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This study presents an X-ray computed nanotomography (nano-CT) based, high-resolution imaging technique. Thanks to a voxel resolution of 540 nm, this novel technique is suitable for observing the 3D morphology of soft biopolymeric scaffolds seeded with stem cells. A sample of highly porous collagen scaffold seeded with contrasted mesenchymal stem cells (MSC) was investigated by using lab-based nano-CT. The whole volume of the sample was analysed without its destruction. To evaluate the potential of nano-CT, a comparison measurement was done using a standard microscopy technique. Scanning electron microscopy (SEM) combined with energy dispersive X-ray analysis (EDX) established an extension and local accumulation of the contrasting agent - heavy metallic osmium tetroxide. The presented imaging technique is novel as it will help to understand better the behaviour of cells while interacting with three-dimensional biomaterials. This is crucial for both experimental and clinical tissue engineering applications in order to limit the risk of uncontrolled cell growth, and potentially tumour formation. LAY DESCRIPTION: Biomaterials play a crucial role in tissue engineering by serving as 3D scaffolds for cellular attachment, proliferation, and in growth ultimately leading to new tissue formation. Cell morphology and proliferation inside the 3D scaffold are necessary to know for assessing cell viability. However, these studies are usually negatively affected by the limitations of imaging techniques. We demonstrate that X-ray computed nanotomography (nano-CT), based on high-resolution imaging technique providing voxel resolution of 540 nm, is a suitable method for observing the 3D morphology of soft biopolymeric scaffolds seeded with stem cells. A sample of highly porous collagen scaffold seeded with contrasted mesenchymal stem cells (MSC) was investigated by using a lab-based nano-CT. The whole volume of the sample was analysed without its destruction. To evaluate the potential of nano-CT, a comparison measurement was done using a standard microscopy technique. Scanning electron microscopy in a combination with energy dispersive X-ray analysis established an extension and local accumulation of the contrasting agent - heavy metallic osmium tetroxide. The presented imaging technique is novel as it will help to understand better the behaviour of cells while interacting with three-dimensional biomaterials. This is crucial for both experimental and clinical tissue engineering applications in order to limit the risk of uncontrolled cell growth, and potentially tumour formation.

Additive manufacturing, also called 3D printing, is an effective method for preparing scaffolds with defined structure and porosity. The disadvantage of the technique is the excessive smoothness of the printed fibers, which does not support cell adhesion. In the present study, a 3D printed scaffold was combined with electrospun classic or structured nanofibers to promote cell adhesion. Structured nanofibers were used to improve the infiltration of cells into the scaffold. Electrospun layers were connected to 3D printed fibers by gluing, thus enabling the fabrication of scaffolds with unlimited thickness. The composite 3D printed/nanofibrous scaffolds were seeded with primary chondrocytes and tested in vitro for cell adhesion, proliferation and differentiation. The experiment showed excellent cell infiltration, viability, and good cell proliferation. On the other hand, partial chondrocyte dedifferentiation was shown. Other materials supporting chondrogenic differentiation will be investigated in future studies.

Materials combining meltblown and electrospinning products with hydroxyapatite powder as potential scaffolds for bone tissue engineering are presented here. The combination of these technologies and parameters and final micro-nanofibrous products are introduced too. The in-vitro testing compared meltblown material, meltblown material with sputtered particles, meltblown material combined with electrospun fibers, meltblown material combined with electrospun fibers and sputtered particles. All the fibrous materials are produced from polycaprolactone. The first in-vitro tests showed the high potential of developed composite materials in bone tissue engineering. The structure of the tested materials allows osteoblasts to proliferate into the sample inner structure with the significant contribution of nanofiber content to cell proliferation.

A three-dimensional scaffold of type I collagen and hydroxyapatite enriched with polycaprolactone nanofibers (Coll/HA/PCL), autologous mesenchymal stem cells (MSCs) in osteogenic media, and thrombocyte-rich solution (TRS) was an optimal implant for bone regeneration in vivo in white rabbits. Nanofibers optimized the viscoelastic properties of the Coll/HA scaffold for bone regeneration. MSCs and TRS in the composite scaffold improved bone regeneration. Three types of Coll/HA/PCL scaffold were prepared: an MSC-enriched scaffold, a TRS-enriched scaffold, and a scaffold enriched with both MSCs and TRS. These scaffolds were implanted into femoral condyle defects 6 mm in diameter and 10-mm deep. Untreated defects were used as a control. Macroscopic and histological analyses of the regenerated tissue from all groups were performed 12 weeks after implantation. The highest volume and most uniform distribution of newly formed bone occurred in defects treated with scaffolds enriched with both MSCs and TRS compared with that in defects treated with scaffolds enriched by either component alone. The modulus of elasticity in compressive testing was significantly higher in the Coll/HA/PCL scaffold than those without nanofibers. The composite Coll scaffold functionalized with PCL nanofibers and enriched with MSCs and TRS appears to be a novel treatment for bone defects.

A wide range of drug-delivery systems are currently attracting the attention of researchers. Nanofibers are very interesting carriers for drug delivery. This is because nanofibers are versatile, flexible, nanobiomimetic and similar to extracellular matrix components, possible to be functionalized both on their surface as well as in their core, and also because they can be produced easily and cost effectively. There have been increasing attempts to use nanofibers in the construction of a range of tissues, including cartilage and bone. Nanofibers have also been favorably engaged as a drug-delivery system in cell-free scaffolds. This short overview is devoted to current applications and to further perspectives of nanofibers as drug-delivery devices in the field of cartilage and bone regeneration, and also in osteochondral reconstruction.

Our aim was to use stereology to quantify the volume fraction of osteocyte lacunes, volume fraction of large blood vessels, numerical density of osteocyte lacunes, volume of osteocyte lacunae and bone surface in series of micro-CT images representing samples of spongy and compact bone of human tibia. The spongy bone had a smaller volume fraction of osteocyte lacunes, a greater numerical density of bone lacunes, a smaller volume of the lacunes within the same bone volume and a greater bone surface density when compared to the compact bone. Stereology provided us with data on hierarchical organization of bone structural heterogeneity with reasonable time costs.

Cell infiltration is a critical parameter for the successful development of 3D matrices for tissue engineering. Application of electrospun nanofibers in tissue engineering has recently attracted much attention. Notwithstanding several of their advantages, small pore size and small thickness of the electrospun layer limit their application for development of 3D scaffolds. Several methods for the pore size and/or electrospun layer thickness increase have been recently developed. Nevertheless, tissue engineering still needs emerging of either novel nanofiber-enriched composites or new techniques for 3D nanofiber fabrication. Forcespinning(®) seems to be a promising alternative. The potential of the Forcespinning(®) method is illustrated in preliminary experiment with mesenchymal stem cells.

Objectives We prepared 3D poly (e-caprolactone) (PCL) nanofibre scaffolds and tested their use for seeding, proliferation, differentiation and migration of mesenchymal stem cell (MSCs). Materials and methods 3D nanofibres were prepared using a special collector for common electrospinning; simultaneously, a 2D PCL nanofibre layer was prepared using a classic plain collector. Both scaffolds were seeded with MSCs and biologically tested. MSC adhesion, migration, proliferation and osteogenic differentiation were investigated. Results The 3D PCL scaffold was characterized by having better biomechanical properties, namely greater elasticity and resistance against stress and strain, thus this scaffold will be able to find broad applications in tissue engineering. Clearly, while nanofibre layers of the 2D scaffold prevented MSCs from migrating through the conformation, cells infiltrated freely through the 3D scaffold. MSC adhesion to the 3D nanofibre PCL layer was also statistically more common than to the 2D scaffold (P

Collagen/hydroxyapatite (HA) composite scaffolds are known to be suitable scaffolds for seeding with mesenchymal stem cells (MSCs) differentiated into osteoblasts and for the in vitro production of artificial bones. However, the optimal collagen/HA ratio remains unclear. Our study confirmed that a higher collagen content increased scaffold stiffness but that a greater stiffness was not sufficient for bone tissue formation, a complex process evidently also dependent on scaffold porosity. We found that the scaffold pore diameter was dependent on the concentration of collagen and HA and that it could play a key role in cell seeding. In conclusion, the optimal scaffold for new bone formation and cell proliferation was found to be a composite scaffold formed from 50 wt % HA in 0.5 wt % collagen I solution.

Hydrogels prepared from a mixture of fibrin and high-molecular weight (MW) hyaluronic acid (HA) were found to be suitable scaffolds for chondrocyte seeding and pig knee cartilage regeneration. Collagen in the hydrogels is not necessary for the formation of biomechanically stable tissue. Regenerated cartilage showed very good biomechanical and histological properties only 6 months after implantation. Notably, the quality of the healing process was dependent on the initial chondrocyte concentration of the scaffolds. These experiments were performed according to good laboratory practice (GLP).

Fibrous scaffolds are desired in tissue engineering applications for their ability to mimic extracellular matrix. In this study we compared fibrous scaffolds prepared from polycaprolactone using three different fabrication methods, electrospinning (ES), electro-blowing and melt-blown combined with ES. Scaffolds differed in morphology, fiber diameters and pore sizes. Mesenchymal stem cell adhesion, proliferation and osteogenic differentiation on scaffolds was evaluated. The most promising scaffold was shown to be melt-blown in combination with ES which combined properties of both technologies. Microfibers enabled good cell infiltration and nanofibers enhanced cell adhesion. This scaffold was used for further testing in critical sized defects in rabbits. New bone tissue formation occurred from the side of the treated defects, compared to a control group where only fat tissue was present. Polycaprolactone fibrous scaffold prepared using a combination of melt-blown and ES technology seems to be promising for bone regeneration. The practical application of results is connected with enormous production capacity and low cost of materials produced by melt-blown technology, compared to other bone scaffold fabrication methods.

Our aim was to show the benefits and limitations of histological assessment of healing supported by implantable biomaterials. We reviewed and showed photographs of the histological and immunohistochemical methods applicable for the assessment of desirable and undesirable effects of biomaterials on the healing of hard and soft tissues. Currently used methods for evaluating the microscopic effects of bioengineered materials on the recipient tissue are reviewed. For histopathological analysis, semiquantitative scoring systems can be used. Alternatively, the main tissue constituents may be quantified using continuous variables giving the numerical densities of cells, lengths of microvessels or connective tissue fibres, area surfaces, area and volumes fractions, or clustering and colocalization of microscopic objects. Using systematic uniform random sampling strategies at the level of tissue blocks, sections, and image fields leads to a reasonable low variability of the quantitative results.

Martin Plencner,1,2 Eva Prosecká,1,2 Michala Rampichová,2,3 Barbora East,4 Matej Buzgo,2,3 Lucie Vysloužilová,3 Jiří Hoch,4 Evžen Amler1,2,5 1Institute of Biophysics, 2nd Faculty of Medicine, Charles University in Prague, Prague, 2Laboratory of Tissue Engineering, Institute of Experimental Medicine, Academy of Sciences of the Czech Republic, Prague, 3University Center for Energy Efficient Buildings (UCEEB), The Czech Technical University in Prague, Bustehrad, 4Department of Surgery, 2nd Faculty of Medicine, Charles University in Prague, Prague, 5Faculty of Biomedical Engineering, Czech Technical University in Prague, Kladno, Czech Republic Abstract: Incisional hernia is the most common postoperative complication, affecting up to 20% of patients after abdominal surgery. Insertion of a synthetic surgical mesh has become the standard of care in ventral hernia repair. However, the implementation of a mesh does not reduce the risk of recurrence and the onset of hernia recurrence is only delayed by 2–3 years. Nowadays, more than 100 surgical meshes are available on the market, with polypropylene the most widely used for ventral hernia repair. Nonetheless, the ideal mesh does not exist yet; it still needs to be developed. Polycaprolactone nanofibers appear to be a suitable material for different kinds of cells, including fibroblasts, chondrocytes, and mesenchymal stem cells. The aim of the study reported here was to develop a functionalized scaffold for ventral hernia regeneration. We prepared a novel composite scaffold based on a polypropylene surgical mesh functionalized with poly-ε-caprolactone (PCL) nanofibers and adhered thrombocytes as a natural source of growth factors. In extensive in vitro tests, we proved the biocompatibility of PCL nanofibers with adhered thrombocytes deposited on a polypropylene mesh. Compared with polypropylene mesh alone, this composite scaffold provided better adhesion, growth, metabolic activity, proliferation, and viability of mouse fibroblasts in all tests and was even better than a polypropylene mesh functionalized with PCL nanofibers. The gradual release of growth factors from biocompatible nanofiber-modified scaffolds seems to be a promising approach in tissue engineering and regenerative medicine. Keywords: nanofibers, growth factors, polypropylene mesh, hernia regeneration, in vitro

Martin Plencner,1,2,* Barbora East,3,* Zbynek Tonar,4 Martin Otáhal,5 Eva Prosecká,1,2 Michala Rampichová,2,6 Tomáš Krejčí,3 Andrej Litvinec,2,7 Matej Buzgo,2,6 Andrea Míčková,1,2,6 Alois Nečas,8 Jirí Hoch,3 Evžen Amler1,2,9 1Institute of Biophysics, Second Faculty of Medicine, Charles University in Prague, Prague, 2Laboratory of Tissue Engineering, Institute of Experimental Medicine, Academy of Sciences of the Czech Republic, Prague, 3Department of Surgery, Second Faculty of Medicine, Charles University in Prague, Prague, 4Biomedical Centre, Faculty of Medicine in Pilsen, Charles University in Prague, Pilsen, 5Department of Anatomy and Biomechanics, Faculty of Physical Education and Sport, Charles University in Prague, Prague, 6University Center for Energy Efficient Buildings, Czech Technical University in Prague, Buštehrad, 7Department of Breeding and Zoohygiene of Laboratory Animals, Institute of Physiology, Academy of Sciences of the Czech Republic, Prague, 8Department of Surgery and Orthopedics, Small Animal Clinic, Faculty of Veterinary Medicine, University of Veterinary and Pharmaceutical Science Brno, Central European Institute of Technology, Brno, 9Faculty of Biomedical Engineering, Czech Technical University in Prague, Kladno, Czech Republic *These authors contributed equally to this work Abstract: Incisional hernia affects up to 20% of patients after abdominal surgery. Unlike other types of hernia, its prognosis is poor, and patients suffer from recurrence within 10years of the operation. Currently used hernia-repair meshes do not guarantee success, but only extend the recurrence-free period by about 5years. Most of them are nonresorbable, and these implants can lead to many complications that are in some cases life-threatening. Electrospun nanofibers of various polymers have been used as tissue scaffolds and have been explored extensively in the last decade, due to their low cost and good biocompatibility. Their architecture mimics the natural extracellular matrix. We tested a biodegradable polyester poly-Ԑ-caprolactone in the form of nanofibers as a scaffold for fascia healing in an abdominal closure-reinforcement model for prevention of incisional hernia formation. Both in vitro tests and an experiment on a rabbit model showed promising results. Keywords: nanofibers, growth factors, surgical mesh, hernia regeneration, in vivo

The aim of the study was to evaluate the effect of a cell-free hyaluronate/type I collagen/fibrin composite scaffold containing polyvinyl alcohol (PVA) nanofibers enriched with liposomes, basic fibroblast growth factor (bFGF) and insulin on the regeneration of osteochondral defects. A novel drug delivery system was developed on the basis of the intake effect of liposomes encapsulated in PVA nanofibers. Time-controlled release of insulin and bFGF improved MSC viability in vitro. Nanofibers functionalized with liposomes also improved the mechanical characteristics of the composite gel scaffold. In addition, time-controlled release of insulin and bFGF stimulated MSC recruitment from bone marrow in vivo. Cell-free composite scaffolds containing PVA nanofibers enriched with liposomes, bFGF, and insulin were implanted into seven osteochondral defects of miniature pigs. Control defects were left untreated. After 12 weeks, the composite scaffold had enhanced osteochondral regeneration towards hyaline cartilage and/or fibrocartilage compared with untreated defects that were filled predominantly with fibrous tissue. The cell-free composite scaffold containing PVA nanofibers, liposomes and growth factors enhanced migration of the cells into the defect, and their differentiation into chondrocytes; the scaffold was able to enhance the regeneration of osteochondral defects in minipigs.

Advanced biomechanical models of biological tissues should be based on statistical morphometry of tissue architecture. A quantitative description of the microscopic properties of real tissue samples is an advantage when devising computer models that are statistically similar to biological tissues in physiological or pathological conditions. The recent development of X-ray microtomography (micro-CT) has introduced resolution similar to that of routine histology. The aim of this chapter is to review and discuss both automatic image processing and interactive, unbiased stereological tools available for micro-CT scans and histological micrographs. We will demonstrate the practical usability of micro-CT in two different types of three-dimensional (3-D) ex vivo samples: (i) bone scaffolds used in tissue engineering and (ii) microvascular corrosion casts.

Pulsed laser deposition was proved as a suitable method for hydroxyapatite (HA) coating of coaxial poly-ɛ-caprolactone/polyvinylalcohol (PCL/PVA) nanofibers. The fibrous morphology of PCL/PVA nanofibers was preserved, if the nanofiber scaffold was coated with thin layers of HA (200 nm and 400 nm). Increasing thickness of HA, however, resulted in a gradual loss of fibrous character. In addition, biomechanical properties were improved after HA deposition on PCL/PVA nanofibers as the value of Young's moduli of elasticity significantly increased. Clearly, thin-layer hydroxyapatite deposition on a nanofiber surface stimulated mesenchymal stem cell viability and their differentiation into osteoblasts. The optimal depth of HA was 800 nm.

Non-woven textile mesh from polyglycolic acid (PGA) was found as a proper material for chondrocyte adhesion but worse for their proliferation. Neither hyaluronic acid nor chitosan nor polyvinyl alcohol (PVA) increased chondrocyte adhesion. However, chondrocyte proliferation suffered from acidic byproducts of PGA degradation. However, the addition of PVA and/or chitosan into a wet-laid non-woven textile mesh from PGA improved chondrocyte proliferation seeded in vitro on the PGA-based composite scaffold namely due to a diminished acidification of their microenvironment. This PVA/PGA composite mesh used in combination with a proper hydrogel minimized the negative effect of PGA degradation without dropping positive parameters of the PGA wet-laid non-woven textile mesh. In fact, presence of PVA and/or chitosan in the PGA-based wet-laid non-woven textile mesh even advanced the PGA-based wet-laid non-woven textile mesh for chondrocyte seeding and artificial cartilage production due to a positive effect of PVA in such a scaffold on chondrocyte proliferation.