Poor Cell Attachment Research Articles

Manufacturing of 3D-Printed Hybrid Scaffolds with Polyelectrolyte Multilayer Coating in Static and Dynamic Culture Conditions.

Three-dimensional printing (3DP) has emerged as a promising method for creating intricate scaffold designs. This study assessed three 3DP scaffold designs fabricated using biodegradable poly(lactic) acid (PLA) through fused deposition modelling (FDM): mesh, two channels (2C), and four channels (4C). To address the limitations of PLA, such as hydrophobic properties and poor cell attachment, a post-fabrication modification technique employing Polyelectrolyte Multilayers (PEMs) coating was implemented. The scaffolds underwent aminolysis followed by coating with SiCHA nanopowders dispersed in hyaluronic acid and collagen type I, and finally crosslinked the outermost coated layers with EDC/NHS solution to complete the hybrid scaffold production. The study employed rotating wall vessels (RWVs) to investigate how simulating microgravity affects cell proliferation and differentiation. Human mesenchymal stem cells (hMSCs) cultured on these scaffolds using proliferation medium (PM) and osteogenic media (OM), subjected to static (TCP) and dynamic (RWVs) conditions for 21 days, revealed superior performance of 4C hybrid scaffolds, particularly in OM. Compared to commercial hydroxyapatite scaffolds, these hybrid scaffolds demonstrated enhanced cell activity and survival. The pre-vascularisation concept on 4C hybrid scaffolds showed the proliferation of both HUVECs and hMSCs throughout the scaffolds, with a positive expression of osteogenic and angiogenic markers at the early stages.

Proliferation and differential regulation of osteoblasts cultured on surface-phosphorylated cellulose nanofiber scaffolds

Phosphorus-containing polymers have received much attention for their excellent ability to regulate bone cell differentiation and calcification. Given the increasing concern about environmental issues, it is promising to utilize “green” biomaterials to construct novel cell culture scaffolds for bone tissue engineering. Herein, surface-phosphorylated cellulose nanofibers (P-CNFs) were fabricated as a novel green candidate for osteoblast culture. Compared with native CNF, P-CNFs possessed shorter fiber morphology with tunable phosphate group content (0–1.42 mmol/g). The zeta-potential values of CNFs were enhanced after phosphorylation, resulting in the formation of uniform and stable scaffolds. The cell culture behavior of mouse osteoblast (MC3T3-E1) cells showed a clear phosphate content-dependent cell proliferation. The osteoblast cells adhered well and proliferated efficiently on P-CNF0.78 and P-CNF1.05, with phosphate contents of 0.78 and 1.05 mmol/g, respectively, whereas the cells grown on native CNF substrate formed aggregates due to poor cell attachment and exhibited limited cell proliferation. In addition, the P-CNF substrates with optimal phosphate content provided a favorable cellular microenvironment and significantly promoted osteogenic differentiation and calcification, even in the absence of a differentiation inducer. The bio-based P-CNFs are expected to mimic the bone components and provide a means to regulate osteoblast proliferation and differentiation in bone tissue engineering.

Mechanically enhanced and osteobioactive synthetic periosteum via development of poly(ε-caprolactone)/microtantalum composite

Periosteum, the thin layer covering adjacent to bone containing specific architecture, is important for functional bone regeneration and remodeling. Synthetic periosteum investigated presently lacks the resemblance of natural periosteum, suffering from poor mechanical strength and cell attachment. Here, we report a newly-developed biomimetic film to function as synthetic periosteum. Based on poly(ε-caprolactone) (PCL), where surface wettability of the synthetic periosteum is enhanced by microtantalum (mTa) particle blending and after a cold drawing process, further obtains topographical anisotropy without any involvement of solvent. This new blend shows mechanical enhancement over pure PCL, with yield stress and elastic strain approaching the natural periosteum. A distinct degradation mechanism is proposed for the blend, and by seeding with mouse calvarial preosteoblasts, cell proliferation is promoted on surface of the drawn PCL but delayed on the mTa-blended PCL. However, cell mineralization is accelerated on the mTa-blended surface. This is less on the drawn PCL. The synergistical integration of cellular proliferation, alignment and osteogenic enhancement suggest that the cold drawn PCL/Ta blend has unique potential for developing into a synthetic periosteum and other tissue-engineering products.

PCL/Agarose 3D-printed scaffold for tissue engineering applications: fabrication, characterization, and cellular activities.

Biomaterials, scaffold manufacturing, and design strategies with acceptable mechanical properties are the most critical challenges facing tissue engineering. In this study, polycaprolactone (PCL) scaffolds were fabricated through a novel three-dimensional (3D) printing method. The PCL scaffolds were then coated with 2% agarose (Ag) hydrogel. The 3D-printed PCL and PCL/Ag scaffolds were characterized for their mechanical properties, porosity, hydrophilicity, and water absorption. The construction and morphology of the printed scaffolds were evaluated via Fourier-Transform infrared spectroscopy (FTIR) and scanning electron microscopy (SEM). The attachment and proliferation of L929 cells cultured on the scaffolds were investigated through MTT assay on the cell culture study upon the 1st, 3rd, and 7th days. The incorporation of Ag hydrogel with PCL insignificantly decreased the mechanical strength of the scaffold. The presence of Ag enhanced the hydrophilicity and water absorption of the scaffolds, which could positively influence their cell behavior compared to the PCL scaffolds. Regarding cell morphology, the cells on the PCL scaffolds had a more rounded shape and less cell spreading, representing poor cell attachment and cell-scaffold interaction due to the hydrophobic nature of PCL. Conversely, the cells on the PCL/Ag scaffolds were elongated with a spindle-shaped morphology indicating a positive cell-scaffold interaction. PCL/Ag scaffolds can be considered appropriate for tissue-engineering applications.

A Biocompatible Dual‐Sided Hernia Mesh with Side‐Specific Properties

AbstractPolypropylene (PP) based hernia mesh often shows multiple post‐surgery complications due to lack of biocompatibility, poor cell attachment, and unwanted tissue adhesion. These limitations can be addressed by material designing and surface modification of a mesh with side‐specific properties such as the visceral side (facing intestine) with low protein and cell attachment and the parietal side (facing incision) with improved cell attachment properties for normal healing. However, the development of dual‐sided mesh is very challenging because of its porous structure. Herein, a dual‐sided biocompatible mesh with protein anti‐adsorption and cell attachment properties on two different sides is developed by grafting highly hydrophilic 2‐methcryloyloxyethyl phosphorylcholine polymer (PMPC) on the plasma‐activated visceral side, while the parietal side is coated with bioactive chitosan and functionalized nanodiamond (Chi/FND) using a temporary polyvinyl alcohol (PVA) mold. The PMPC‐grafted side demonstrated excellent resistance to protein adsorption (96% reduction compared to PP) and cell attachment. However, the bioactive coating on the parietal side has significantly improved cell attachment and proliferation properties. In addition, both sides confirmed the presence of the respective biomaterials after an accelerated degradation study for 28 days. Hence, the newly developed dual‐sided mesh by semi‐solid polymer mold (SSPM) method is a promising candidate to address the long‐existing multiple issues of hernia mesh.

Coating of 3D printed PCL/TCP scaffolds using homogenized-fibrillated collagen

BackgroundPoly(3-caprolactone) (PCL)/β-tricalcium phosphate (β-TCP) composite scaffolds fabricated by three-dimensional (3D) printing are one of the common scaffolds for bone tissue regeneration. However, the main challenge of these 3D printed PCL/β-TCP scaffolds is the fact that many cells pass from porosities during in vitro cell seeding, leading to poor initial cell attachment. This study aimed to demonstrate the fabrication of a new collagen coating process for optimizing the hydrophilic property and cell-substrate interactions. This method may be used for coating collagen on any relevant biomedical constructs made of synthetic polymers to increase their biocompatibility and cell attachment. Materials and methodsPorous composite scaffolds fabricated by 3D printing were coated with collagen by a novel method and compared to traditional methods. After plasma treatment, samples were inverted in a homogenized collagen solution, freeze-dried, stabilized by crosslinking, freeze-dried again, and fibrillated using a defined salt concentration. Samples were characterized by a 3D laser microscope, cytocompatibility assay, attenuated total reflectance Fourier-transform infrared (ATR-FTIR) spectroscopy, water absorption, protein absorption, and bioactivity assay. ResultsHomogenized collagen at pH= 7 resulted in a very uniform layer on the surface of scaffolds with significantly higher cell proliferation (p < 0.05). Collagen-coated scaffolds showed significantly higher water absorption, protein absorption, and bioactivity compared to non-coated samples (p < 0.05). ConclusionThe results demonstrate that both the pH and collagen structure influence the coating of scaffolds, while the concentrations used in this study do not have a significant difference in this aspect. The combination of homogenization and fibrillization makes scaffolds more biocompatible and desirable for bone tissue engineering.

3D-Printed PCL Scaffolds Coated with Nanobioceramics Enhance Osteogenic Differentiation of Stem Cells.

With advances in bone tissue engineering, various materials and methods have been explored to find a better scaffold that can help in improving bone growth and regeneration. Three-dimensional (3D) printing by fused deposition modeling can produce customized scaffolds from biodegradable polyesters such as polycaprolactone (PCL). Although the fabricated PCL scaffolds exhibited a lack of bioactivity and poor cell attachment on their surfaces, herein, using a simple postfabrication modification method with hydroxyapatite (HA) and bioglasses (BGs), we obtained better cell proliferation and attachment. Biological behavior and osteosupportive capacity of the 3D-printed scaffolds including PCL, PCL/HA, PCL/BG, and PCL/HA/BG were evaluated in this study, while human adipose tissue-derived mesenchymal stem cells (hADSCs) were cultured on the scaffolds. The cell morphology, attachment, and proliferation were investigated using scanning electron microscopy (SEM), 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl tetrazolium bromide (MTT) assay, and 4′,6-diamidino-2-phenylindole (DAPI) staining. In the next step, the ability of stem cells to differentiate into osteoblasts was evaluated by measuring alkaline phosphatase (ALP) activity, calcium deposition, and bone-related gene and protein expression. In the end, the expression levels of miR-20a, miR-125a, and their target genes were also investigated as positive and negative regulators in osteogenesis pathways. The results showed that the coated scaffolds with bioceramics present a more appropriate surface for cell adhesion and proliferation, as well as efficient potential in inducing osteoconduction and osteointegration compared to PCL alone and control. The PCL/HA/BG scaffold exhibited higher in vitro cell viability and bone formation compared to the other groups, which can be due to the synergistic effect of HA and BG. On the whole, this tricomponent 3D-printing scaffold has a promising prospect for bone tissue engineering applications.

StemBond hydrogels control the mechanical microenvironment for pluripotent stem cells

Studies of mechanical signalling are typically performed by comparing cells cultured on soft and stiff hydrogel-based substrates. However, it is challenging to independently and robustly control both substrate stiffness and extracellular matrix tethering to substrates, making matrix tethering a potentially confounding variable in mechanical signalling investigations. Moreover, unstable matrix tethering can lead to poor cell attachment and weak engagement of cell adhesions. To address this, we developed StemBond hydrogels, a hydrogel in which matrix tethering is robust and can be varied independently of stiffness. We validate StemBond hydrogels by showing that they provide an optimal system for culturing mouse and human pluripotent stem cells. We further show how soft StemBond hydrogels modulate stem cell function, partly through stiffness-sensitive ERK signalling. Our findings underline how substrate mechanics impact mechanosensitive signalling pathways regulating self-renewal and differentiation, indicating that optimising the complete mechanical microenvironment will offer greater control over stem cell fate specification.

Characterizing the Effects of Synergistic Thermal and Photo-Cross-Linking during Biofabrication on the Structural and Functional Properties of Gelatin Methacryloyl (GelMA) Hydrogels.

Gelatin methacryloyl (GelMA) hydrogels have emerged as promising and versatile biomaterial matrices with applications spanning drug delivery, disease modeling, and tissue engineering and regenerative medicine. GelMA exhibits reversible thermal cross-linking at temperatures below 37 °C due to the entanglement of constitutive polymeric chains, and subsequent ultraviolet (UV) photo-cross-linking can covalently bind neighboring chains to create irreversibly cross-linked hydrogels. However, how these cross-linking modalities interact and can be modulated during biofabrication to control the structural and functional characteristics of this versatile biomaterial is not well explored yet. Accordingly, this work characterizes the effects of synergistic thermal and photo-cross-linking as a function of GelMA solution temperature and UV photo-cross-linking duration during biofabrication on the hydrogels' stiffness, microstructure, proteolytic degradation, and responses of NIH 3T3 and human adipose-derived stem cells (hASC). Smaller pore size, lower degradation rate, and increased stiffness are reported in hydrogels processed at lower temperature or prolonged UV exposure. In hydrogels with low stiffness, the cells were found to shear the matrix and cluster into microspheroids, while poor cell attachment was noted in high stiffness hydrogels. In hydrogels with moderate stiffness, ones processed at lower temperature demonstrated better shape fidelity and cell proliferation over time. Analysis of gene expression of hASC encapsulated within the hydrogels showed that, while the GelMA matrix assisted in maintenance of stem cell phenotype (CD44), a higher matrix stiffness resulted in higher pro-inflammatory marker (ICAM1) and markers for cell-matrix interaction (ITGA1 and ITGA10). Analysis of constructs with ultrasonically patterned hASC showed that hydrogels processed at higher temperature possessed lower structural fidelity but resulted in more cell elongation and greater anisotropy over time. These findings demonstrate the significant impact of GelMA material formulation and processing conditions on the structural and functional properties of the hydrogels. The understanding of these material-process-structure-function interactions is critical toward optimizing the functional properties of GelMA hydrogels for different targeted applications.

Microporosity mediated proliferation of preosteoblast cells on 3D printed bone scaffolds

AbstractMicroporous structure plays a significant role in bone tissue engineering, to influence inductive bone formation and elevate bone ingrowth inside microporous scaffolds. We hereby fabricated microporous scaffolds by 3D printing with mixture of porogens and photopolymerizing resin. Our method can tune the microporosity of scaffolds from 0% to 36% by varying the porogen concentration in the inks from zero to 60 vol%. The microporosity and microspore size of scaffolds can affect in vitro expansion of preosteoblast cells. We evaluated the attachment, spreading and proliferation of MC3T3‐E1 mouse preosteoblast cells on the printed porous scaffolds. Our studies revealed that preosteoblast cells’ in vitro adhesion and proliferation were significantly mediated by the printed scaffolds. Cells proliferation on scaffolds with 20%–30% microporosity showed much higher rate than on other scaffolds. In this microporosity range, scaffolds also showed much better cell spreading and morphologies. At a low level of microporosity (&lt; 7%), the cell proliferation rate was even lower than the solid scaffolds, which indicates that the microporous structure is “toxic” for cells at low microporosity range. A higher microporosity (&gt; 35%) led to very poor cell attachment and is unfavorable for the proliferation of MC3T3‐E1 cells. Furthermore, the printed dual macro‐/micro‐porous scaffolds showed higher proliferation rate of MC3T3‐E1 cells as compared to mono‐pore sized scaffolds, either the macro‐porous or microporous structure.

Recent Trends in Three-Dimensional Bioinks Based on Alginate for Biomedical Applications.

Three-dimensional (3D) bioprinting is an appealing and revolutionary manufacturing approach for the accurate placement of biologics, such as living cells and extracellular matrix (ECM) components, in the form of a 3D hierarchical structure to fabricate synthetic multicellular tissues. Many synthetic and natural polymers are applied as cell printing bioinks. One of them, alginate (Alg), is an inexpensive biomaterial that is among the most examined hydrogel materials intended for vascular, cartilage, and bone tissue printing. It has also been studied pertaining to the liver, kidney, and skin, due to its excellent cell response and flexible gelation preparation through divalent ions including calcium. Nevertheless, Alg hydrogels possess certain negative aspects, including weak mechanical characteristics, poor printability, poor structural stability, and poor cell attachment, which may restrict its usage along with the 3D printing approach to prepare artificial tissue. In this review paper, we prepare the accessible materials to be able to encourage and boost new Alg-based bioink formulations with superior characteristics for upcoming purposes in drug delivery systems. Moreover, the major outcomes are discussed, and the outstanding concerns regarding this area and the scope for upcoming examination are outlined.

Functionalisation of a heat-derived and bio-inert albumin hydrogel with extracellular matrix by air plasma treatment

Albumin-based hydrogels are increasingly attractive in tissue engineering because they provide a xeno-free, biocompatible and potentially patient-specific platform for tissue engineering and drug delivery. The majority of research on albumin hydrogels has focused on bovine serum albumin (BSA), leaving human serum albumin (HSA) comparatively understudied. Different gelation methods are usually employed for HSA and BSA, and variations in the amino acid sequences of HSA and BSA exist; these account for differences in the hydrogel properties. Heat-induced gelation of aqueous HSA is the easiest method of synthesizing HSA hydrogels however hydrogel opacity and poor cell attachment limit their usefulness in downstream applications. Here, a solution to this problem is presented. Stable and translucent HSA hydrogels were created by controlled thermal gelation and the addition of sodium chloride. The resulting bio-inert hydrogel was then subjected to air plasma treatment which functionalised its surface, enabling the attachment of basement membrane matrix (Geltrex). In vitro survival and proliferation studies of foetal human osteoblasts subsequently demonstrated good biocompatibility of functionalised albumin hydrogels compared to untreated samples. Thus, air plasma treatment enables functionalisation of inert heat-derived HSA hydrogels with extracellular matrix proteins and these may be used as a xeno-free platform for biomedical research or cell therapy.

Ammonium hydroxide modification of polylactic acid and polyglycolic acid monofilaments for acupoint catgut embedding therapy

Polylactic acid (PLA) and polyglycolic acid (PGA) monofilaments have attracted much attention for their wide usage in acupoint catgut embedding therapy (ACET). Their application is restricted, however, by their poor hydrophilicity and cell attachment properties. In this study, PLA and PGA monofilaments were produced from polymer chips, and then modified by the application of an ammonium hydroxide solution to the surface. The modified PLA and PGA monofilaments were fully characterized with respect to their structural, mechanical, and in-vitro properties. The results showed that the surface roughness and hydrophilicity of the materials were greatly increased; surface modified samples of both materials exhibited the smallest contact angle values: 79.2° ± 2.5° (sample PLA2) and 75.9° ± 1.4° (sample PGA2). The weights and diameters and the tensile and flexibility properties of the materials changed little with surface modification, but their swelling ratios increased significantly. All the prepared samples were non-toxic (more than 75% of cells being viable). Surface modification also enhanced cell attachment: PLA2 (48.15% ± 2.16%) and PGA2 (59.43% ± 3.18%) showed the largest cell attachment values (cultured for 48 h) among the samples. In summary, the study proves the feasibility of ammonium hydroxide modification of PLA and PGA, which is beneficial for guiding future work on developing functional PLA or PGA materials for ACET.

Co–Cr–Mo alloy binding peptide as molecular glue for constructing biomedical surfaces

The mechanical properties of Co–Cr–Mo (CCM) alloys are advantageous in various biomedical applications. However, because of their bioinert surface, CCM alloys exhibit poor endothelial cell attachment properties; thus, problems of biocompatibility remain. In this study, we aimed to improve the biocompatibility of the CCM alloy surface using solid-binding peptides. We selected peptides with high binding affinity for cast CCM alloy surfaces through in vitro evolution by the phage display method. The peptides were functionalized on the CCM alloy surfaces by simple immersion in the peptide solution. The peptide bound to both cast and 3D-printed CCMs with the same affinity. The peptides linked to the amino acid motif that promotes cell adhesion, and improved the attachment of endothelial cells on the 3D-printed CCM in serum and serum-free conditions. Hence, CCM-binding peptides are attractive tools for constructing a biofunctional surface on CCM-based biodevices.

Improving cell distribution on 3D additive manufactured scaffolds through engineered seeding media density and viscosity

In order to ensure the long-term in vitro and in vivo functionality of cell-seeded 3D scaffolds, an effective and reliable method to control cell seeding efficiency and distribution is crucial. Static seeding on 3D additive manufactured scaffolds made of synthetic polymers still remains challenging, as it often results in poor cell attachment, high cell sedimentation and non-uniform cell distribution, due to gravity and to the intrinsic macroporosity and surface chemical properties of the scaffolds. In this study, the biocompatible macromolecules dextran and Ficoll (Ficoll-Paque) were used for the first time as temporary supplements to alter the viscosity and density of the seeding media, respectively, and improve the static seeding output. The addition of these macromolecules drastically reduced the cell sedimentation velocities, allowing for homogeneous cell attachment to the scaffold filaments. Both dextran and Ficoll-Paque -based seeding methods supported human mesenchymal stromal cells viability and osteogenic differentiation post-seeding. Interestingly, the improved cell distribution led to increased matrix production and mineralization compared to scaffolds seeded by conventional static method. These results suggest a simple and universal method for an efficient seeding of 3D additive manufactured scaffolds, independent of their material and geometrical properties, and applicable for bone and various other tissue regeneration. Statement of significanceAdditive manufacturing has emerged as one of the desired technologies to fabricate complex and patient-specific 3D scaffolds for bone regeneration. Along with the technology, new synthetic polymeric materials have been developed to meet processability requirements, as well as the mechanical properties and biocompatibility necessary for the application. Yet, there is still lack of methodology for a universal cell seeding method applicable to all additive manufactured 3D scaffolds regardless of their characteristics. We believe that our simple and reliable method, which is based on adjusting the cell settling velocity to aid cell attachment, could potentially help to maximize the efficiency, and therefore, functionality of cell-seeded constructs. This is of great importance when aiming for both in vitro and future clinical applications.

Hydroxyapatite nano bioceramics optimized 3D printed poly lactic acid scaffold for bone tissue engineering application

To achieve optimum functionality and mechanical properties of advanced manufacturing-based scaffolds for biomedical application, it is important to study their mechanical strength by 3D-printing at different orientations. This study examined the effects of printing at different orientations on the mechanical properties of synthesized 3D-polylactic acid (PLA) and hydroxyapatite-modified PLA (PLA-HAp) scaffolds. A total number of 30 samples were printed in three orientations on the XY plane: 0°, 45°, and 90°. Finite element modeling and simulation was employed to identify the strongest scaffold in terms of compression strength, which is the primary criterion for load bearing bone tissue scaffolds. These findings indicate that 3D-printing at an orientation of 90° on the XY plane resulted in a scaffold with the highest compression strength. Moreover, the fabricated PLA scaffolds showed very poor cell attachment and proliferation on their surface, which is not suitable for their biomedical application. This study additionally showed the optimization of a very simple post-fabrication modification technique with nano HAp for better cell attachment and proliferation with enhanced mechanical properties. The post-fabrication modification of PLA scaffolds by nano-HAp results in excellent cell attachment property with enhanced mechanical strength and stability of up to 47.16% for 90° 3D-printed PLA-HAp scaffolds.

GPTMS-Modified Bredigite/PHBV Nanofibrous Bone Scaffolds with Enhanced Mechanical and Biological Properties.

Bioceramic nanoparticles with high specific surface area often tend to agglomerate in the polymer matrix, which results in undesirable mechanical properties of the composites and poor cell spreading and attachment. In the present work, bredigite (BR) nanoparticles were modified with an organosilane coupling agent, 3-glycidoxypropyltrimethoxysilane (GPTMS), to enhance its dispersibility in the polymer matrix. The polyhydroxybutyrate-co-hydroxyvaletare (PHBV) nanofibrous scaffolds containing either bredigite or GPTMS-modified bredigite (G-BR) nanoparticles were fabricated using electrospinning technique and characterized using scanning electron microscopy, transmission electron microscopy, and tensile strength. Results demonstrated that modification of bredigite was effective in enhancing nanoparticle dispersion in the PHBV matrix. PHBV/G-BR scaffold showed improved mechanical properties compared to PHBV and PHBV/BR, especially at the higher concentration of nanoparticles. In vitro bioactivity assay performed in the simulated body fluid (SBF) indicated that composite PHBV scaffolds were able to induce the formation of apatite deposits after incubation in SBF. From the results of in vitro biological assay, it is concluded that the synergetic effect of BR and GPTMS provided an enhanced hFob cells attachment and proliferation. The developed PHBV/G-BR nanofibrous scaffolds may be considered for application in bone tissue engineering.

Tunable Volumetric Density and Porous Structure of Spherical Poly-ε-caprolactone Microcarriers, as Applied in Human Mesenchymal Stem Cell Expansion.

Polymeric microspheres may serve as microcarrier (MC) matrices, for the expansion of anchorage-dependent stem cells. They require surface properties that promote both initial cell adhesion and the subsequent spreading of cells, which is a prerequisite for successful expansion. When implemented in a three-dimensional culture environment, under agitation, their suspension under low shear rates depends on the MCs having a modest negative buoyancy, with a density of 1.02-1.05 g/cm3. Bioresorbable poly-ε-caprolactone (PCL), with a density of 1.14 g/cm3, requires a reduction in volumetric density, for the microspheres to achieve high cell viability and yields. Uniform-sized droplets, from solutions of PCL dissolved in dichloromethane (DCM), were generated by coaxial microfluidic geometry. Subsequent exposure to ethanol rapidly extracted the DCM solvent, solidifying the droplets and yielding monodisperse microspheres with a porous structure, which was demonstrated to have tunable porosity and a hollow inner core. The variation in process parameters, including the molecular weight of PCL, its concentration in DCM, and the ethanol concentration, served to effectively alter the diffusion flux between ethanol and DCM, resulting in a broad spectrum of volumetric densities of 1.04-1.11 g/cm3. The solidified microspheres are generally covered by a smooth thin skin, which provides a uniform cell culture surface and masks their internal porous structure. When coated with a cationic polyelectrolyte and extracellular matrix protein, monodisperse microspheres with a diameter of approximately 150 μm and densities ranging from 1.05-1.11 g/cm3 are capable of supporting the expansion of human mesenchymal stem cells (hMSCs). Validation of hMSC expansion was carried out with a positive control of commercial Cytodex 3 MCs and a negative control of uncoated low-density PCL MCs. Static culture conditions generated more than 70% cell attachment and similar yields of sixfold cell expansion on all coated MCs, with poor cell attachment and growth on the negative control. Under agitation, coated porous microspheres, with a low density of 1.05 g/cm3, achieved robust cell attachment and resulted in high cell yields of ninefold cell expansion, comparable with those generated by commercial Cytodex 3 MCs.

Bi-layered constructs of poly(glycerol-sebacate)-β-tricalcium phosphate for bone-soft tissue interface applications

This study aims to establish a facile protocol for the preparation of a bi-layered poly(glycerol-sebacate) (PGS)/β-tricalcium phosphate (β-TCP) construct and to investigate its potential for bone-soft tissue engineering applications. The layered structure was prepared by distributing the ceramic particles within a prepolymer synthesized in a microwave reactor followed by a cross-linking of the final construct in vacuum (<10mbar). The vacuum stage led to the separation of cross-linked elastomer (top) and ceramic (bottom) phases. Results showed that addition of β-TCP particles to the elastomer matrix after the polymerization led to an increase in compression strength (up to 14±2.3MPa). Tensile strength (σ), Young's modulus (E), and elongation at break (%) values were calculated as 0.29±0.03MPa and 0.21±0.03; 0.38±0.02 and 1.95±0.4; and 240±50% and 24±2% for PGS and PGS/β-TCP bi-layered constructs, respectively. Morphology was characterized by using Scanning Electron Microscopy (SEM) and micro-computed tomography (μ-CT). Tomography data revealed an open porosity of 35% for the construct, mostly contributed from the ceramic phase since the elastomer side has no pore. Homogeneous β-TCP distribution within the elastomeric structure was observed. Cell culture studies confirmed biocompatibility with poor elastomer-side and good bone-side cell attachment. In a further study to investigate the osteogenic properties, the construct were loaded with BMP-2 and/or TGF-β1. The PGS/β-TCP bi-layered constructs with improved mechanical and biological properties have the potential to be used in bone-soft tissue interface applications where soft tissue penetration is a problem.

Cell sheets prepared via gel-sol transition of calcium RGD-Alginate

Cell sheets prepared via gel-sol transition of calcium RGD-Alginate