Hydrops fetalis caused by a complex congenital heart defect with concurrent hypoplasia of pulmonary blood vessels and lungs visualized by micro-CT in a French Bulldog.

BACKGROUND: Hydrops fetalis (HF) is fluid accumulation in fetus body cavities and subcutaneous tissue. The condition has been described in various farm and companion animal species, including dogs. Most of cases result from a heart defect. Exact nature of this defect is rarely clarified. CASE PRESENTATION: A newborn, male French bulldog puppy with severe HF underwent a full anatomopathological examination to diagnose the primary cause of HF. Based on the anatomopathological examination, fetal ultrasound, and micro-computed tomography, transposition of the great arteries with hypoplasia of the ascending aorta, aortic arch interruption, ostium secundum atrial septal defect, severe tricuspid valve dysplasia, as well as hypoplasia of pulmonary vessels and lungs were diagnosed. CONCLUSIONS: This is the first report of HF caused by severe, complex congenital heart defects with concurrent pulmonary vessel and lung hypoplasia.

Petrophysical Analyses of Rock Construction Materials from a Roman Rural Settlement in Podsilo Bay on Rab Island (North-East Adriatic, Croatia).

This article presents the results of petrophysical analyses of limestones and sandstones used for the construction of the wall structures of a Roman rural settlement located in Podsilo Bay on Rab Island (Croatia). An on-site analysis of the walls indicated the use of different lithotypes, which is an uncommon case in the area. So far, no petrophysical properties of the applied materials have been tested, and their provenance has not been specified. The aim of this research was to determine their usability as construction materials in an attempt to determine the possible reasons behind the usage of multiple lithotypes and their suitability as building materials. The following procedure was used to address these issues: (1) determination of the petrographic characteristics of the rocks, (2) performance of tests to characterise the mechanical properties in a complex stress state of uniaxial tension followed by uniaxial and triaxial compression, and, finally, (3) determination of the internal structure of the rocks using methods based on X-ray imaging. Multi-proxy characteristics of the materials including numerous observations and methods were performed: optical microscopy used to characterise rock petrography and mineralogy, scanning electron microscopy (SEM) coupled with EDS, as well as grinding tests; furthermore, mechanical properties were determined on cylindrical samples in accordance with European standards. X-ray microtomography using the XµCT method enabled microscopic observations and determination of the orientation of discontinuities and the rock structure. The performed analyses allowed us to distinguish three lithological types of sandstone and two types of limestone among the examined stone blocks. Stone blocks of fine- and medium-grained sandstone with carbonate binders, as well as sparitic limestone and mudstone with calcite veins, were used to build the studied structures. The analysed blocks showed traces of partial edge processing. Despite the defects in the material structure identified using XµCT, all the types of rock were characterised by high or very high strength. High values of longitudinal wave velocity confirmed the good quality of the material. These results contribute to a better understanding of the construction process and the related technological choices, and they provide the first dataset which can be used for the reconstruction of the building's original appearance in the future.

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.

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.

Composite microgranular scaffolds with surface modifications for improved initial osteoblastic cell proliferation.

Polyester-based granular scaffolds are a potent material for tissue engineering due to their porosity, controllable pore size, and potential to be molded into various shapes. Additionally, they can be produced as composite materials, e.g., mixed with osteoconductive ß-tricalcium phosphate or hydroxyapatite. Such polymer-based composite materials often happen to be hydrophobic, which disrupts cell attachment and decreases cell growth on the scaffold, undermining its primary function. In this work, we propose the experimental comparison of three modification techniques for granular scaffolds to increase their hydrophilicity and cell attachment. Those techniques include atmospheric plasma treatment, polydopamine coating, and polynorepinephrine coating. Composite polymer/ß-tricalcium phosphate granules have been produced in a solution-induced phase separation (SIPS) process using commercially available biomedical polymers: poly(lactic acid), poly(lactic-co-glycolic acid), and polycaprolactone. We used thermal assembly to prepare cylindrical scaffolds from composite microgranules. Atmospheric plasma treatment, polydopamine coating, and polynorepinephrine coating showed similar effects on polymer composites' hydrophilic and bioactive properties. All modifications significantly increased human osteosarcoma MG-63 cell adhesion and proliferation in vitro compared to cells cultured on unmodified materials. In the case of polycaprolactone/ß-tricalcium phosphate scaffolds, modifications were the most necessary, as unmodified polycaprolactone-based material disrupted the cell attachment. Modified polylactide/ß-tricalcium phosphate scaffold supported excellent cell growth and showed ultimate compressive strength exceeding this of human trabecular bone. This suggests that all investigated modification techniques can be used interchangeably for increasing wettability and cell attachment properties of various scaffolds for medical applications, especially those with high surface and volumetric porosity, like granular scaffolds.

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.

Synchrotron XRF and Histological Analyses Identify Damage to Digestive Tract of Uranium NP-Exposed Daphnia magna.

Micro- and nanoscopic X-ray techniques were used to investigate the relationship between uranium (U) tissue distributions and adverse effects to the digestive tract of aquatic model organism Daphnia magna following uranium nanoparticle (UNP) exposure. X-ray absorption computed tomography measurements of intact daphnids exposed to sublethal concentrations of UNPs or a U reference solution (URef) showed adverse morphological changes to the midgut and the hepatic ceca. Histological analyses of exposed organisms revealed a high proportion of abnormal and irregularly shaped intestinal epithelial cells. Disruption of the hepatic ceca and midgut epithelial tissues implied digestive functions and intestinal barriers were compromised. Synchrotron-based micro X-ray fluorescence (XRF) elemental mapping identified U co-localized with morphological changes, with substantial accumulation of U in the lumen as well as in the epithelial tissues. Utilizing high-resolution nano-XRF, 400-1000 nm sized U particulates could be identified throughout the midgut and within hepatic ceca cells, coinciding with tissue damages. The results highlight disruption of intestinal function as an important mode of action of acute U toxicity in D. magna and that midgut epithelial cells as well as the hepatic ceca are key target organs.

Controlled formation of highly porous polylactic acid­calcium phosphate granules with defined structure.

Synthetic bone repair materials are becoming increasingly popular in tissue engineering as a replacement for autografts and human/animal-based bone grafts. The biomedical application requires precise control over the material composition and structure, as well as over the size of granulate used for filling the bone defects, as the pore size and interconnectivity affect the regeneration process. This paper proposes a process of alloplastic and biodegradable polylactic acid/ß-tricalcium phosphate granulates preparation and its parameters described. Using solvent-induced phase separation technique, porous spheres have been obtained in various sizes and morphologies. The design of the experiment's approach generated an experimental plan for further statistical modeling using the resulting data. The statistical modeling approach to the data from conducting a designed set of experiments allowed analysis of the influence of process parameters on the properties of the resulting granules. We confirmed that the content of ß-tricalcium phosphate plays the most significant role in the size distribution of prepared granulate. The shape of the particles becomes less spherical with higher phosphate concentration in the emulsion. The proposed technique allows preparing porous granulates in the 0.2-1.8 mm diameter range, where granules' mean diameter and sphericity are tunable with polymer and phosphate concentrations. The granulate created a potentially implantable scaffold for resected bone regeneration, as cytotoxicity tests assured the material is non-cytotoxic in vitro, and human mesenchymal stem cells have been cultured on the surface of granulates. Results from cell cultures seeded on the Resomer LR 706S granulates were the most promising.

Novel optical photothermal infrared (O-PTIR) spectroscopy for the noninvasive characterization of heritage glass-metal objects.

Optical photothermal infrared (O-PTIR) is a recently developed molecular spectroscopy technique that allows to noninvasively obtain chemical information on organic and inorganic samples at a submicrometric scale. The high spatial resolution (≈450 nm), lack of sample preparation, and comparability of the spectral results to traditional Fourier transform infrared spectroscopy make it a promising candidate for the analysis of cultural heritage. In this work, the potential of O-PTIR for the noninvasive characterization of small heritage objects (few cubic centimeters) is demonstrated on a series of degraded 16th century brass and glass decorative elements. These small and challenging samples, typically encountering limitations with existing noninvasive methods such as macroscopic x-ray powder diffraction and µRaman, were successfully characterized by O-PTIR, ultimately identifying the markers of glass-induced metal corrosion processes. The results clearly demonstrate how O-PTIR can be easily implemented in a noninvasive multianalytical strategy for the study of heritage materials, making it a fundamental tool for cultural heritage analyses.

Modified Histopathological Protocol for Poly-ɛ-Caprolactone Scaffolds Preserving Their Trabecular, Honeycomb-like Structure.

Poly-ɛ-caprolactone (PCL) is now widely studied in relation to the engineering of bone, cartilage, tendons, and other tissues. Standard histological protocols can destroy the carefully created trabecular and honeycomb-like architecture of PCL scaffolds, and could lead to scaffold fibers swelling, resulting in the displacement or compression of tissues inside the scaffold. The aim of this study was to modify a standard histopathological protocol for PCL scaffold preparation and evaluate it on porous cylindrical PCL scaffolds in a rat model. In 16 inbred Wag rats, 2 PCL scaffolds were implanted subcutaneously to both inguinal areas. Two months after implantation, harvested scaffolds were first subjected to µCT imaging, and then to histopathological analysis with standard (left inguinal area) and modified histopathological protocols (right inguinal area). To standardize the results, soft tissue percentages (STPs) were calculated on scaffold cross-sections obtained from both histopathological protocols and compared with corresponding µCT cross-sections. The modified protocol enabled the assessment of almost 10× more soft tissues on the scaffold cross-section than the standard procedure. Moreover, STP was only 1.5% lower than in the corresponding µCT cross-sections assessed before the histopathological procedure. The presented modification of the histopathological protocol is cheap, reproducible, and allows for a comprehensive evaluation of PCL scaffolds while maintaining their trabecular, honeycomb-like structure on cross-sections.

Ultrashort Sintering and Near Net Shaping of Zr-Based AMZ4 Bulk Metallic Glass.

The GeniCore Upgraded Field Assisted Sintering Technology U-FAST was applied to the sintering of a commercial Zr-based bulk metallic glass powder AMZ4. The XRD, SEM and DSC analysis of the sintered compacts showed the benefit of the U-FAST method as an enabler for the production of fully amorphous samples with 100% relative density when sintering at 420 °C/480 s (693 K/480 s) and 440 °C/ 60 s (713 K/480 s). The hardness values for fully amorphous samples, over HV1 519, surpass cast materials and 1625 MPa compressive strengths are comparable to commercial cast products. The advantage of the U-FAST technology in this work is attributed to the high heating and cooling rates inherent to ultra-short pulses, which allow to maintain metastable structures and achieve better temperature control during the process. Increasing sintering temperature and time led to the crystallization of the materials. The geometry and material of the dies and punch determine the thermal inertia and pressure distribution inside the compacts, thus affecting the properties of the near net shape NNS compacts made using the U-FAST device.

Naturally Formed Chitinous Skeleton Isolated from the Marine Demosponge Aplysina fistularis as a 3D Scaffold for Tissue Engineering.

Tissue engineering (TE) is a field of regenerative medicine that has been experiencing a special boom in recent years. Among various materials used as components of 3D scaffolds, naturally formed chitinous materials seem to be especially attractive because of their abundance, non-toxic and eco-friendly character. In this study, chitinous skeleton isolated from the marine sponge Aplysina fistularis (phylum: Porifera) was used for the first time as a support for the cultivation of murine fibroblasts (Balb/3T3), human dermal fibroblasts (NHDF), human keratinocyte (HaCaT), and human neuronal (SH-SY5Y) cells. Characterization techniques such as ATR FTIR, TGA, and µCT, clearly indicate that an interconnected macro-porous, thermostable, pure α-chitin scaffold was obtained after alkali-acid treatment of air-dried marine sponge. The biocompatibility of the naturally formed chitin scaffolds was confirmed by cell attachment and proliferation determined by various microscopic methods (e.g., SEM, TEM, digital microscopy) and specific staining. Our observations show that fibroblasts and keratinocytes form clusters on scaffolds that resemble a skin structure, including the occurrence of desmosomes in keratinocyte cells. The results obtained here suggest that the chitinous scaffold from the marine sponge A. fistularis is a promising biomaterial for future research about tissues regeneration.

From Matrix Vesicles to Miniature Rocks: Evolution of Calcium Deposits in Calf Costochondral Junctions.

OBJECTIVE: Initial stages of cartilage matrix calcification depend on the activity of matrix vesicles. The purpose of the study was to describe how calcified matrix vesicles join into larger structures, to present their up-to-date undescribed 3-dimensional image, and to observe how calcified matrix relates to chondrocyte lacunae. DESIGN: Calcified cartilage was obtained from the zone of provisional calcification of calf costochondral junctions, then enzymatically isolated and studied by microtomography, scanning electron microscopy, atomic force microscopy and X-ray diffraction, and Fourier transform infrared spectroscopy. RESULTS: Hyaluronidase digestion released packets of granules surrounded by the cartilage matrix. Further digestion, with collagenase and trypsin, removed matrix and exposed granules with dimensions within 50 to 150 nm range, which we consider as equivalent of calcified matrix vesicles. Granules joined into larger groups with dimensions of 0.5 to 2 µm, which we call globular units. Certain matrix vesicles appeared well connected but contained globular units that had spaces filled with electron lucent material, presumably matrix or chondrocyte remnants. Globular units were organized into massive structures taking the shape of oval plates. Comparison of these plates with lacunae containing isogenous groups of chondrocytes from proliferative zone of costochondral junction suggests that the cells from a single lacuna were responsible for the formation of one plate. The plates were connected with each other and extended over provisional calcification zone. CONCLUSIONS: The outcome showed how particular calcified matrix vesicles associate into globular units, which organize into massive structures assuming the shape of oval plates and eventually cover large areas of cartilage matrix.

Functionalization of 3D Chitinous Skeletal Scaffolds of Sponge Origin Using Silver Nanoparticles and Their Antibacterial Properties.

Chitin, as one of nature's most abundant structural polysaccharides, possesses worldwide, high industrial potential and a functionality that is topically pertinent. Nowadays, the metallization of naturally predesigned, 3D chitinous scaffolds originating from marine sponges is drawing focused attention. These invertebrates represent a unique, renewable source of specialized chitin due to their ability to grow under marine farming conditions. In this study, the development of composite material in the form of 3D chitin-based skeletal scaffolds covered with silver nanoparticles (AgNPs) and Ag-bromide is described for the first time. Additionally, the antibacterial properties of the obtained materials and their possible applications as a water filtration system are also investigated.

Analysis of Microstructure and Properties of a Ti-AlN Composite Produced by Selective Laser Melting.

Selective Laser Melting (SLM) is a manufacturing technique that is currently used for the production of functional parts that are difficult to form by the traditional methods such as casting or CNC (Computer Numerical Control) cutting from a wide range of metallic materials. In our study, a mixture of commercially pure titanium (Ti) and 15% at. aluminum nitride (AlN) was Selective Laser Melted to form three-dimensional objects. The obtained 4 mm edge cubes with an energy density that varied from 70 to 140 J/mm3 were examined in terms of their microstructure, chemical and phase composition, porosity, and Vickers microhardness. Scanning Electron Microscopy (SEM) observations of the etched samples showed inhomogeneities in the form of pores and unmelted and partly melted AlN particles in the fine-grained dendritic matrix, which is typical for titanium nitrides and titanium aluminum nitrides. The AlN particles remained unmelted in samples, but no porosity was observed in the interface area between them and the dendritic matrix. Additionally, samples fabricated with the presintering step had zones with different sizes of dendrites, suggesting a differing chemical composition of the matrix and the possibility of the formation of the phases forming an Ti-Al-N ternary system. The chemical composition in the microareas of the samples was determined using Energy Dispersive X-Ray Spectroscopy (EDS) and revealed differences in the homogeneity of the samples depending on the SLM process parameters and the additional presintering step. The phase composition, examined using X-ray Diffraction analysis (XRD), showed that samples were formed from Ti, TiN, and AlN phases. Porosity tests carried out using a computer microtomography revealed porosities in a range from 7% to 17.5%. The formed material was characterized by a relatively high hardness exceeding 700 HV0.2 over the entire cross-section, which depended on the manufacturing conditions.

Micro-analytical characterization of thorium-rich aggregates from Norwegian NORM sites (Fen Complex, Telemark).

In this study we performed microscopic characterization of mineral particles that were collected in the thorium-rich Fen Complex in Norway and identified and isolated based on autoradiography in function of their radioactivity. For this we combined information obtained with X-ray absorption µ-CT, µ-XRF and µ-XRD, both in bi- and in three-dimensional (tomographic) mode. We demonstrate that radionuclides and metals are heterogeneously distributed both within soil samples and within individual Th-enriched aggregates, which are characterised as low-density mineral bulk particles with high density material inclusions, where Th as well as several metals are highly concentrated. For these sites, it is important to take into account how these inhomogeneous distributions could affect the overall environmental behaviour of Th and progeny upon weathering due to human or environmental factors. Moreover, the estimated size of the Th-containing inclusions as determined in this work represents information of importance for the characterization of radionuclides and toxic metals exposure, as well as for assessing the viability of mining for Th and rare-earth metals in the Fen Complex and the associated environmental impact.

Scaffold vascularization method using an adipose-derived stem cell (ASC)-seeded scaffold prefabricated with a flow-through pedicle.

BACKGROUND: Vascularization is important for the clinical application of tissue engineered products. Both adipose-derived stem cells (ASCs) and surgical prefabrication can be used to induce angiogenesis in scaffolds. Our aim was to compare the angiogenic potential of ASC-seeded scaffolds combined with scaffold prefabrication with that of non-seeded, non-prefabricated scaffolds. METHODS: For prefabrication, functional blood vessels were introduced into the scaffold using a flow-through pedicle system. ASCs were isolated from rat fat deposits. Three-dimensional-printed cylindrical poly-ε-caprolactone scaffolds were fabricated by fused deposition modelling. Three groups, each containing six rats, were investigated by using non-seeded, ASC-seeded, and osteogenic induced ASC-seeded scaffolds. In each group, one rat was implanted with two scaffolds in the inguinal region. On the right side, a scaffold was implanted subcutaneously around the inferior epigastric vessels (classic prefabrication group). On the left side, the inferior epigastric vessels were placed inside the prefabricated scaffold in the flow-through pedicle system (flow-through prefabrication group). The vessel density and vascular architecture were examined histopathologically and by µCT imaging, respectively, at 2 months after implantation. RESULTS: The mean vessel densities were 10- and 5-fold higher in the ASC-seeded and osteogenic induced ASC-seeded scaffolds with flow-through prefabrication, respectively, than in the non-seeded classic prefabricated group (p < 0.001). µCT imaging revealed functional vessels within the scaffold. CONCLUSION: ASC-seeded scaffolds with prefabrication showed significantly improved scaffold vasculogenesis and could be useful for application to tissue engineering products in the clinical settings.

3D bioprinting of hydrogel constructs with cell and material gradients for the regeneration of full-thickness chondral defect using a microfluidic printing head.

Osteochondral (OC) tissue is a biphasic material comprised of articular cartilage integrated atop subchondral bone. Damage to this tissue is highly problematic, owing to its intrinsic inability to regenerate functional tissue in response to trauma or disease. Further, the function of the tissue is largely conferred by its compartmentalized zonal microstructure and composition. Current clinical treatments fail to regenerate new tissue that recapitulates this zonal structure. Consequently, regenerated tissue often lacks long-term stability. To address this growing problem, we propose the development of tissue engineered biomaterials that mimic the zonal cartilage organization and extracellular matrix composition through the use of a microfluidic printing head bearing a mixing unit and incorporated into an extrusion-based bioprinter. The system is devised so that multiple bioinks can be delivered either individually or at the same time and rapidly mixed to the extrusion head, and finally deposited through a coaxial nozzle. This enables the deposition of either layers or continuous gradients of chemical, mechanical and biological cues and fabrication of scaffolds with very high shape fidelity and cell viability. Using such a system we bioprinted cell-laden hydrogel constructs recapitulating the layered structure of cartilage, namely, hyaline and calcified cartilage. The construct was assembled out of two bioinks specifically formulated to mimic the extracellular matrices present in the targeted tissues and to ensure the desired biological response of human bone marrow-derived mesenchymal stem cells and human articular chondrocytes. Homogeneous and gradient constructs were thoroughly characterized in vitro with respect to long-term cell viability and expression of hyaline and hypertrophic markers by means of real-time quantitative PCR and immunocytochemical staining. After 21 days of in vitro culture, we observed production of zone-specific matrix. The PCR analysis demonstrated upregulated expression of hypertrophic markers in the homogenous equivalent of calcified cartilage but not in the gradient heterogeneous construct. The regenerative potential was assessed in vivo in a rat model. The histological analysis of surgically damaged rat trochlea revealed beneficial effect of the bioprinted scaffolds on regeneration of OC defect when compared to untreated control.

3D bioprinted hydrogel model incorporating ß-tricalcium phosphate for calcified cartilage tissue engineering.

One promising strategy to reconstruct osteochondral defects relies on 3D bioprinted three-zonal structures comprised of hyaline cartilage, calcified cartilage, and subchondral bone. So far, several studies have pursued the regeneration of either hyaline cartilage or bone in vitro while-despite its key role in the osteochondral region-only few of them have targeted the calcified layer. In this work, we present a 3D biomimetic hydrogel scaffold containing ß-tricalcium phosphate (TCP) for engineering calcified cartilage through a co-axial needle system implemented in extrusion-based bioprinting process. After a thorough bioink optimization, we showed that 0.5% w/v TCP is the optimal concentration forming stable scaffolds with high shape fidelity and endowed with biological properties relevant for the development of calcified cartilage. In particular, we investigate the effect induced by ceramic nano-particles over the differentiation capacity of bioprinted bone marrow-derived human mesenchymal stem cells in hydrogel scaffolds cultured up to 21 d in chondrogenic media. To confirm the potential of the presented approach to generate a functional in vitro model of calcified cartilage tissue, we evaluated quantitatively gene expression of relevant chondrogenic (COL1, COL2, COL10A1, ACAN) and osteogenic (ALPL, BGLAP) gene markers by means of RT-qPCR and qualitatively by means of fluorescence immunocytochemistry.