Printing of 3D biomimetic structures for the study of bone metastasis: A review.

Bone metastasis primarily occurs when breast, prostate, or lung cancers disseminate tumoral cells into bone tissue, leading to a range of complications in skeletal tissues and, in severe cases, paralysis resulting from spinal cord compression. Unfortunately, our understanding of pathophysiological mechanisms is incomplete and the translation of bone metastasis research into the clinic has been slow, mainly due to the lack of credible ex vivo and in vivo models to study the disease progression. Development of reliable and rational models to study how tumor cells become circulating cells and then invade and sequentially colonize the bone are in great need. Advances in tissue engineering technologies offers reliable 3D tissue alternatives which answer relevant research questions towards the understanding of cancer evolution and key functional properties of metastasis progression as well as prognosis of therapeutic approach. Here we performed an overview of cellular mechanisms involved in bone metastasis including a short summary of normal bone physiology and metastasis initiation and progression. Also, we comprehensively summarized current advances and methodologies in fabrication of reliable bone tumor models based on state-of-the-art printing technologies which recapitulate structural and biological features of native tissue. STATEMENT OF SIGNIFICANCE: This review provides a comprehensive summary of the collective findings in relation to various printed bone metastasis models utilized for investigating specific bone metastasis diseases, related characteristic functions and chemotherapeutic drug screening. These tumoral models are comprehensively evaluated and compared, in terms of their ability to recapitulate physiological metastasis microenvironment. Various biomaterials (natural and synthetic polymers and ceramic based substrates) and printing strategies and design architecture of models used for printing of 3D bone metastasis models are discussed here. This review clearly out-lines current challenges and prospects for 3D printing technologies in bone metastasis research by focusing on the required perspective models for clinical application of these technologies in chemotherapeutic drug screening.

Numerical design of open-porous titanium scaffolds for Powder Bed Fusion using Laser Beam (PBF-LB).

The paper concerns the numerical design of novel three-dimensional titanium scaffolds with complex open-porous structures and desired mechanical properties for the Powder Bed Fusion using Laser Beam (PBF-LB). The 60 structures with a broad range of porosity (38-78%), strut diameters (0.70-1.15 mm), and coefficients of pore volume variation, CV(Vp), 0.35-5.35, were designed using the Laguerre-Voronoi tessellations (LVT). Their Young's moduli and Poisson's ratios were calculated using Finite Element Model (FEM) simulations. The experimental verification was performed on the representative designs additively manufactured (AM) from commercially pure titanium (CP Ti) which, after chemical polishing, were subjected to uniaxial compression tests. Scanning Electron Microscopy (SEM) observations and microtomography (µ-CT) confirmed the removal of the support structures and unmelted powder particles. PBF-LB structures after chemical polishing were in close agreement with the CAD models' dimensions having 4-12% more volume. The computational and experimental results show that elastic properties were predicted in very close agreement for the low CV(Vp), and with even 30-40% discrepancies for CV(Vp) higher than 4.0, mainly due to PBF-LB scaffold architecture drawbacks rather than CAD inaccuracy. Our research demonstrates the possibility of designing the open-porous scaffolds with pore volume diversity and tuning their elastic properties for biomedical applications.

Indocyanine green and iohexol loaded hydroxyapatite in poly(L-lactide-co-caprolactone)-based composite for bimodal near-infrared fluorescence- and X-ray-based imaging.

This study aimed to develop material for multimodal imaging by means of X-ray and near-infrared containing FDA- and EMA-approved iohexol and indocyanine green (ICG). The mentioned contrast agents (CAs) are hydrophilic and amphiphilic, respectively, which creates difficulties in fabrication of functional polymeric composites for fiducial markers (FMs) with usage thereof. Therefore, this study exploited for the first time the possibility of enhancing the radiopacity and introduction of the NIR fluorescence of FMs by adsorption of the CAs on hydroxyapatite (HAp) nanoparticles. The particles were embedded in the poly(L-lactide-co-caprolactone) (P[LAcoCL]) matrix resulting in the composite material for bimodal near-infrared fluorescence- and X-ray-based imaging. The applied method of material preparation provided homogenous distribution of both CAs with high iohexol loading efficiency and improved fluorescence signal due to hindered ICG aggregation. The material possessed profound contrasting properties for both imaging modalities. Its stability was evaluated during in vitro experiments in phosphate-buffered saline (PBS) and foetal bovine serum (FBS) solutions. The addition of HAp nanoparticles had significant effect on the fluorescence signal. The X-ray radiopacity was stable within minimum 11 weeks, even though the addition of ICG contributed to a faster release of iohexol. The stiffness of the material was not affected by iohexol or ICG, but incorporation of HAp nanoparticles elevated the values of bending modulus by approximately 70%. Moreover, the performed cell study revealed that all tested materials were not cytotoxic. Thus, the developed material can be successfully used for fabrication of FMs.

Tuning Physical Properties of GelMA Hydrogels through Microarchitecture for Engineering Osteoid Tissue.

Gelatin methacryloyl (GelMA) hydrogels have gained significant attention due to their biocompatibility and tunable properties. Here, a new approach to engineer GelMA-based matrices to mimic the osteoid matrix is provided. Two cross-linking methods were employed to mimic the tissue stiffness: standard cross-linking (SC) based on visible light exposure (VL) and dual cross-linking (DC) involving physical gelation, followed by VL. It was demonstrated that by reducing the GelMA concentration from 10% (G10) to 5% (G5), the dual-cross-linked G5 achieved a compressive modulus of ∼17 kPa and showed the ability to support bone formation, as evidenced by alkaline phosphatase detection over 3 weeks of incubation in osteogenic medium. Moreover, incorporating poly(ethylene) oxide (PEO) into the G5 and G10 samples was found to hinder the fabrication of highly porous hydrogels, leading to compromised cell survival and reduced osteogenic differentiation, as a consequence of incomplete PEO removal.

Mesoporous Particle Embedded Nanofibrous Scaffolds Sustain Biological Factors for Tendon Tissue Engineering.

In recent years, fiber-based systems have been explored in the frame of tissue engineering due to their robustness in recapitulating the architecture and mechanical properties of native tissues. Such scaffolds offer anisotropic architecture capable of reproducing the native collagen fibers' orientation and distribution. Moreover, fibrous constructs might provide a biomimetic environment for cell encapsulation and proliferation as well as influence their orientation and distribution. In this work, we combine two fiber fabrication techniques, such as electrospinning and wet-spinning, in order to obtain novel cell-laden 3D fibrous layered scaffolds which can simultaneously provide: (i) mechanical support; (ii) suitable microenvironment for 3D cell encapsulation; and (iii) loading and sustained release of growth factors for promoting the differentiation of human bone marrow-derived mesenchymal stem cells (hB-MSCs). The constructs are formed from wet-spun hydrogel fibers loaded with hB-MSCs deposited on a fibrous composite electrospun matrix made of polycaprolactone, polyamide 6, and mesoporous silica nanoparticles enriched with bone morphogenetic protein-12 (BMP-12). Morphological and mechanical characterizations of the structures were carried out, and the growth factor release was assessed. The biological response in terms of cell viability, alignment, differentiation, and extracellular matrix production was investigated. Ex vivo testing of the layered structure was performed to prove the layers' integrity when subjected to mechanical stretching in the physiological range. The results reveal that 3D layered scaffolds can be proposed as valid candidates for tendon tissue engineering.

On the Effect of Non-Thermal Atmospheric Pressure Plasma Treatment on the Properties of PET Film.

The aim of the work was to investigate the effect of non-thermal plasma treatment of an ultra-thin polyethylene terephthalate (PET) film on changes in its physicochemical properties and biodegradability. Plasma treatment using a dielectric barrier discharge plasma reactor was carried out in air at room temperature and atmospheric pressure twice for 5 and 15 min, respectively. It has been shown that pre-treatment of the PET surface with non-thermal atmospheric plasma leads to changes in the physicochemical properties of this polymer. After plasma modification, the films showed a more developed surface compared to the control samples, which may be related to the surface etching and oxidation processes. After a 5-min plasma exposure, PET films were characterized by the highest wettability, i.e., the contact angle decreased by more than twice compared to the untreated samples. The differential scanning calorimetry analysis revealed the influence of plasma pretreatment on crystallinity content and the melt crystallization behavior of PET after soil degradation. The main novelty of the work is the fact that the combined action of two factors (i.e., physical and biological) led to a reduction in the content of the crystalline phase in the tested polymeric material.

Investigating bevacizumab and its fragments sustained release from intravitreal administrated PLGA Microspheres: A modeling approach.

Intravitreal administrated bevacizumab has emerged as an effective antibody for suppressing VEGF expression in age-related macular degeneration (AMD) therapy. This study discusses certain issues related to the sustained release of bevacizumab from intravitreal poly(lactic-co-glycolic acid) (PLGA) microspheres. A computational model elucidating the ocular kinetics of bevacizumab is demonstrated, wherein the release of the drug from PLGA microspheres is modeled using the Koizumi approach, complemented by an empirical model that links the kinetics of bevacizumab release to a size-dependent hydrolytic degradation of the drug-loaded polymeric microparticles. The results of the simulation were then rigorously validated against experimental data. The as-developed model proved remarkably accurate in predicting the time-concentration profiles obtained following the intravitreal injection of PLGA microspheres of significantly different sizes. Notably, the time-concentration profiles of bevacizumab in distinct ocular tissues were almost unaffected by the size of the intravitreally administered PLGA microparticles. Furthermore, the model successfully predicted the retinal concentration of bevacizumab and its fragments (e.g., ranibizumab) administrated in the form of a solution. As such, this model for drug sustained release and ocular transport holds tremendous potential for facilitating the reliable evaluation of planned anti-VEGF therapies.

On the Mechanical Properties of Microfibre-Based 3D Chitinous Scaffolds from Selected Verongiida Sponges.

Skeletal constructs of diverse marine sponges remain to be a sustainable source of biocompatible porous biopolymer-based 3D scaffolds for tissue engineering and technology, especially structures isolated from cultivated demosponges, which belong to the Verongiida order, due to the renewability of their chitinous, fibre-containing architecture focused attention. These chitinous scaffolds have already shown excellent and promising results in biomimetics and tissue engineering with respect to their broad diversity of cells. However, the mechanical features of these constructs have been poorly studied before. For the first time, the elastic moduli characterising the chitinous samples have been determined. Moreover, nanoindentation of the selected bromotyrosine-containing as well as pigment-free chitinous scaffolds isolated from selected verongiids was used in the study for comparative purposes. It was shown that the removal of bromotyrosines from chitin scaffolds results in a reduced elastic modulus; however, their hardness was relatively unaffected.

Effect of adipose-derived stem cells seeding and surgical prefabrication on composite scaffold vascularization.

The study aimed to evaluate an angiogenic effect of adipose-derived stem cells (ASCs) seeding and surgical prefabrication (placing a vascular pedicle inside the scaffold) on developed composite scaffolds made of poly-ε-caprolactone (PCL), ß-tricalcium phosphate (ß-TCP), and poly (lactic-co-glycolic acid) (PLGA) (PCL+ß-TCP+PLGA). Moreover, we aimed to compare our data with previously tested PCL scaffolds to assess whether the new material has better angiogenic properties. The study included 18 inbred male WAG rats. There were three scaffold groups (six animals each): with non-seeded PCL+ß-TCP+PLGA scaffolds, with PCL+ß-TCP+PLGA scaffolds seeded with ASCs and with PCL+ß-TCP+PLGA scaffolds seeded with ASCs and osteogenic-induced. Each rat was implanted with two scaffolds in the inguinal region (one prefabricated and one non-prefabricated). After 2 months from implantation, the scaffolds were explanted, and vessel density was determined by histopathological examination. Prefabricated ASC-seeded PCL+ß-TCP+PLGA scaffolds promoted greater vessel formation than non-seeded scaffolds (19.73 ± 5.46 vs 12.54 ± 0.81; p = .006) and those seeded with osteogenic-induced ASCs (19.73 ± 5.46 vs 11.87±2.21; p = .004). The developed composite scaffold promotes vessel formation more effectively than the previously described PCL scaffold.

3D-printed wound dressings containing a fosmidomycin-derivative prevent Acinetobacter baumannii biofilm formation.

Acinetobacter baumannii causes a wide range of infections, including wound infections. Multidrug-resistant A. baumannii is a major healthcare concern and the development of novel treatments against these infections is needed. Fosmidomycin is a repurposed antimalarial drug targeting the non-mevalonate pathway, and several derivatives show activity toward A. baumannii. We evaluated the antimicrobial activity of CC366, a fosmidomycin prodrug, against a collection of A. baumannii strains, using various in vitro and in vivo models; emphasis was placed on the evaluation of its anti-biofilm activity. We also developed a 3D-printed wound dressing containing CC366, using melt electrowriting technology. Minimal inhibitory concentrations of CC366 ranged from 1 to 64 µg/mL, and CC366 showed good biofilm inhibitory and moderate biofilm eradicating activity in vitro. CC366 successfully eluted from a 3D-printed dressing, the dressings prevented the formation of A. baumannnii wound biofilms in vitro and reduced A. baumannii infection in an in vivo mouse model.

Deep eutectic solvent-assisted fabrication of bioinspired 3D carbon-calcium phosphate scaffolds for bone tissue engineering.

Tissue engineering is a burgeoning field focused on repairing damaged tissues through the combination of bodily cells with highly porous scaffold biomaterials, which serve as templates for tissue regeneration, thus facilitating the growth of new tissue. Carbon materials, constituting an emerging class of superior materials, are currently experiencing remarkable scientific and technological advancements. Consequently, the development of novel 3D carbon-based composite materials has become significant for biomedicine. There is an urgent need for the development of hybrids that will combine the unique bioactivity of ceramics with the performance of carbonaceous materials. Considering these requirements, herein, we propose a straightforward method of producing a 3D carbon-based scaffold that resembles the structural features of spongin, even on the nanometric level of their hierarchical organization. The modification of spongin with calcium phosphate was achieved in a deep eutectic solvent (choline chloride : urea, 1 : 2). The holistic characterization of the scaffolds confirms their remarkable structural features (i.e., porosity, connectivity), along with the biocompatibility of α-tricalcium phosphate (α-TCP), rendering them a promising candidate for stem cell-based tissue-engineering. Culturing human bone marrow mesenchymal stem cells (hMSC) on the surface of the biomimetic scaffold further verifies its growth-facilitating properties, promoting the differentiation of these cells in the osteogenesis direction. ALP activity was significantly higher in osteogenic medium compared to proliferation, indicating the differentiation of hMSC towards osteoblasts. However, no significant difference between C and C-αTCP in the same medium type was observed.

Combining rotary wet-spinning biofabrication and electro-mechanical stimulation for thein vitroproduction of functional myo-substitutes.

In this work, we present an innovative, high-throughput rotary wet-spinning biofabrication method for manufacturing cellularized constructs composed of highly-aligned hydrogel fibers. The platform is supported by an innovative microfluidic printing head (MPH) bearing a crosslinking bath microtank with a co-axial nozzle placed at the bottom of it for the immediate gelation of extruded core/shell fibers. After a thorough characterization and optimization of the new MPH and the fiber deposition parameters, we demonstrate the suitability of the proposed system for thein vitroengineering of functional myo-substitutes. The samples produced through the described approach were first characterizedin vitroand then used as a substrate to ascertain the effects of electro-mechanical stimulation on myogenic maturation. Of note, we found a characteristic gene expression modulation of fast (MyH1), intermediate (MyH2), and slow (MyH7) twitching myosin heavy chain isoforms, depending on the applied stimulation protocol. This feature should be further investigated in the future to biofabricate engineered myo-substitutes with specific functionalities.

3D Bioprinting in Microgravity: Opportunities, Challenges, and Possible Applications in Space.

3D bioprinting has developed tremendously in the last couple of years and enables the fabrication of simple, as well as complex, tissue models. The international space agencies have recognized the unique opportunities of these technologies for manufacturing cell and tissue models for basic research in space, in particular for investigating the effects of microgravity and cosmic radiation on different types of human tissues. In addition, bioprinting is capable of producing clinically applicable tissue grafts, and its implementation in space therefore can support the autonomous medical treatment options for astronauts in future long term and far-distant space missions. The article discusses opportunities but also challenges of operating different types of bioprinters under space conditions, mainly in microgravity. While some process steps, most of which involving the handling of liquids, are challenging under microgravity, this environment can help overcome problems such as cell sedimentation in low viscous bioinks. Hopefully, this publication will motivate more researchers to engage in the topic, with publicly available bioprinting opportunities becoming available at the International Space Station (ISS) in the imminent future.

Calcification alters the viscoelastic properties of tendon fascicle bundles depending on matrix content.

Tendon fascicle bundles are often used as biological grafts and thus must meet certain quality requirements, such as excluding calcification, which alters the biomechanical properties of soft tissues. In this work, we investigate the influence of early-stage calcification on the mechanical and structural properties of tendon fascicle bundles with varying matrix content. The calcification process was modeled using sample incubation in concentrated simulated body fluid. Mechanical and structural properties were investigated using uniaxial tests with relaxation periods, dynamic mechanical analysis, as well as magnetic resonance imaging and atomic force microscopy. Mechanical tests showed that the initial phase of calcification causes an increase in the elasticity, storage, and loss modulus, as well as a drop in the normalized value of hysteresis. Further calcification of the samples results in decreased modulus of elasticity and a slight increase in the normalized value of hysteresis. Analysis via MRI and scanning electron microscopy showed that incubation alters fibrillar relationships within the tendon structure and the flow of body fluids. In the initial stage of calcification, calcium phosphate crystals are barely visible; however, extending the incubation time for the next 14 days results in the appearance of calcium phosphate crystals within the tendon structure and leads to damage in its structure. Our results show that the calcification process modifies the collagen-matrix relationships and leads to a change in their mechanical properties. These findings will help to understand the pathogenesis of clinical conditions caused by calcification process, leading to the development of effective treatments for these conditions. STATEMENT OF SIGNIFICANCE: This study investigates how calcium mineral deposition in tendons affects their mechanical response and which processes are responsible for this phenomenon. By analyzing the elastic and viscoelastic properties of animal fascicle bundles affected by calcification induced via incubation in concentrated simulated body fluid, the study sheds light on the relationship between structural and biochemical changes in tendons and their altered mechanical response. This understanding is crucial for optimizing tendinopathy treatment and preventing tendon injury. The findings provide insights into the calcification pathway and its resulting changes in the biomechanical behaviors of affected tendons, which have been previously unclear.

In vitro and in vivo degradation behavior of Mg-0.45Zn-0.45Ca (ZX00) screws for orthopedic applications.

Magnesium (Mg) alloys have become a potential material for orthopedic implants due to their unnecessary implant removal, biocompatibility, and mechanical integrity until fracture healing. This study examined the in vitro and in vivo degradation of an Mg fixation screw composed of Mg-0.45Zn-0.45Ca (ZX00, in wt.%). With ZX00 human-sized implants, in vitro immersion tests up to 28 days under physiological conditions, along with electrochemical measurements were performed for the first time. In addition, ZX00 screws were implanted in the diaphysis of sheep for 6, 12, and 24 weeks to assess the degradation and biocompatibility of the screws in vivo. Using scanning electron microscopy (SEM) coupled with energy dispersive X-ray spectroscopy (EDX), micro-computed tomography (µCT), X-ray photoelectron spectroscopy (XPS), and histology, the surface and cross-sectional morphologies of the corrosion layers formed, as well as the bone-corrosion-layer-implant interfaces, were analyzed. Our findings from in vivo testing demonstrated that ZX00 alloy promotes bone healing and the formation of new bone in direct contact with the corrosion products. In addition, the same elemental composition of corrosion products was observed for in vitro and in vivo experiments; however, their elemental distribution and thicknesses differ depending on the implant location. Our findings suggest that the corrosion resistance was microstructure-dependent. The head zone was the least corrosion-resistant, indicating that the production procedure could impact the corrosion performance of the implant. In spite of this, the formation of new bone and no adverse effects on the surrounding tissues demonstrated that the ZX00 is a suitable Mg-based alloy for temporary bone implants.

Toward osteomimetic formation of calcium phosphate coatings with carbonated hydroxyapatite.

Biomimetic production of coatings on various types of scaffolds is based mainly on simulated body fluid precipitation (SBF) of apatites, or, if the HCO3- is present, carbonated apatites. Recently, we proposed formation of calcium phosphates (CaP) precipitates by alkaline phosphatase (ALP) hydrolysing glycerophosphate in presence of calcium ions as an alternative to SBF. Since apatites synthesized in bone by the ALP activity contain carbonate anions, it was tempting to investigate whether the phosphatase method could be advanced into osteomimetic one. Therefore, taking example from the SBF studies, phosphatase incubation medium was enriched with carbonate ions at 4.2 and 27 mM concentration. X-ray diffraction of the precipitates disclosed peaks typical for hydroxyapatite (HAP). FTIR analysis showed that at both concentration of carbonate ions, apatites underwent both B and A substitution, more extensive at higher concentration. Thus, osteomimetic approach produced carbonated hydroxyapatites of the type encountered in bone tissue even at HCO3- concentration as low as 4.2 mM. Composite plates made of poly(ε-caprolactone) and mixture of ß-tricalcium phosphate and hydroxyapatite at mass ratio of 1:0.5:0.5, respectively, were covered by CaP coatings, i.e., CaP-0, CaP-4.2, CaP-27, by incubation in phosphatase medium containing 0, 4.2 or 27 mM of NaHCO3, respectively. Pristine or coated PCL50 plates were used to study release of calcium and adsorption/desorption of proteins, or seeded with human bone marrow mesenchymal stem cells (hMSC) for study of cell adhesion, spreading and osteogenic differentiation. Introduction of carbonate into the CaP coatings significantly increased release of Ca2+ in a carbonate concentration-dependent manner; the release was up to 4 times higher, when compared to CaP-0 coating, and reached 0.41 ± 0.01 mM for CaP-27 after first 24 h. Coating CaP-4.2 yielded significantly higher adsorption of bovine serum albumin and cytochrome C than CaP-0. All of the CaP coatings improved significantly hMSC adhesion, however, only CaP-4.2 provided 2 times higher cell number than PCL50 after 2 weeks of culture. Interestingly, ALP activity calculated per cell number was the highest on pristine plates, presumably because hMSC differentiate preferentially into osteoblasts at lower seeding densities. It appears, therefore, that the osteomimetic approach may be useful for production of carbonated hydroxyapatite coatings, but requires further studies and replacing intestinal phosphatase used in this work with one originating from bone.

Magnesium implant degradation provides immunomodulatory and proangiogenic effects and attenuates peri-implant fibrosis in soft tissues.

Implants made of magnesium (Mg) are increasingly employed in patients to achieve osteosynthesis while degrading in situ. Since Mg implants and Mg2+ have been suggested to possess anti-inflammatory properties, the clinically observed soft tissue inflammation around Mg implants is enigmatic. Here, using a rat soft tissue model and a 1-28 d observation period, we determined the temporo-spatial cell distribution and behavior in relation to sequential changes of pure Mg implant surface properties and Mg2+ release. Compared to nondegradable titanium (Ti) implants, Mg degradation exacerbated initial inflammation. Release of Mg degradation products at the tissue-implant interface, culminating at 3 d, actively initiated chemotaxis and upregulated mRNA and protein immunomodulatory markers, particularly inducible nitric oxide synthase and toll-like receptor-4 up to 6 d, yet without a cytotoxic effect. Increased vascularization was demonstrated morphologically, preceded by high expression of vascular endothelial growth factor. The transition to appropriate tissue repair coincided with implant surface enrichment of Ca and P and reduced peri-implant Mg2+ concentration. Mg implants revealed a thinner fibrous encapsulation compared with Ti. The detailed understanding of the relationship between Mg material properties and the spatial and time-resolved cellular processes provides a basis for the interpretation of clinical observations and future tailoring of Mg implants.

Aerobic exercise and scaffolds with hierarchical porosity synergistically promote functional recovery post volumetric muscle loss.

Volumetric muscle loss (VML), which refers to a composite skeletal muscle defect, most commonly heals by scarring and minimal muscle regeneration but substantial fibrosis. Current surgical interventions and physical therapy techniques are limited in restoring muscle function following VML. Novel tissue engineering strategies may offer an option to promote functional muscle recovery. The present study evaluates a colloidal scaffold with hierarchical porosity and controlled mechanical properties for the treatment of VML. In addition, as VML results in an acute decrease in insulin-like growth factor 1 (IGF-1), a myogenic factor, the scaffold was designed to slowly release IGF-1 following implantation. The foam-like scaffold is directly crosslinked onto remnant muscle without the need for suturing. In situ 3D printing of IGF-1-releasing porous muscle scaffold onto VML injuries resulted in robust tissue ingrowth, improved muscle repair, and increased muscle strength in a murine VML model. Histological analysis confirmed regeneration of new muscle in the engineered scaffolds. In addition, the scaffolds significantly reduced fibrosis and increased the expression of neuromuscular junctions in the newly regenerated tissue. Exercise training, when combined with the engineered scaffolds, augmented the treatment outcome in a synergistic fashion. These data suggest highly porous scaffolds and exercise therapy, in combination, may be a treatment option following VML.

The combined effect of zinc and calcium on the biodegradation of ultrahigh-purity magnesium implants.

Magnesium (Mg)-based implants are promising candidates for orthopedic interventions, because of their biocompatibility, good mechanical features, and ability to degrade completely in the body, eliminating the need for an additional removal surgery. In the present study, we synthesized and investigated two Mg-based materials, ultrahigh-purity ZX00 (Mg-Zn-Ca; <0.5 wt% Zn and <0.5 wt% Ca, in wt%; Fe-content <1 ppm) and ultrahigh-purity Mg (XHP-Mg, >99.999 wt% Mg; Fe-content <1 ppm), in vitro and in vivo in juvenile healthy rats to clarify the effect of the alloying elements Zn and Ca on mechanical properties, microstructure, cytocompatibility and degradation rate. Potential differences in bone formation and bone in-growth were also assessed and compared with state-of-the-art non-degradable titanium (Ti)-implanted, sham-operated, and control (non-intervention) groups, using micro-computed tomography, histology and scanning electron microscopy. At 6 and 24 weeks after implantation, serum alkaline phosphatase (ALP), calcium (Ca), and Mg level were measured and bone marrow stromal cells (BMSCs) were isolated for real-time PCR analysis. Results show that ZX00 implants have smaller grain size and superior mechanical properties than XHP-Mg, and that both reveal good biocompatibility in cytocompatibilty tests. ZX00 homogenously degraded with an increased gas accumulation 12 and 24 weeks after implantation, whereas XHP-Mg exhibited higher gas accumulation already at 2 weeks. Serum ALP, Ca, and Mg levels were comparable among all groups and both Mg-based implants led to similar relative expression levels of Alp, Runx2, and Bmp-2 genes at weeks 6 and 24. Histologically, Mg-based implants are superior for new bone tissue formation and bone in-growth compared to Ti implants. Furthermore, by tracking the sequence of multicolor fluorochrome labels, we observed higher mineral apposition rate at week 2 in both Mg-based implants compared to the control groups. Our findings suggest that (i) ZX00 and XHP-Mg support bone formation and remodeling, (ii) both Mg-based implants are superior to Ti implants in terms of new bone tissue formation and osseointegration, and (iii) ZX00 is more favorable due to its lower degradation rate and moderate gas accumulation.

In vitro functional models for human liver diseases and drug screening: beyond animal testing.

Liver is one of the most important and complex organs in the human body, being characterized by a sophisticated microarchitecture and responsible for key physiological functions. Despite its remarkable ability to regenerate, acute liver failure and chronic liver diseases are major causes of morbidity and mortality worldwide. Therefore, understanding the molecular mechanisms underlying such liver disorders is critical for the successful development of novel therapeutics. In this frame, preclinical animal models have been portrayed as the most commonly used tool to address such issues. However, due to significant species differences in liver architecture, regenerative capacity, disease progression, inflammatory markers, metabolism rates, and drug response, animal models cannot fully recapitulate the complexity of human liver metabolism. As a result, translational research to model human liver diseases and drug screening platforms may yield limited results, leading to failure scenarios. To overcome this impasse, over the last decade, 3D human liver in vitro models have been proposed as an alternative to pre-clinical animal models. These systems have been successfully employed for the investigation of the etiology and dynamics of liver diseases, for drug screening, and - more recently - to design patient-tailored therapies, resulting in potentially higher efficacy and reduced costs compared to other methods. Here, we review the most recent advances in this rapidly evolving field with particular attention to organoid cultures, liver-on-a-chip platforms, and engineered scaffold-based approaches.