD and bioprinting bacterial cellulose (BC) nanofibers with a high‐throughput optimized micropattern screening microchip, a total IVD is created that contained type II collagen‐based nucleus pulposus (NP) and hierarchically organized and micropatterned BC‐based annulus fibrosus (AF), mimicking native IVD tissue. The artificial NP contains rat NP cells, whereas the AF contains concentrically arranged BC layers with aligned micropatterns and attached AF cells in +/?30° alternate directions between adjacent layers. Long‐term (3 months) implantation experiments on rats demonstrate the excellent structural (shape maintenance, hydration, tissue integration) and functional (mechanical support and flexibility) performance of the artificial IVD. This study provides a novel strategy for creating highly sophisticated artificial tissues.     

Evaluation of synovium-derived mesenchymal stem cells and 3D printed nanocomposite scaffolds for tissue engineering

Stem cells and scaffolds play a very important role in tissue engineering. Here, we isolated synovium-derived mesenchymal stem cells (SMSCs) from synovial membrane tissue and characterized stem-cell properties. Gelatin nanoparticles (NP) were prepared using a two-step desolvation method and then pre-mixed into different host matrix (silk fibroin (SF), gelatin (Gel), or SF–Gel mixture) to generate various 3D printed nanocomposite scaffolds (NP/SF, NP/SF–Gel, NP/Gel-1, and NP/Gel-2). The microstructure was examined by scanning electron microscopy. Biocompatibility assessment was performed through CCK-8 assay by coculturing with SMSCs at 1, 3, 7 and 14 days. According to the results, SMSCs are similar to other MSCs in their surface epitope expression, which are negative for CD45 and positive for CD44, CD90, and CD105. After incubation in lineage-specific medium, SMSCs could differentiate into chondrocytes, osteocytes and adipocytes. 3D printed nanocomposite scaffolds exhibited a good biocompatibility in the process of coculturing with SMSCs and had no negative effect on cell behavior. The study provides a strategy to obtain SMSCs and fabricate 3D printed nanocomposite scaffolds, the combination of which could be used for practical applications in tissue engineering.     

Chitosan/gelatin scaffolds support bone regeneration

Chitosan/Gelatin (CS:Gel) scaffolds were fabricated by chemical crosslinking with glutaraldehyde or genipin by freeze drying. Both crosslinked CS:Gel scaffold types with a mass ratio of 40:60% form a gel-like structure with interconnected pores. Dynamic rheological measurements provided similar values for the storage modulus and the loss modulus of the CS:Gel scaffolds when crosslinked with the same concentration of glutaraldehyde vs. genipin. Compared to genipin, the glutaraldehyde-crosslinked scaffolds supported strong adhesion and infiltration of pre-osteoblasts within the pores as well as survival and proliferation of both MC3T3-E1 pre-osteoblastic cells after 7 days in culture, and human bone marrow mesenchymal stem cells (BM-MSCs) after 14 days in culture. The levels of collagen secreted into the extracellular matrix by the pre-osteoblasts cultured for 4 and 7 days on the CS:Gel scaffolds, significantly increased when compared to the tissue culture polystyrene (TCPS) control surface. Human BM-MSCs attached and infiltrated within the pores of the CS:Gel scaffolds allowing for a significant increase of the osteogenic gene expression of RUNX2, ALP, and OSC. Histological data following implantation of a CS:Gel scaffold into a mouse femur demonstrated that the scaffolds support the formation of extracellular matrix, while fibroblasts surrounding the porous scaffold produce collagen with minimal inflammatory reaction. These results show the potential of CS:Gel scaffolds to support new tissue formation and thus provide a promising strategy for bone tissue engineering.     

Construction of collagen II/hyaluronate/chondroitin-6-sulfate tri-copolymer scaffold for nucleus pulposus tissue engineering and preliminary analysis of its physico-chemical properties and biocompatibility

To construct a novel scaffold for nucleus pulposus (NP) tissue engineering, The porous type II collagen (CII)/hyaluronate (HyA)–chondroitin-6-sulfate (6-CS) scaffold was prepared using 1-ethyl-3-(3-dimethylaminopropyl)-carbodiimide (EDC) and N-hydroxysuccinimide (NHS) cross-linking system. The physico-chemical properties and biocompatibility of CII/HyA–CS scaffolds were evaluated. The results suggested CII/HyA–CS scaffolds have a highly porous structure (porosity: 94.8 ± 1.5%), high water-binding capacity (79.2 ± 2.8%) and significantly improved mechanical stability by EDC/NHS crosslinking (denaturation temperature: 74.6 ± 1.8 and 58.1 ± 2.6°C, respectively, for the crosslinked scaffolds and the non-crosslinked; collagenase degradation rate: 39.5 ± 3.4 and 63.5 ± 2.0%, respectively, for the crosslinked scaffolds and the non-crosslinked). The CII/HyA–CS scaffolds also showed satisfactory cytocompatibility and histocompatibility as well as low immunogenicity. These results indicate CII/HyA–CS scaffolds may be an alternative material for NP tissue engineering due to the similarity of its composition and physico-chemical properties to those of the extracellular matrices (ECM) of native NP.     

Evaluation of synovium-derived mesenchymal stem cells and 3D printed nanocomposite scaffolds for tissue engineering

Abstract

Stem cells and scaffolds play a very important role in tissue engineering. Here, we isolated synovium-derived mesenchymal stem cells (SMSCs) from synovial membrane tissue and characterized stem-cell properties. Gelatin nanoparticles (NP) were prepared using a two-step desolvation method and then pre-mixed into different host matrix (silk fibroin (SF), gelatin (Gel), or SF–Gel mixture) to generate various 3D printed nanocomposite scaffolds (NP/SF, NP/SF–Gel, NP/Gel-1, and NP/Gel-2). The microstructure was examined by scanning electron microscopy. Biocompatibility assessment was performed through CCK-8 assay by coculturing with SMSCs at 1, 3, 7 and 14 days. According to the results, SMSCs are similar to other MSCs in their surface epitope expression, which are negative for CD45 and positive for CD44, CD90, and CD105. After incubation in lineage-specific medium, SMSCs could differentiate into chondrocytes, osteocytes and adipocytes. 3D printed nanocomposite scaffolds exhibited a good biocompatibility in the process of coculturing with SMSCs and had no negative effect on cell behavior. The study provides a strategy to obtain SMSCs and fabricate 3D printed nanocomposite scaffolds, the combination of which could be used for practical applications in tissue engineering.     

Loading of VEGF to the heparin cross-linked demineralized bone matrix improves vascularization of the scaffold

Deficient vascularization is one of the prominent shortcomings of porous tissue-engineering scaffolds, which results in insufficient oxygen and nutrients transportation. Here, heparin cross-linked demineralized bone matrices (HC-DBM) pre-loaded with vascular endothelial growth factor (VEGF) were designed to promote cells and new microvessels invasion into the matrices. After being chemical crosslinked with heparin by N-hydroxysuccinimide and N-(3-di-methylaminopropyl)-N’-ethylcarbodiimide, the scaffold could bind more VEGF than the non-crosslinked one and achieve localized and sustained delivery. The biological activity of VEGF binding on heparinized collagen was demonstrated by promoting endothelial cells proliferation. Evaluation of the angiogenic potential of heparinized DBM loaded with VEGF was further investigated by subcutaneous implantation. Improved angiogenesis of heparinized DBM loaded with VEGF was observed from haematoxylin-eosin staining and immunohistochemistry examination. The results demonstrated that heparin cross-linked DBM binding VEGF could be a useful strategy to stimulate cells and blood vessels invasion into the scaffolds.     

Optimising collagen scaffold architecture for enhanced periodontal ligament fibroblast migration

Design of cell-free scaffolds for endogenous cell recruitment requires an intimate knowledge of precise relationships between structure and biological function. Here, we use morphological analysis by Micro-CT to identify the key structural features necessary for periodontal ligament fibroblast recruitment into collagen scaffolds. By the combined use of time-lapse imaging and end-point invasion analysis, we distinguish the influences of pore size, pore wall alignment, and pore transport pathways (percolation diameter) on the individual cell migration and bulk invasion characteristics of these fibroblasts. Whereas maximising percolation diameter increased individual cell speed, elongation and directionality, and produced the most rapid bulk cell invasion, a pore size of 100?μm was found to be necessary to ensure an even distribution of cells across the scaffold cross-section. These results demonstrate that control of percolation diameter and pore size may be used respectively to tune the efficiency and uniformity of invasion through macroporous scaffolds. Crucially, however, these observations were subject to the condition of pore wall alignment, with low alignment in the direction of travel producing relatively low cell speeds and limited invasion in all cases. Pore wall alignment should therefore be carefully optimised in the design of scaffolds for cell recruitment, such as that required for periodontal ligament regeneration, as a key determining factor for cell movement.

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Polyurethane (PU) scaffolds prepared by solvent casting/particulate leaching (SCPL) combined with centrifugation

This article reports an enhanced solvent casting/particulate (salt) leaching (SCPL) method developed for preparing three-dimensional porous polyurethane (PU) scaffolds for cardiac tissue engineering. The solvent for the preparation of the PU scaffolds was a mixture of dimethylformamide (DFM) and tetrahydrofuran (THF). The enhanced method involved the combination of a conventional SCPL method and a step of centrifugation, with the centrifugation being employed to improve the pore uniformity and the pore interconnectivity of scaffolds. Highly porous three-dimensional scaffolds with a well interconnected porous structure could be achieved at the polymer solution concentration of up to 20% by air or vacuum drying to remove the solvent. When the salt particle sizes of 212–295, 295–425, or 425–531 µm and a 15% w/v polymer solution concentration were used, the porosity of the scaffolds was between 83–92% and the compression moduli of the scaffolds were between 13 kPa and 28 kPa. Type I collagen acidic solution was introduced into the pores of a PU scaffold to coat the collagen onto the pore walls throughout the whole PU scaffold. The human aortic endothelial cells (HAECs) cultured in the collagen-coated PU scaffold for 2 weeks were observed by scanning electron microscopy (SEM). It was shown that the enhanced SCPL method and the collagen coating resulted in a spatially uniform distribution of cells throughout the collagen-coated PU scaffold.     

Cartilage replacement by use of hybrid systems of autologous cells and polyethylene: an experimental study

This study used porous polyethylene (PE) as a scaffold in an animal model system. The surface of the scaffolds was either modified with collagen II coating or first functionalized by oxygen plasma treatment and then coated with collagen II. The specimens were inoculated with autologous chondrocytes and transplanted into the concha of guinea pigs. Bare scaffolds were used as controls. Periods of 1, 6, and 12 months after implantation, samples of cells containing specimens and control samples were evaluated microscopically. As a result, the pre-seeded specimens were better integrated into the surrounding tissue than cell-free PE-specimens. Also a weaker immune reaction and an improved cartilage generation could be detected in the pre-seeded specimen. Compared to the other surface modifications, no further improvement of cartilage development was observed in the long term in vivo animal experimental study.     

Effect of multiple unconfined compression on cellular dense collagen scaffolds for bone tissue engineering

Plastic compression of hydrated collagen gels rapidly produces biomimetic scaffolds of improved mechanical properties. These scaffolds can potentially be utilised as cell seeded systems for bone tissue engineering. This work investigated the influence of multiple unconfined compression on the biocompatibility and mechanical properties of such systems. Single and double compressed dense collagen matrices were produced and characterised for protein dry weight, morphology and mechanical strength. Compression related maintenance of the seeded HOS TE85 cell line viability in relation to the extent of compression was evaluated up to 10 days in culture using the TUNEL assay. Fluorescence Live/Dead assay was conducted to examine overall cell survival and morphology. Cell induced structural changes in the dense collagenous scaffolds were assessed by routine histology. The mechanical properties of the cellular scaffolds were also evaluated as a function of time in culture. It is clear that a single plastic compression step produced dense collagenous scaffolds capable of maintaining considerable cell viability and function as signs of matrix remodeling, and maintenance of mechanical properties were evident. Such scaffolds should therefore be further developed as systems for bone tissue regeneration.     

Fabrication and evaluation of biomimetic scaffolds by using collagen–alginate fibrillar gels for potential tissue engineering applications

Pore architecture and its stable functionality under cell culturing of three dimensional (3D) scaffolds are of great importance for tissue engineering purposes. In this study, alginate was incorporated with collagen to fabricate collagen–alginate composite scaffolds with different collagen/alginate ratios by lyophilizing the respective composite gels formed via collagen fibrillogenesis in vitro and then chemically crosslinking. The effects of alginate amount and crosslinking treatment on pore architecture, swelling behavior, enzymatic degradation and tensile property of composite scaffolds were systematically investigated. The relevant results indicated that the present strategy was simple but efficient to fabricate highly interconnected strong biomimetic 3D scaffolds with nanofibrous surface. NIH3T3 cells were used as a model cell to evaluate the cytocompatibility, attachment to the nanofibrous surface and porous architectural stability in terms of cell proliferation and infiltration within the crosslinked scaffolds. Compared with the mechanically weakest crosslinked collagen sponges, the cell-cultured composite scaffolds presented a good porous architecture, thus permitting cell proliferation on the top surface as well as infiltration into the inner part of 3D composite scaffolds. These composite scaffolds with pore size ranging from 150 to 300 μm, over 90% porosity, tuned biodegradability and water-uptake capability are promising for tissue engineering applications.     

Preparation and characterization of malonic acid cross-linked chitosan and collagen 3D scaffolds: an approach on non-covalent interactions

The present study emphasizes the influence of non-covalent interactions on the mechanical and thermal properties of the scaffolds of chitosan/collagen origin. Malonic acid (MA), a bifuncitonal diacid was chosen to offer non-covalent cross-linking. Three dimensional scaffolds was prepared using chitosan at 1.0% (w/v) and MA at 0.2% (w/v), similarly collagen 0.5% (w/v) and MA 0.2% (w/v) and characterized. Results on FT-IR, TGA, DSC, SEM and mechanical properties (tensile strength, stiffness, Young’s modulus, etc.) assessment demonstrated the existence of non-covalent interaction between MA and chitosan/collagen, which offered flexibility and high strength to the scaffolds suitable for tissue engineering research. Studies using NIH 3T3 fibroblast cells suggested biocompatibility nature of the scaffolds. Docking simulation study further supports the intermolecular hydrogen bonding interactions between MA and chitosan/collagen.     

Preparation and characterization of collagen/PLA,chitosan/PLA,and collagen/chitosan/PLA hybrid scaffolds for cartilage tissue engineering

In this study, three-dimensional (3D) porous scaffolds were developed for the repair of articular cartilage defects. Novel collagen/polylactide (PLA), chitosan/PLA, and collagen/chitosan/PLA hybrid scaffolds were fabricated by combining freeze-dried natural components and synthetic PLA mesh, where the 3D PLA mesh gives mechanical strength, and the natural polymers, collagen and/or chitosan, mimic the natural cartilage tissue environment of chondrocytes. In total, eight scaffold types were studied: four hybrid structures containing collagen and/or chitosan with PLA, and four parallel plain scaffolds with only collagen and/or chitosan. The potential of these types of scaffolds for cartilage tissue engineering applications were determined by the analysis of the microstructure, water uptake, mechanical strength, and the viability and attachment of adult bovine chondrocytes to the scaffolds. The manufacturing method used was found to be applicable for the manufacturing of hybrid scaffolds with highly porous 3D structures. All the hybrid scaffolds showed a highly porous structure with open pores throughout the scaffold. Collagen was found to bind water inside the structure in all collagen-containing scaffolds better than the chitosan-containing scaffolds, and the plain collagen scaffolds had the highest water absorption. The stiffness of the scaffold was improved by the hybrid structure compared to plain scaffolds. The cell viability and attachment was good in all scaffolds, however, the collagen hybrid scaffolds showed the best penetration of cells into the scaffold. Our results show that from the studied scaffolds the collagen/PLA hybrids are the most promising scaffolds from this group for cartilage tissue engineering.     

Electrospun antimicrobial microfibrous scaffold for annulus fibrosus tissue engineering

In this study, polycaprolactone (PCL) microfibrous scaffolds with berberine were fabricated to mimic the natural extracellular matrix (ECM) architecture and provide antimicrobial activity for annulus fibrosus tissue engineering. Morphological characterization showed that there was a significant decrease of the average fiber diameter in the berberine-loaded microfibrous scaffolds (B-MFS, 0.40 ± 0.02 μm) compared with that of the non-drug-loaded microfibrous scaffolds (MFS, 1.89 ± 0.15 μm). The antimicrobial activity, drug release profile, and biocompatibility of the scaffolds were evaluated. The B-MFS displayed excellent antimicrobial activities against Gram-positive bacteria (S. aureus 6538), Gram-negative bacteria (E. coli 15597), fungus (C. albicans 10231) and drug-resistant bacteria (methicillin-resistant S. aureus BAA-811, or MRSA BAA-811). After seeding with porcine AF cells, the in vitro biocompatibility of the scaffolds was determined by measuring cell attachment, cell proliferation, and ECM production. Total cell number, sGAG and collagen content gradually increased from day 1 to day 7 in both groups. When compared to MFS, the B-MFS group displayed higher levels of cell proliferation throughout the experimental period. These results indicate that PCL microfibrous loaded with berberine are novel biocompatible scaffolds with a broad-spectrum antimicrobial activity for AF tissue engineering.     

Surface modified electrospun nanofibrous scaffolds for nerve tissue engineering

The development of biodegradable polymeric scaffolds with surface properties that dominate interactions between the material and biological environment is of great interest in biomedical applications. In this regard, poly-ε-caprolactone (PCL) nanofibrous scaffolds were fabricated by an electrospinning process and surface modified by a simple plasma treatment process for enhancing the Schwann cell adhesion, proliferation and interactions with nanofibers necessary for nerve tissue formation. The hydrophilicity of surface modified PCL nanofibrous scaffolds (p-PCL) was evaluated by contact angle and x-ray photoelectron spectroscopy studies. Naturally derived polymers such as collagen are frequently used for the fabrication of biocomposite PCL/collagen scaffolds, though the feasibility of procuring large amounts of natural materials for clinical applications remains a concern, along with their cost and mechanical stability. The proliferation of Schwann cells on p-PCL nanofibrous scaffolds showed a 17% increase in cell proliferation compared to those on PCL/collagen nanofibrous scaffolds after 8 days of cell culture. Schwann cells were found to attach and proliferate on surface modified PCL nanofibrous scaffolds expressing bipolar elongations, retaining their normal morphology. The results of our study showed that plasma treated PCL nanofibrous scaffolds are a cost-effective material compared to PCL/collagen scaffolds, and can potentially serve as an ideal tissue engineered scaffold, especially for peripheral nerve regeneration.     

Proteolytic surface functionalization enhances in vitro magnetic nanoparticle mobility through extracellular matrix

Steric barriers such as collagen I sharply limit interstitial delivery of macromolecular and nanoparticle (NP) based therapeutic agents. Collagenase-linked superparamagnetic NPs overcame these barriers and moved through in vitro extracellular matrix (ECM) at 90 microm h(-1), a rate similar to invasive cells, under the influence of a magnetic field. NP migration in ECM diminished linearly over 5 days. The collagenase-NP construct overcame two of the most significant barriers to nano- and microscale therapeutics deployment: proteolytic enzyme stability was maintained during a clinically useful time frame by immobilization on the NP surface and degradation of interstitial barriers to tissue biodistribution was enabled by the conjugated microbial protease.     

Cell response to collagen-calcium phosphate cement scaffolds investigated for nonviral gene delivery

Collagen-hydroxyapatite (HA) scaffolds for the non-viral delivery of a plasmid encoding the osteoinductive protein bone morphogenetic protein (BMP)-7 were developed. The collagen-HA was obtained by the combination of calcium phosphate cement in a collagen template. The effect on cell behavior of increasing amounts of HA in the scaffolds was evaluated. Collagen-HA scaffolds containing 13, 23 or 83 wt% HA were prepared. Cell proliferation was reduced in the 83% HA scaffold after 1 day compared to 13 and 23% HA, but by 14 days the number of cells in 83% HA considerably increased. Alkaline phosphatase (ALP) activity was 8 times higher for the 83% HA scaffolds. BMP-7 plasmid was incorporated into the 83% HA scaffold. The transfection was low, although significant levels of BMP7 were expressed, associated with an increase in cell proliferation.