MgCa-Based Alloys Modified with Zn- and Ga-Doped CaP Coatings Lead to Controlled Degradation and Enhanced Bone Formation in a Sheep Cranium Defect Model.

Mg-based biodegradable metallic implants are gaining increased attraction for applications in orthopedics and dentistry. However, their current applications are hampered by their high rate of corrosion, degradation, and rapid release of ions and gas bubbles into the physiological medium. The aim of the present study is to investigate the osteogenic and angiogenic potential of coated Mg-based implants in a sheep cranial defect model. Although their osteogenic potential was studied to some extent, their potential to regenerate vascularized bone formation was not studied in detail. We have studied the potential of magnesium-calcium (MgCa)-based alloys modified with zinc (Zn)- or gallium (Ga)-doped calcium phosphate (CaP) coatings as a strategy to control their degradation rate while enhancing bone regeneration capacity. MgCa and its implants with CaP coatings (MgCa/CaP) as undoped or as doped with Zn or Ga (MgCa/CaP + Zn and MgCa/CaP + Ga, respectively) were implanted in bone defects created in the sheep cranium. MgCa implants degraded faster than the others at 4 weeks postop and the weight loss was ca. 50%, while it was ca. 15% for MgCa/CaP and <10% in the presence of Zn and Ga with CaP coating. Scanning electron microscopy (SEM) analysis of the implant surfaces also revealed that the MgCa implants had the largest degree of structural breakdown of all the groups. Radiological evaluation revealed that surface modification with CaP to the MgCa implants induced better bone regeneration within the defects as well as the enhancement of bone-implant surface integration. Bone volume (%) within the defect was ca. 25% in the case of MgCa/CaP + Ga, while it was around 15% for undoped MgCa group upon micro-CT evaluation. This >1.5-fold increase in bone regeneration for MgCa/CaP + Ga implant was also observed in the histopathological examination of the H&E- and Masson's trichrome-stained sections. Immunohistochemical analysis of the bone regeneration (antiosteopontin) and neovascularization (anti-CD31) at the defect sites revealed >2-fold increase in the expression of the markers in both Ga- and Zn-doped, CaP-coated implants. Zn-doped implants further presented low inflammatory reaction, notable bone regeneration, and neovascularization among all the implant groups. These findings indicated that Ga- and Zn-doped CaP coating is an important strategy to control the degradation rate as well as to achieve enhanced bone regeneration capacity of the implants made of Mg-based alloys.

Advances in In Vitro Blood-Air Barrier Models and the Use of Nanoparticles in COVID-19 Research.

Respiratory infections caused by coronaviruses (CoVs) have become a major public health concern in the past two decades as revealed by the emergence of SARS-CoV in 2002, MERS-CoV in 2012, and SARS-CoV-2 in 2019. The most severe clinical phenotypes commonly arise from exacerbation of immune response following the infection of alveolar epithelial cells localized at the pulmonary blood-air barrier. Preclinical rodent models do not adequately represent the essential genetic properties of the barrier, thus necessitating the use of humanized transgenic models. However, existing monolayer cell culture models have so far been unable to mimic the complex lung microenvironment. In this respect, air-liquid interface models, tissue engineered models, and organ-on-a-chip systems, which aim to better imitate the infection site microenvironment and microphysiology, are being developed to replace the commonly used monolayer cell culture models, and their use is becoming more widespread every day. On the contrary, studies on the development of nanoparticles (NPs) that mimic respiratory viruses, and those NPs used in therapy are progressing rapidly. The first part of this review describes in vitro models that mimic the blood-air barrier, the tissue interface that plays a central role in COVID-19 progression. In the second part of the review, NPs mimicking the virus and/or designed to carry therapeutic agents are explained and exemplified.

Development of a Tri-Layered Vascular Construct and In Vitro Evaluation of Endothelization.

Advances in the development of vascular substitutes for small-sized arteries are ongoing because the present grafts do not entirely meet the requirements of native equivalents and are suboptimal in clinical performance. This study aims to develop a tri-layered vascular construct mimicking natural tissue using polyester blends and to investigate its endothelization through in vitro studies as a potential small-caliber vascular graft. The innermost layer is obtained by dip coating as a tubular porous film with a lumen diameter of 3 mm and a pore size of ≤8 µm. Circumferentially aligned electrospun fiber (diameter 100-800 nm) with a deviation angle of 15° are deposited over the porous film forming the intermediate layer. The random electrospun fibers (diameter 100-1100 nm) deviating at different angles are wrapped as the outermost layer. The mechanical properties of the tri-layered vascular construct are determined to be 44.80 ± 14.80 MPa for Young's modulus and 4.25 ± 0.75 MPa for ultimate tensile strength. MTS and cell behavior studies show that the isolated human umbilical cord vein endothelial cells proliferate and line the lumen of the vascular substitute. The vascular construct developed, with its biomimetic architecture, mechanical features, size, and endothelization, can be tested with in vivo studies.

Is 3D Printing Promising for Osteochondral Tissue Regeneration?

Osteochondral tissue regeneration is quite difficult to achieve due to the complexity of its organization. In the design of these complex multilayer structures, a fabrication method, 3D printing, started to be employed, especially by using extrusion, stereolithography and inkjet printing approaches. In this paper, the designs are discussed including biphasic, triphasic, and gradient structures which aim to mimic the cartilage and the calcified cartilage and the whole osteochondral tissue closely. In the first section of the review paper, 3D printing of hydrogels including gelatin methacryloyl (GelMa), alginate, and polyethylene glycol diacrylate (PEGDA) are discussed. However, their physical and biological properties need to be augmented, and this generally is achieved by blending the hydrogel with other, more durable, less hydrophilic, polymers. These scaffolds are very suitable to carry growth factors, such as TGF-ß1, to further stimulate chondrogenesis. The bone layer is mimicked by printing calcium phosphates (CaPs) or bioactive glasses together with the hydrogels or as a component of another polymer layer. The current research findings indicate that polyester (i.e. polycaprolactone (PCL), polylactic acid (PLA) and poly(lactide-co-glycolide) (PLGA)) reinforced hydrogels may more successfully mimic the complex structure of osteochondral tissue. Moreover, more recent printing methods such as melt electrowriting (MEW), are being used to integrate polyester fibers to enhance the mechanical properties of hydrogels. Additionally, polyester scaffolds that are 3D printed without hydrogels are discussed after the hydrogel-based scaffolds. In this review paper, the relevant studies are analyzed and discussed, and future work is recommended with support of tables of designed scaffolds. The outcome of the survey of the field is that 3D printing has significant potential to contribute to osteochondral tissue repair.

Nuclear Deformability of Breast Cells Analyzed from Patients with Malignant and Benign Breast Diseases.

Breast cancer is a heterogeneous and dynamic disease, in which cancer cells are highly responsive to alterations in the microenvironment. Today, conventional methods of detecting cancer give a rather static image of the condition of the disease, so dynamic properties such as invasiveness and metastasis are difficult to capture. In this study, conventional molecular-level evaluations of the patients with breast adenocarcinoma were combined with in vitro methods on micropatterned poly(methyl methacrylate) (PMMA) biomaterial surfaces that deform cells. A correlation between deformability of the nuclei and cancer stemness, invasiveness, and metastasis was sought. Clinical patient samples were from regions of the breast with different proximities to the tumor. Responses at the single-cell level toward the micropatterned surfaces were studied using CD44/24, epithelial cell adhesion marker (EpCAM), MUC1, and PCK. Results showed that molecular markers and shape descriptors can discriminate the cells from different proximities to the tumor center and from different patients. The cells with the most metastatic and invasive properties showed both the highest deformability and the highest level of metastatic markers. In conclusion, by using a combination of molecular markers together with nuclear deformation, it is possible to improve detection and separation of subpopulations in heterogenous breast cancer specimens at the single-cell level.

Zn doped CaP coatings used for controlling the degradation rate of MgCa1 alloy: In vitro anticorrosive properties, sterilization and bacteria/cell-material interactions.

The purpose of this study was to investigate the effect of Zn doped CaP coatings prepared by micro-arc oxidation method, as a possible approach to control MgCa1 alloy degradation. All the prepared coatings comprised a calcium deficient CaP phase. The control in this evaluation was performed with undoped CaP coating in SBF solution at body temperature (37 ± 0.5°C). The investigation involved determination of microchemical, mechanical, morphological, properties along with anticorrosive, cytocompatibility and antibacterial efficacy. The effect of sterilization process on the properties of the surfaces was also investigated. The results showed that the addition of Zn into CaP increased the corrosion resistance of MgCa1 alloy. Moreover, the adhesion strength of the coatings to MgCa1 alloy was enhanced by Zn addition. In cytotoxicity testing of the samples, extracts of the samples in MEM were incubated with L929 cells and malformation, degeneration and lysis of the cells were examined microscopically after 72 h. The results showed that all samples were cytocompatible. The degradation of MgCa1 alloy in the simulated body fluids (SBF) or DMEM was decreased by coating with CaP. Moreover, the degradation rate of CaP was further decreased by adding a small amount of Zn into the CaP matrix. The samples having CaP coatings and Zn doped CaP coating demonstrated antibacterial efficacy against E.coli. As a result, coating of magnesium alloy with Zn-doped CaP decreased the degradation rate, increased the corrosion resistance, cytocompatibility and the antibacterial effects of the alloys.

Injectable methacrylated gelatin/thiolated pectin hydrogels carrying melatonin/tideglusib-loaded core/shell PMMA/silk fibroin electrospun fibers for vital pulp regeneration.

Use of injectable hydrogels attract attention in the regeneration of dental pulp due to their ability to fill non-uniform voids such as pulp cavities. Here, gelatin methacrylate/thiolated pectin hydrogels (GelMA/PecTH) carrying electrospun core/shell fibers of melatonin (Mel)-polymethylmethacrylate (PMMA)/Tideglusib (Td)-silk fibroin (SF) were designed as an injectable hydrogel for vital pulp regeneration, through prolonged release of Td and Mel to induce proliferation and odontoblastic differentiation of dental pulp stem cells (DPSC). H NMR and FTIR confirmed methacrylation of Gel and thiolation of Pec. Addition of PMMA/SF increased degradation and water retention capacities of GelMA/PecTH. Rheological analyses and syringe tests proved the injectability of the hydrogel systems. Release studies indicated that Td and Mel were released from the fibers inside the hydrogels sequentially due to their specific locations. This release pattern from the hydrogels resulted in DPSC proliferation and odontogenic differentiation in vitro. Gene expression studies showed that the upregulation of DMP1, DSPP, and Axin-2 genes was promoted by GelMA/PecTH carrying PMMA/SF loaded with Mel (50 µg/mL) and Td (200 nM), respectively. Our results suggest that this hydrogel system holds promise for use in the regeneration of pulp tissue.

Coaxial electrospinning of composite mats comprised of core/shell poly(methyl methacrylate)/silk fibroin fibers for tissue engineering applications.

Mimicking extracellular matrix (ECM) of native tissue by tissue-engineered constructs is critical to induce regeneration of the damaged site. In this study, coaxial electrospinning of core/shell poly(methyl methacrylate) (PMMA)/silk fibroin (SF) fibers was optimized for the first time to provide ECM-like microenvironment for new tissue formation by utilization of a new collector design for obtaining homogeneously deposited mats from the collector screen. SF-shell was produced to increase cell-affinity of fiber surfaces whereas PMMA-core was designed to support the tissue mechanically during regeneration. PMMA/SF membranes were characterized. Morphology of core/shell PMMA/SF fibers resembled neat SF (ribbon-like) fibers rather than neat PMMA (cylindrical) fibers since SF constituted the shell part. The average diameter of PMMA/SF fibers (2.51 µm) lied in between the neat counterparts (PMMA: 2.40 µm and SF: 2.84 µm). The morphological and chemical properties affected the water contact angle and porosity of the mats, leading to the highest hydrophilicity for SF mats and the highest porosity for PMMA mats among the groups. X-ray photoelectron spectroscopy (XPS) and Fourier transform infrared spectroscopy (FTIR) confirmed the core/shell structure of PMMA/SF fibers. The combination of these remarkably different polymers (synthetic, hydrophobic, brittle PMMA and natural, hydrophilic, flexible SF) resulted in intermediate mechanical properties of PMMA/SF mats both in dry and wet conditions by preserving fibrous and porous structures in the core/shell form unlike the neat mats. Thermogravimetric analyses (TGA) showed the highest mass loss for PMMA/SF mats which lost 13.9% of their initial weight unlike the neat counterparts. In vitro hydrolytic & enzymatic degradation studies revealed that PMMA/SF had the weight loss between those observed for SF and PMMA mats in the presence and absence of enzymes while possessing the highest water uptake capacity. SEM examinations of mats after 14 days of hydrolytic degradation demonstrated the SF-shell of the fibers were fused at the intercept points of the PMMA/SF network while the PMMA-core acted as a separating backbone and preserved fibrous, and hence porous architecture of the mats. Cell culture studies demonstrated that human dental pulp stem cells (DPSC) were able to attach and proliferated on PMMA/SF mats while a lower degree of cell spreading on PMMA mats was observed. DPSC adhesion was improved by SF-shell in PMMA/SF group. In conclusion, electrospun composite mats composed of core/shell PMMA/SF fibers could be considered a promising candidate for tissue engineering applications and drug delivery strategies.

A Cell Culture Chip with Transparent, Micropillar-Decorated Bottom for Live Cell Imaging and Screening of Breast Cancer Cells.

In the recent years, microfabrication technologies have been widely used in cell biology, tissue engineering, and regenerative medicine studies. Today, the implementation of microfabricated devices in cancer research is frequent and advantageous because it enables the study of cancer cells in controlled microenvironments provided by the microchips. Breast cancer is one of the most common cancers in women, and the way breast cancer cells interact with their physical microenvironment is still under investigation. In this study, we developed a transparent cell culture chip (Ch-Pattern) with a micropillar-decorated bottom that makes live imaging and monitoring of the metabolic, proliferative, apoptotic, and morphological behavior of breast cancer cells possible. The reason for the use of micropatterned surfaces is because cancer cells deform and lose their shape and acto-myosin integrity on micropatterned substrates, and this allows the quantification of the changes in morphology and through that identification of the cancerous cells. In the last decade, cancer cells were studied on micropatterned substrates of varying sizes and with a variety of biomaterials. These studies were conducted using conventional cell culture plates carrying patterned films. In the present study, cell culture protocols were conducted in the clear-bottom micropatterned chip. This approach adds significantly to the current knowledge and applications by enabling low-volume and high-throughput processing of the cell behavior, especially the cell-micropattern interactions. In this study, two different breast cancer cell lines, MDA-MB-231 and MCF-7, were used. MDA-MB-231 cells are invasive and metastatic, while MCF-7 cells are not metastatic. The nuclei of these two cell types deformed to distinctly different levels on the micropatterns, had different metabolic and proliferation rates, and their cell cycles were affected. The Ch-Pattern chips developed in this study proved to have significant advantages when used in the biological analysis of live cells and highly beneficial in the study of screening breast cancer cell-substrate interactions in vitro.

In vitro evaluation of injectable Tideglusib-loaded hyaluronic acid hydrogels incorporated with Rg1-loaded chitosan microspheres for vital pulp regeneration.

Injectable systems receive attention in endodontics due to the complicated and irregular anatomical structure of root canals. Here, injectable Tideglusib (Td)-loaded hyaluronic acid hydrogels (HAH) incorporated with Rg1-loaded chitosan microspheres (CSM) were developed for vital pulp regeneration, providing release of Td and Rg1 to trigger odontoblastic differentiation of human dental pulp stem cells (DPSC) by Td and vascularization of pulp by Rg1. The optimal concentrations were determined as 90 nM and 50 µg/mL for Td and Rg1, and loaded in HA and CSM in HAH, respectively. Odontogenic (COL1A1, ALP, OCN, Axin-2, DSPP, and DMP1) and angiogenic (VEGFA, VEGFR2, and eNOS) differentiation of DPSC cultured in the presence of hydrogels was shown at gene expression level. Our results suggest that our injectable hydrogel formulation has potential to improve strategies for vital pulp regeneration. In vivo evaluations are needed to test the feasibility and potential of these hydrogels for vital pulp regeneration.

Corrosion Resistance and Cytocompatibility of Magnesium-Calcium Alloys Modified with Zinc- or Gallium-Doped Calcium Phosphate Coatings.

In orthopedic surgery, metals are preferred to support or treat damaged bones due to their high mechanical strength. However, the necessity for a second surgery for implant removal after healing creates problems. Therefore, biodegradable metals, especially magnesium (Mg), gained importance, although their extreme susceptibility to galvanic corrosion limits their applications. The focus of this study was to control the corrosion of Mg and enhance its biocompatibility. For this purpose, surfaces of magnesium-calcium (MgCa1) alloys were modified with calcium phosphate (CaP) or CaP doped with zinc (Zn) or gallium (Ga) via microarc oxidation. The effects of surface modifications on physical, chemical, and mechanical properties and corrosion resistance of the alloys were studied using surface profilometry, goniometry, scanning electron microscopy (SEM), energy-dispersive X-ray spectroscopy (EDS), nanoindentation, and electrochemical impedance spectroscopy (EIS). The coating thickness was about 5-8 µm, with grain sizes of 43.1 nm for CaP coating and 28.2 and 58.1 nm for Zn- and Ga-doped coatings, respectively. According to EIS measurements, the capacitive response (Yc) decreased from 11.29 to 8.72 and 0.15 Ω-1 cm-2 sn upon doping with Zn and Ga, respectively. The Ecorr value, which was -1933 mV for CaP-coated samples, was found significantly electropositive at -275 mV for Ga-doped ones. All samples were cytocompatible according to indirect tests. In vitro culture with Saos-2 cells led to changes in the surface compositions of the alloys. The numbers of cells attached to the Zn-doped (2.6 × 104 cells/cm2) and Ga-doped (6.3 × 104 cells/cm2) coatings were higher than that on the surface of the undoped coating (1.0 × 103 cells/cm2). Decreased corrosivity and enhanced cell affinity of the modified MgCa alloys (CaP coated and Zn and Ga doped, with Ga-doped ones having the greatest positive effect) make them novel and promising candidates as biodegradable metallic implant materials for the treatment of bone damages and other orthopedic applications.

Fabrication of a 3D Printed PCL Nerve Guide: In Vitro and In Vivo Testing.

Nerve guides are medical devices designed to guide proximal and distal ends of injured peripheral nerves in order to assist regeneration of the damaged nerves. A 3D-printed polycaprolactone (PCL) nerve guide using an aligned gelatin-poly(3-hydroxybutyrate-co-3-hydroxyvalerate) electrospun mat, seeded with PC12 and Schwann cells (SCs) is produced. During characterization with microCT and SEM porosity (55%), pore sizes (675 ± 40 µm), and fiber diameters (382 ± 25 µm) are determined. Electrospun fibers have degree of alignment of 7°, indicating high potential for guidance. On Day 14, PC12 cells migrated from proximal to distal end of nerve guide when SCs are seeded on the guide. After 28 days, over 95% of PC12 are alive and aligned. PC12 cells express early differentiation marker beta-tubulin 10 times more than late marker NeuN. In a 10 mm rat sciatic nerve injury, functional recovery evaluated by using static sciatic index (SSI) is observed in mat-free guides and guides containing mat and SCs. Nerve conduction velocities are also improved in these groups. Histological stainings showed tissue growth around nerve guides with highest new tissue organization being observed with mat and cell-free guides. These suggest 3D-printed PCL nerve guides have significant potential for treatment of peripheral nerve injuries.

The role of biomaterials and scaffolds in immune responses in regenerative medicine: macrophage phenotype modulation by biomaterial properties and scaffold architectures.

Scaffolds are an integral part of the regenerative medicine field. The contact of biomaterials with tissue, as was clearly observed over the years, induces immune reactions in a material and patient specific manner, where both surface and bulk properties of scaffolds, together with their 3D architecture, have a significant influence on the outcome. This review presents an overview of the reactions to the biomaterials with a specific focus on clinical complications with the implants in the context of immune reactions and an overview of the studies involving biomaterial properties and interactions with innate immune system cells. We emphasize the impact of these studies on scaffold selection and upscaling of microenvironments created by biomaterials from 2D to 3D using immune cell encapsulation, seeding in a 3D scaffold and co-culture with relevant tissue cells. 3D microenvironments are covered with a specific focus on innate cells since a large proportion of these studies used innate immune cells. Finally, the recent studies on the incorporation of adaptive immune cells in immunomodulatory systems are covered in this review. Biomaterial-immune cell interactions are a critical part of regenerative medicine applications. Current efforts in establishing the ground rules for such interactions following implantation can control immune response during all phases of inflammation. Thus, in the near future for complete functional recovery, tissue engineering and control over biomaterials must be considered at the first step of immune modulation and this review covers these interactions, which have remained elusive up to now.

Evaluation of a collagen-bioaggregate composite scaffold in the repair of sheep pulp tissue.

PURPOSE: This study aimed to compare the effects of the collagen-BioAggregate mixture (CBA-M) and collagen-BioAggregate composite (CBA-C) sponge as a scaffolding material on the reparative dentin formation. MATERIALS AND METHODS: CBA-C sponge (10:1 w/w) was obtained and characterized by Scanning Electron Microscopy (SEM) and Mercury Porosimetry. Cytotoxicity of the CBA-C sponge was tested by using the L929 mouse fibroblast cell line. Dental pulp stem cells (DPSCs) were isolated from the pulp tissue of sheep teeth and characterized by flow cytometry for the presence of mesenchymal stem cell marker, CD44. The osteogenic differentiation capability of isolated DPSCs was studied by Alizarin Red staining. The cells were then used to study for the compatibility of CBA-C sponge with cell proliferation and calcium phosphate deposition. The effect of CBA-C sponge and CBA-M on the induction of dentin regeneration was studied in the perforated teeth of sheep for the eight-week period. All the analyses were performed with appropriate statistical hypothesis tests. RESULTS: CBA-C sponge was found to be biocompatible for DPSCs. The DPSCs seeded on the CBA-C sponge were able to differentiate into the osteoblastic lineage and deposit calcium phosphate crystals in vitro. Reparative dentin formation was observed after the second week in the CBA-C sponge applied group. At the end of eight weeks, a complete reparative dentin structure was formed in the CBA-C sponge applied group, whereas necrotic tissue residues were observed in groups treated with the CBA-M. CONCLUSION: CBA-C sponge represents a better microenvironment for reparative dentin formation probably due to maintaining DPSCs and allowing their osteogenic differentiation and thus calcium phosphate deposition.

3D printed hybrid bone constructs of PCL and dental pulp stem cells loaded GelMA.

Fabrication of scaffolds using polymers and then cell seeding is a routine protocol of tissue engineering applications. Synthetic polymers have adequate mechanical properties to substitute for some bone tissue, but they are generally hydrophobic and have no specific cell recognition sites, which leads to poor cell affinity and adhesion. Some natural polymers, have high cell affinity but are mechanically weak and do not have the strength required as a bone supporting material. In the present study, 3D printed hybrid scaffolds were fabricated using PCL and GelMA carrying dental pulp stem cells (DPSCs), which is printed in the gaps between the PCL struts. This cell loaded GelMA was shown to support osteoinductivity, while the PCL provided mechanical strength needed to mimic the bone tissue. 3D printed PCL/GelMA and GelMA scaffolds were highly stable during 21 days of incubation in PBS. The compressive moduli of the hybrid scaffolds were in the range of the compressive moduli of trabecular bone. DPSCs were homogeneously distributed throughout the entire hydrogel component and exhibited high cell viability in both scaffolds during 21 days of incubation. Upon osteogenic differentiation DPSCs expressed two key matrix proteins, osteopontin and osteocalcin. Alizarin red staining showed mineralized nodules, which demonstrates osteogenic differentiation of DPSCs within GelMA. This construct yielded a very high cell viability, osteogenic differentiation and mineralization comparable to cell culture without compromising mechanical strength suitable for bone tissue engineering applications. Thus, 3D printed, cell loaded PCL/GelMA hybrid scaffolds have a great potential for use in bone tissue engineering applications.

Evaluating Oxygen Tensions Related to Bone Marrow and Matrix for MSC Differentiation in 2D and 3D Biomimetic Lamellar Scaffolds.

The physiological O2 microenvironment of mesenchymal stem cells (MSCs) and osteoblasts and the dimensionality of a substrate are known to be important in regulating cell phenotype and function. By providing the physiologically normoxic environments of bone marrow (5%) and matrix (12%), we assessed their potential to maintain stemness, induce osteogenic differentiation, and enhance the material properties in the micropatterned collagen/silk fibroin scaffolds that were produced in 2D or 3D. Expression of osterix (OSX) and vascular endothelial growth factor A (VEGFA) was significantly enhanced in the 3D scaffold in all oxygen environments. At 21% O2, OSX and VEGFA expressions in the 3D scaffold were respectively 13,200 and 270 times higher than those of the 2D scaffold. Markers for assessing stemness were significantly more pronounced on tissue culture polystyrene and 2D scaffold incubated at 5% O2. At 21% O2, we measured significant increases in ultimate tensile strength (p < 0.0001) and Young's modulus (p = 0.003) of the 3D scaffold compared to the 2D scaffold, whilst 5% O2 hindered the positive effect of cell seeding on tensile strength. In conclusion, we demonstrated that the 3D culture of MSCs in collagen/silk fibroin scaffolds provided biomimetic cues for bone progenitor cells toward differentiation and enhanced the tensile mechanical properties.

Micropatterned Surfaces Expose the Coupling between Actin Cytoskeleton-Lamin/Nesprin and Nuclear Deformability of Breast Cancer Cells with Different Malignancies.

Mechanotransduction proteins transfer mechanical stimuli through nucleo-cytoskeletal coupling and affect the nuclear morphology of cancer cells. However, the contribution of actin filament integrity has never been studied directly. It is hypothesized that differences in nuclear deformability of cancer cells are influenced by the integrity of actin filaments. In this study, transparent micropatterned surfaces as simple tools to screen cytoskeletal and nuclear distortions are presented. Surfaces decorated with micropillars are used to culture and image breast cancer cells and quantify their deformation using shape descriptors (circularity, area, perimeter). Using two drugs (cytochalasin D and jasplakinolide), actin filaments are disrupted. Deformation of cells on micropillars is decreased upon drug treatment as shown by increased circularity. However, the effect is much smaller on benign MCF10A than on malignant MCF7 and MDAMB231 cells. On micropatterned surfaces, molecular analysis shows that Lamin A/C and Nesprin-2 expressions decreased but, after drug treatment, increased in malignant cells but not in benign cells. These findings suggest that Lamin A/C, Nesprin-2 and actin filaments are critical in mechanotransduction of cancer cells. Consequently, transparent micropatterned surfaces can be used as image analysis platforms to provide robust, high throughput measurements of nuclear deformability of cancer cells, including the effect of cytoskeletal elements.

3D cellular alignment and biomimetic mechanical stimulation enhance human adipose-derived stem cell myogenesis.

Determination of a stem cell source with sufficient myogenic differentiation capacity that can be easily obtained in large quantities is of great importance in skeletal muscle regeneration therapies. Adipose-derived stem cells (ASCs) are readily available, can be isolated from fat tissue with high yield and possess myogenic differentiation capacity. Consequently, ASCs have high applicability in muscle regenerative therapies. However, a key challenge is their low differentiation efficiency. In this study, we have explored the potential of mimicking the natural microenvironment of the skeletal muscle tissue to enhance ASC myogenesis by inducing 3D cellular alignment and using dynamic biomimetic culture. ASCs were entrapped and 3D aligned in parallel within fibrin-based microfibers and subjected to uniaxial cyclic stretch. 3D cell alignment was shown to be necessary for achieving and maintaining the stiffness of the construct mimicking the natural tissue (12 ± 1 kPa), where acellular aligned fibers and cell-laden random fibers had stiffness values of 4 ± 1 and 5 ± 2 kPa, respectively, at the end of 21 d. The synergistic effect of 3D cell alignment and biomimetic dynamic culture was evaluated on cell proliferation, viability and the expression of muscle-specific markers (immunofluorescent staining for MyoD1, myogenin, desmin and myosin heavy chain). It was shown that the myogenic markers were only expressed on the aligned-dynamic culture samples on day 21 of dynamic culture. These results demonstrate that 3D skeletal muscle grafts can be developed using ASCs by mimicking the structural and physiological muscle microenvironment.

A Circulating Bioreactor Reprograms Cancer Cells Toward a More Mesenchymal Niche.

Cancer is a complex and heterogeneous disease, and cancer cells dynamically interact with the mechanical microenvironment such as hydrostatic pressure, fluid shear, and interstitial flow. These factors play an essential role in cell fate and circulating tumor cell heterogeneity, and can influence the cellular phenotype. In this study, a peristaltic continuous flow reactor is designed and applied to HCT-116 colorectal carcinoma cells to mimic the fluid dynamics of circulation. With this intervention, a CD44/CD24-cell subpopulation emerges, and 100 genes are significantly regulated. The expression of cells at 4 h in the flow reactor is very similar to TGF-ß treatment, which is an inducer of epithelial-mesenchymal transition. ATF3 and SERPINE1 are significantly upregulated in these groups, suggesting that the mesenchymal transition is induced through this signaling pathway. This flow reactor model is satisfactory on its own to reprogram colorectal cancer cells toward a more mesenchymal niche mimicking circulation of the blood.

A two-compartment bone tumor model to investigate interactions between healthy and tumor cells.

We produced a novel three-dimensional (3D) bone tumor model (BTM) to study the interactions between healthy and tumor cells in a tumor microenvironment, the migration tendency of the tumor cells, and the efficacy of an anticancer drug, Doxorubicin, on the cancer cells. The model consisted of two compartments: (a) a healthy bone tissue mimic, made of poly(lactic acid-co-glycolic acid) (PLGA)/beta-tricalcium phosphate (ß-TCP) sponge seeded with human fetal osteoblastic cells (hFOB) and human umbilical vein endothelial cells (HUVECs), and (b) a tumor mimic, made of lyophilized collagen sponge seeded with human osteosarcoma cells (Saos-2). The tumor mimic component was placed into a central cavity created in the healthy bone mimic and together they constituted the complete 3D bone tumor model (3D-BTM). The porosities of both sponges were higher than 85% and the diameters of the pores were 199 ± 52 µm for the PLGA/TCP and 50-150 µm for the collagen scaffolds. The compression Young's modulus of the PLGA/TCP and the collagen sponges were determined to be 4.76 MPa and 140 kPa, respectively. Cell proliferation, morphology, calcium phosphate forming capacity and alkaline phosphatase production were studied separately on both the healthy and tumor mimics. All cells demonstrated cellular extensions and spread well in porous scaffolds indicating good cell-material interactions. Confocal microscopy analysis showed direct contact between the cells present in different parts of the 3D-BTM. Migration of HUVECs from the healthy bone mimic to the tumor compartment was confirmed by the increase in the levels of angiogenic factors vascular endothelial growth factor, basic fibroblast growth factor, and interleukin 8 in the tumor component. Doxorubicin (2.7 µg.ml-1) administered to the 3D-BTM caused a seven-fold decrease in the cell number after 24 h of interaction with the anticancer drug. Caspase-3 enzyme activity assay results demonstrated apoptosis of the osteosarcoma cells. This novel 3D-BTM has a high potential for use in studying the metastatic capabilities of cancer cells, and in determining the effective drug types and combinations for personalized treatments.