The Phenotype of Mesenchymal Stromal Cell and Articular Chondrocyte Cocultures on Highly Porous Bilayer Poly-L-Lactic Acid Scaffolds Produced by Thermally Induced Phase Separation and Supplemented with Hydroxyapatite.

Bilayer scaffolds could provide a suitable topology for osteochondral defect repair mimicking cartilage and subchondral bone architecture. Hence, they could facilitate the chondro- and osteogenic lineage commitment of multipotent mesenchymal stromal cells (MSCs) with hydroxyapatite, the major inorganic component of bone, stimulating osteogenesis. Highly porous poly-L-lactic acid (PLLA) scaffolds with two layers of different pore sizes (100 and 250 µm) and hydroxyapatite (HA) supplementation were established by thermally induced phase separation (TIPS) to study growth and osteogenesis of human (h) MSCs. The topology of the scaffold prepared via TIPS was characterized using scanning electron microscopy (SEM), a microCT scan, pycnometry and gravimetric analysis. HMSCs and porcine articular chondrocytes (pACs) were seeded on the PLLA scaffolds without/with 5% HA for 1 and 7 days, and the cell attachment, survival, morphology, proliferation and gene expression of cartilage- and bone-related markers as well as sulfated glycosaminoglycan (sGAG) synthesis were monitored. All scaffold variants were cytocompatible, and hMSCs survived for the whole culture period. Cross-sections revealed living cells that also colonized inner scaffold areas, producing an extracellular matrix (ECM) containing sGAGs. The gene expression of cartilage and bone markers could be detected. HA represents a cytocompatible supplement in PLLA composite scaffolds intended for osteochondral defects.

Three-dimensional (3D) polylactic acid gradient scaffold to study the behavior of osteosarcoma cells under dynamic conditions.

This study adopts an in vitro method to recapitulate the behavior of Saos-2 cells, using a system composed of a perfusion bioreactor and poly-L-lactic acid (PLLA) scaffold fabricated using the low-cost thermally-induced phase separation (TIPS) technique. Four distinct scaffold morphologies with different pore sizes were fabricated, characterized by Scanning electron microscopy and micro-CT analysis and tested with osteosarcoma cells under static and dynamic environments to identify the best morphology for cellular growth. In order to accomplish this purpose, cell growth and matrix deposition of the Saos-2 osteosarcoma cell line were assessed using Picogreen and OsteoImage assays. The obtained data allowed us to identify the morphology that better promotes Saos-2 cellular activity in static and dynamic conditions. These findings provided valuable insights into scaffold design and fabrication strategies, emphasizing the importance of the dynamic culture to recreate an appropriate 3D osteosarcoma model. Remarkably, the gradient scaffold exhibits promise for osteosarcoma applications, offering the potential for targeted tissue engineering approaches.

Behavior of Calcium Phosphate-Chitosan-Collagen Composite Coating on AISI 304 for Orthopedic Applications.

Calcium phosphate/chitosan/collagen composite coating on AISI 304 stainless steel was investigated. Coatings were realized by galvanic coupling that occurs without an external power supply because it begins with the coupling between two metals with different standard electrochemical potentials. The process consists of the co-deposition of the three components with the calcium phosphate crystals incorporated into the polymeric composite of chitosan and collagen. Physical-chemical characterizations of the samples were executed to evaluate morphology and chemical composition. Morphological analyses have shown that the surface of the stainless steel is covered by the deposit, which has a very rough surface. XRD, Raman, and FTIR characterizations highlighted the presence of both calcium phosphate compounds and polymers. The coatings undergo a profound variation after aging in simulated body fluid, both in terms of composition and structure. The tests, carried out in simulated body fluid to scrutinize the corrosion resistance, have shown the protective behavior of the coating. In particular, the corrosion potential moved toward higher values with respect to uncoated steel, while the corrosion current density decreased. This good behavior was further confirmed by the very low quantification of the metal ions (practically absent) released in simulated body fluid during aging. Cytotoxicity tests using a pre-osteoblasts MC3T3-E1 cell line were also performed that attest the biocompatibility of the coating.

Effect of Polyhydroxyalkanoate (PHA) Concentration on Polymeric Scaffolds Based on Blends of Poly-L-Lactic Acid (PLLA) and PHA Prepared via Thermally Induced Phase Separation (TIPS).

Hybrid porous scaffolds composed of both natural and synthetic biopolymers have demonstrated significant improvements in the tissue engineering field. This study investigates for the first time the fabrication route and characterization of poly-L-lactic acid scaffolds blended with polyhydroxyalkanoate up to 30 wt%. The hybrid scaffolds were prepared by a thermally induced phase separation method starting from ternary solutions. The microstructure of the hybrid porous structures was analyzed by scanning electron microscopy and related to the blend composition. The porosity and the wettability of the scaffolds were evaluated through gravimetric and water contact angle measurements, respectively. The scaffolds were also characterized in terms of the surface chemical properties via Fourier transform infrared spectroscopy in attenuated total reflectance. The mechanical properties were analyzed through tensile tests, while the crystallinity of the PLLA/PHA scaffolds was investigated by differential scanning calorimetry and X-ray diffraction.

A dynamic air-liquid interface system for in vitro mimicking of the nasal mucosa.

The development of an in vitro 3D model for the growth of the nasal mucosa cells can improve the therapy and the study of pathological states for subjects with chronic airway conditions. We have previously characterized a system consisting of a scaffold with an internal channel and a perfusion bioreactor with two independent flows provided by an external and an internal circuit, respectively. In this paper, this system was designed as a model of the nasal cavity, in which cells, grown on the inner surface of the scaffold channel, would be in contact at the same time with both culture medium, supplied by the external circuit, and air, provided with the internal flow. To ensure adequate nutrient supply to the cells in the scaffold channel, the radial diffusion of the culture medium through the porous matrix was evaluated first in qualitative and, then, in quantitative terms, demonstrating the capability of the system to control the value and direction of this flux. As a preliminary study, the culture of epithelial cells in the scaffold channel is also discussed in static, maintaining the air-liquid interface condition for up to 3 weeks. Despite minor abnormalities, such as a gap between cell layers and some detachments from the scaffold, the scaffold ensured cell survival and growth during the experimental time.

A High-Throughput Mechanical Activator for Cartilage Engineering Enables Rapid Screening of in vitro Response of Tissue Models to Physiological and Supra-Physiological Loads.

Articular cartilage is crucially influenced by loading during development, health, and disease. However, our knowledge of the mechanical conditions that promote engineered cartilage maturation or tissue repair is still incomplete. Current in vitro models that allow precise control of the local mechanical environment have been dramatically limited by very low throughput, usually just a few specimens per experiment. To overcome this constraint, we have developed a new device for the high throughput compressive loading of tissue constructs: the High Throughput Mechanical Activator for Cartilage Engineering (HiT-MACE), which allows the mechanoactivation of 6 times more samples than current technologies. With HiT-MACE we were able to apply cyclic loads in the physiological (e.g., equivalent to walking and normal daily activity) and supra-physiological range (e.g., injurious impacts or extensive overloading) to up to 24 samples in one single run. In this report, we compared the early response of cartilage to physiological and supra-physiological mechanical loading to the response to IL-1ß exposure, a common but rudimentary in vitro model of cartilage osteoarthritis. Physiological loading rapidly upregulated gene expression of anabolic markers along the TGF-ß1 pathway. Notably, TGF-ß1 or serum was not included in the medium. Supra-physiological loading caused a mild catabolic response while IL-1ß exposure drove a rapid anabolic shift. This aligns well with recent findings suggesting that overloading is a more realistic and biomimetic model of cartilage degeneration. Taken together, these findings showed that the application of HiT-MACE allowed the use of larger number of samples to generate higher volume of data to effectively explore cartilage mechanobiology, which will enable the design of more effective repair and rehabilitation strategies for degenerative cartilage pathologies.

Current Strategies for Tracheal Replacement: A Review.

Airway cancers have been increasing in recent years. Tracheal resection is commonly performed during surgery and is burdened from post-operative complications severely affecting quality of life. Tracheal resection is usually carried out in primary tracheal tumors or other neoplasms of the neck region. Regenerative medicine for tracheal replacement using bio-prosthesis is under current research. In recent years, attempts were made to replace and transplant human cadaver trachea. An effective vascular supply is fundamental for a successful tracheal transplantation. The use of biological scaffolds derived from decellularized tissues has the advantage of a three-dimensional structure based on the native extracellular matrix promoting the perfusion, vascularization, and differentiation of the seeded cell typologies. By appropriately modulating some experimental parameters, it is possible to change the characteristics of the surface. The obtained membranes could theoretically be affixed to a decellularized tissue, but, in practice, it needs to ensure adhesion to the biological substrate and/or glue adhesion with biocompatible glues. It is also known that many of the biocompatible glues can be toxic or poorly tolerated and induce inflammatory phenomena or rejection. In tissue and organ transplants, decellularized tissues must not produce adverse immunological reactions and lead to rejection phenomena; at the same time, the transplant tissue must retain the mechanical properties of the original tissue. This review describes the attempts so far developed and the current lines of research in the field of tracheal replacement.

Solution-Based Processing for Scaffold Fabrication in Tissue Engineering Applications: A Brief Review.

The fabrication of 3D scaffolds is under wide investigation in tissue engineering (TE) because of its incessant development of new advanced technologies and the improvement of traditional processes. Currently, scientific and clinical research focuses on scaffold characterization to restore the function of missing or damaged tissues. A key for suitable scaffold production is the guarantee of an interconnected porous structure that allows the cells to grow as in native tissue. The fabrication techniques should meet the appropriate requirements, including feasible reproducibility and time- and cost-effective assets. This is necessary for easy processability, which is associated with the large range of biomaterials supporting the use of fabrication technologies. This paper presents a review of scaffold fabrication methods starting from polymer solutions that provide highly porous structures under controlled process parameters. In this review, general information of solution-based technologies, including freeze-drying, thermally or diffusion induced phase separation (TIPS or DIPS), and electrospinning, are presented, along with an overview of their technological strategies and applications. Furthermore, the differences in the fabricated constructs in terms of pore size and distribution, porosity, morphology, and mechanical and biological properties, are clarified and critically reviewed. Then, the combination of these techniques for obtaining scaffolds is described, offering the advantages of mimicking the unique architecture of tissues and organs that are intrinsically difficult to design.

Core-shell PLA/Kef hybrid scaffolds for skin tissue engineering applications prepared by direct kefiran coating on PLA electrospun fibers optimized via air-plasma treatment.

Over the recent years, there is a growing interest in electrospun hybrid scaffolds composed of synthetic and natural polymers that can support cell attachment and proliferation. In this work, the physical and biological properties of polylactic acid (PLA) electrospun mats coated with kefiran (Kef) were evaluated. Gravimetric, spectroscopic (FTIR-ATR) and morphological investigations via scanning electron microscopy confirmed the effective formation of a thin kefiran layer wrapped on the PLA fibers with an easy-tunable thickness. Air plasma pre-treatment carried out on PLA (P-PLA) affected both the morphology and the crystallinity of Kef coating as confirmed by differential scanning calorimetry and X-ray diffraction analyses. Scaffolds were mechanically characterized with tensile tests to evaluate the reinforcing action of the Kef coating. The water resistance of Kefiran coating in distilled water at 37 °C evaluated on both PLA/Kef and P-PLA/Kef was carried out by gravimetric and morphological analyses. Finally, cell culture assays with embryonic fibroblast cells were conducted on selected hybrid scaffolds to compare the cell proliferation, morphology, and collagen production with PLA and P-PLA electrospun scaffolds. Based on the results, we can demonstrate that direct coating of PLA from Kef/water solutions is an effective approach to prepare hybrid scaffolds with tunable properties and that the plasma pre-treatment enhances the affinity between PLA and Kefiran. In vitro tests demonstrated the great potential of PLA/Kef hybrid scaffolds for skin tissue engineering.

Novel dual-flow perfusion bioreactor for in vitro pre-screening of nanoparticles delivery: design, characterization and testing.

An advanced dual-flow perfusion bioreactor with a simple and compact design was developed and evaluated as a potential apparatus to reduce the gap between animal testing and drug administration to human subjects in clinical trials. All the experimental tests were carried out using an ad hoc Poly Lactic Acid (PLLA) scaffold synthesized via Thermally Induced Phase Separation (TIPS). The bioreactor shows a tunable radial flow throughout the microporous matrix of the scaffold. The radial perfusion was quantified both with permeability tests and with a mathematical model, applying a combination of Darcy's Theory, Bernoulli's Equation, and Poiseuille's Law. Finally, a diffusion test allowed to investigate the efficacy of the radial flow using Polymeric Fluorescent Nanoparticles (FNPs) mimicking drug/colloidal carriers. These tests confirmed the ability of our bioreactor to create a uniform distribution of particles inside porous matrices. All the findings candidate our system as a potential tool for drug pre-screening testing with a cost and time reduction over animal models.

Physical and biological properties of electrospun poly(d,l-lactide)/nanoclay and poly(d,l-lactide)/nanosilica nanofibrous scaffold for bone tissue engineering.

Electrospun scaffolds exhibiting high physical performances with the ability to support cell attachment and proliferation are attracting more and more scientific interest for tissue engineering applications. The inclusion of inorganic nanoparticles such as nanosilica and nanoclay into electrospun biopolymeric matrices can meet these challenging requirements. The silica and clay incorporation into polymeric nanofibers has been reported to enhance and improve the mechanical properties as well as the osteogenic properties of the scaffolds. In this work, for the first time, the physical and biological properties of polylactic acid (PLA) electrospun mats filled with different concentrations of nanosilica and nanoclay were evaluated and compared. The inclusion of the particles was evaluated through morphological investigations and Fourier transform infrared spectroscopy. The morphology of nanofibers was differently affected by the amount and kind of fillers and it was correlated to the viscosity of the polymeric suspensions. The wettability of the scaffolds, evaluated through wet contact angle measurements, slightly increased for both the nanocomposites. The crystallinity of the systems was investigated by differential scanning calorimetry highlighting the nucleating action of both nanosilica and nanoclay on PLA. Scaffolds were mechanically characterized with tensile tests to evaluate the reinforcing action of the fillers. Finally, cell culture assays with pre-osteoblastic cells were conducted on a selected composite scaffold in order to compare the cell proliferation and morphology with that of neat PLA scaffolds. Based on the results, we can convince that nanosilica and nanoclay can be both considered great potential fillers for electrospun systems engineered for bone tissue regeneration.

3D polymeric supports promote the growth and progression of anaplastic thyroid carcinoma.

Anaplastic thyroid carcinoma (ATC) is a rare and aggressive malignancy that accounts for the majority of deaths from all thyroid cancers. ATC exhibits invasiveness and highly resistance to conventional therapies which include cytotoxic chemotherapy, the combination of BRAF and MEK inhibition and, more recently, immunotherapies, that have shown promising but still limited results. A growing knowledge on ATC tumor biology is needed for developing more effective therapies with significant better survival. Researchers have begun to utilize 3D models to culture cancer cells for in vitro studies. In this work, C643 ATC cell line was cultured on polymeric scaffolds with high-interconnected porous matrix. They exhibited distinct viability, proliferation and 3D morphology similar to an in vivo solid tumor mass. We also carried out quantitative real-time PCR experiments for monitoring Cancer Stem Cells enrichment, since they are most probably the cause of tumor resistance, reoccurrence and metastasis. The same tests were performed after cell treatment with the chemotherapic Doxorubicin. An up-regulation of the analyzed stem-cell markers confirmed the high resistance to treatment of these cell line with respect to conventional drugs. In conclusion, 3D scaffolds could be an ideal platform for studying the mechanisms that regulate ACT growth and survival and also improving novel therapeutic approaches for treatment-resistant thyroid cancer.

Effect of hydroxyapatite concentration and size on morpho-mechanical properties of PLA-based randomly oriented and aligned electrospun nanofibrous mats.

The growing demand for nanofibrous biocomposites able to provide peculiar properties requires systematic investigations of processing-structure-property relationships. Understanding the morpho-mechanical properties of an electrospun scaffold as a function of the filler features and mat microstructure can aid in designing these systems. In this work, the reinforcing effect of micrometric and nanometric hydroxyapatite particles in polylactic acid-based electrospun scaffold presenting random and aligned fibers orientation, was evaluated. The particles incorporation was investigated trough Fourier transform infrared spectroscopy in attenuated total reflectance. The morphology of the nanofibers was analyzed through scanning electron microscopy and it was correlated with the viscosity of polymeric solutions studied by rheological measurements. Scaffolds were mechanical characterized with tensile tests in order to find a correlation between the preparation method and the strength of the mats. The influence of hydroxyapatite particles on the crystallinity of the composites was investigated by differential scanning calorimetry. Finally, cell culture assays with pre-osteoblatic cells were conducted on a selected composite scaffold in order to compare the cell proliferation and morphology with that of polylactic acid scaffolds. Based on the results, we can prove that polylactic acid/hydroxyapatite composites can be one of the biomaterials with the greatest potential for bone tissue regeneration.

Improvement of osteogenic differentiation of human mesenchymal stem cells on composite poly l-lactic acid/nano-hydroxyapatite scaffolds for bone defect repair.

Tissue engineering offers new approaches to repair bone defects, which cannot be repaired physiologically, developing scaffolds that mimic bone tissue architecture. Furthermore, biomechanical stimulation induced by bioreactor, provides biomechanical cues that regulate a wide range of cellular events especially required for cellular differentiation and function. The improvement of human mesenchymal stem cells (hMSCs) colonization in poly-l-lactic-acid (PLLA)/nano-hydroxyapatite (nHA) composite scaffold was evaluated in terms of cell proliferation (dsDNA content), bone differentiation (gene expression and protein synthesis) and ultrastructural analysis by comparing static (s3D) and dynamic (d3D) 3D culture conditions at 7 and 21 days. The colonization rate of hMSCs and osteogenic differentiation were amplified by d3D when physical stimulation was provided by a perfusion bioreactor. Increase in dsDNA content (p < 0.0005), up-regulation of RUNX2, ALPL, SPP1 (p < 0.0005) and SOX9 (p < 0.005) gene expression, and more calcium nodule formation (p < 0.0005) were observed in d3D cultures in comparison to s3D ones over time. Dynamic 3D culture, mimicking the mechanical signals of bone environment, improved significantly osteogenic differentiation of hMSCs on PLLA/nHA scaffold, without the addition of growth factors, confirming this composite scaffold suitable for bone regeneration.

Blend scaffolds with polyaspartamide/polyester structure fabricated via TIPS and their RGDC functionalization to promote osteoblast adhesion and proliferation.

Target of this work was to prepare a RGDC functionalized hybrid biomaterial via TIPS technique to achieve a more efficient control of osteoblast adhesion and diffusion on the three-dimensional (3D) scaffolds. Starting from a crystalline poly(l-lactic acid) (PLLA) and an amorphous α,ß-poly(N-2-hydroxyethyl) (2-aminoethylcarbamate)-d,l-aspartamide-graft-polylactic acid (PHEA-EDA-g-PLA) copolymer, blend scaffolds were characterized by an appropriate porosity and pore interconnection. The PHEA-EDA-PLA interpenetration with PLLA improved hydrolytic susceptibility of hybrid scaffolds. The presence of free amino groups on scaffolds allowed to tether the cyclic RGD peptide (RGDC) via Michael addition using the maleimide chemistry. Cell culture test carried out on preosteoblastic cells MC3T3-E1 incubated with scaffolds, has evidenced cell adhesion and proliferation. Furthermore, the presence of distributed bone matrix on all scaffolds was evaluated after 70 days compared to PLLA only samples.

A 3D­scaffold of PLLA induces the morphological differentiation and migration of primary astrocytes and promotes the production of extracellular vesicles.

The present study analyzed the ability of primary rat astrocytes to colonize a porous scaffold, mimicking the reticular structure of the brain parenchyma extracellular matrix, as well as their ability to grow, survive and differentiate on the scaffold. Scaffolds were prepared using poly­L­lactic acid (PLLA) via thermally­induced phase separation. Firstly, the present study studied the effects of scaffold morphology on the growth of astrocytes, evaluating their capability to colonize. Specifically, two different morphologies were tested, which were obtained by changing the polymer concentration in the starting solution. The structures were characterized by scanning electron microscopy, and a pore size of 20 µm (defined as the average distance between the pore walls) was detected. For comparison, astrocytes were also cultured in the traditional 2D culture system that we have been using since 2003. Then the effects of different substrates, such as collagen I and IV, and fibronectin were analyzed. The results revealed that the PLLA scaffolds, coated with collagen IV, served as very good matrices for astrocytes, which were observed to adhere, grow and colonize the matrix, acquiring their typical morphology. In addition, under these conditions, they secreted extracellular vesicles (EVs) that were compatible in size with exosomes. Their ability to produce exosomes was also suggested by transmission electron microscopy pictures which revealed both EVs and intracellular structures that could be interpreted as multivesicular bodies. The fact that these cells were able to adapt to the PLLA scaffold, together with our previous results, which demonstrated that brain capillary endothelial cells can grow and differentiate on the same scaffold, could support the future use of 3D brain cell co­culture systems.

PLLA scaffolds with controlled architecture as potential microenvironment for in vitro tumor model.

The "microenvironment" where a tumor develops plays a fundamental role in determining its progression, the onset of metastasis and, eventually, its resistance to therapies. Tumor cells can be considered more or less invasive depending both on the nature of the cells and on the site where they are located. Commonly adopted laboratory culture protocols for the investigation of tumor cells take usually place on standard two-dimensional supports. However, such cultures do not allow for reproduction of the biophysical properties of the tumor's microenvironment, thus causing the cells to lose most of their relevant characteristics. In this work MDA-MB 231 breast cancer cells were cultivated within Poly-l-Lactic Acid (PLLA) scaffolds produced via Thermally Induced Phase Separation (TIPS). Starting from a ternary solution (polymer-solvent-nonsolvent) we produced scaffolds with different morphologies, porosities and pore architectures. The influence of porosity and average pore size upon cell adhesion and growth were investigated by using Cell Counting Kit-8 (CCK-8) as cell viability test, a fluorescence assay staining cell with DAPI and Scanning Electron Microscopy (SEM). Our study demonstrates that the average pore size of the polymeric scaffolds influences both the cell adhesion and resulting morphology of the growing breast cancer cells. In particular, the reported data corroborate the evidence that an average pore size ranging from 40 to 50 µm induces tumor cell aggregation and the formation of the irregular tumor masses typically observed in-vivo. In addition, TIPS proved to be a suitable manufacturing technique for finely tuning the scaffolds' architecture, relevant to developing the most effective microenvironment for an in-vitro tumor cells growth closely mimicking in-vivo conditions.

Establishment of a pulmonary epithelial barrier on biodegradable poly-L-lactic-acid membranes.

Development of biocompatible and functional scaffolds for tissue engineering is a major challenge, especially for development of polarised epithelia that are critical structures in tissue homeostasis. Different in vitro models of the lung epithelial barrier have been characterized using non-degradable polyethylene terephthalate membranes which limits their uses for tissue engineering. Although poly-L-lactic acid (PLLA) membranes are biodegradable, those prepared via conventional Diffusion Induced Phase Separation (DIPS) lack open-porous geometry and show limited permeability compromising their use for epithelial barrier studies. Here we used PLLA membranes prepared via a modification of the standard DIPS protocol to control the membrane surface morphology and permeability. These were bonded to cell culture inserts for use in barrier function studies. Pulmonary epithelial cells (H441) readily attached to the PLLA membranes and formed a confluent cell layer within two days. This was accompanied by a significant increase in trans-epithelial electrical resistance and correlated with the formation of tight junctions and vectorial cytokine secretion in response to TNFα. Our data suggest that a structurally polarized and functional epithelial barrier can be established on PLLA membranes produced via a non-standard DIPS protocol. Therefore, PLLA membranes have potential utility in lung tissue engineering applications requiring bio-absorbable membranes.

Human nasoseptal chondrocytes maintain their differentiated phenotype on PLLA scaffolds produced by thermally induced phase separation and supplemented with bioactive glass 1393.

Damage of hyaline cartilage such as nasoseptal cartilage requires proper reconstruction, which remains challenging due to its low intrinsic repair capacity. Implantation of autologous chondrocytes in combination with a biomimetic biomaterial represents a promising strategy to support cartilage repair. Despite so far mostly tested for bone tissue engineering, bioactive glass (BG) could exert stimulatory effects on chondrogenesis. The aim of this work was to produce and characterize composite porous poly(L-lactide) (PLLA)/1393BG scaffolds via thermally induced phase separation (TIPS) technique and assess their effects on chondrogenesis of nasoseptal chondrocytes. The PLLA scaffolds without or with 1, 2.5, 5% BG1393 were prepared via TIPS technique starting from a ternary solution (polymer/solvent/non-solvent) in a single step. Scaffolds were characterized by scanning electron microscopy (SEM), X-ray diffraction (XRD) and differential scanning calorimetric analysis (DSC). Human nasoseptal chondrocytes were seeded on the scaffolds with 1 and 2.5% BG for 7 and 14 days and cell survival, attachment, morphology and expression of SOX9 and cartilage-specific extracellular cartilage matrix (ECM) components were monitored. The majority of chondrocytes survived on all PLLA scaffolds functionalized with BG for the whole culture period. Also inner parts of the scaffold were colonized by chondrocytes synthesizing an ECM which contained glycosaminoglycans. Type II collagen and aggrecan gene expression increased significantly in 1% BG scaffolds during the culture. Chondrocyte protein expression for cartilage ECM proteins indicated that the chondrocytes maintained their differentiated phenotype in the scaffolds. BG could serve as a cytocompatible basis for future scaffold composites for osteochondral cartilage defect repair. Abbreviations: AB: alcian blue ACAN: gene coding for aggrecan; BG: Bioactive glass; 2D: two-dimensional; 3D: three-dimensional; COL2A1: gene coding for type II collagen; DAPI: 4',6-diamidino-2-phenylindole; DMEM: Dulbecco's Modified Eagle's Medium; DMMB: dimethylmethylene blue; DSC: Differential scanning calorimetric analysis; ECM: extracellular matrix; EDTA: ethylenediaminetetraacetic acid; EtBr: ethidium bromide; FCS: fetal calf serum; FDA: fluorescein diacetate; GAG: glycosaminoglycans; HDPE: high density polyethylene; HE: hematoxylin and eosin staining; HCA: hydoxylapatite; PBE: phosphate buffered EDTA100 mM Na2HPO4 and 5 mM EDTA, pH8; PBS: phosphate buffered saline; PFA: paraformaldehyde; PG: proteoglycans; PI: propidium iodide; PLLA: Poly-L-Lactic Acid Scaffold; RT: room temperature; SD: standard deviation; SEM: scanning electron microscopy; sGAG: sulfated glycosaminoglycans; SOX9/Sox9: SRY (sex-determining region Y)-box 9 protein; TBS: TRIS buffered saline; TIPS: Thermally Induced Phase Separation; XRD: X-ray diffraction analysis.

Chitosan-Coating Deposition via Galvanic Coupling.

A galvanic method to deposit chitosan coatings on stainless steel substrate is reported. Deposition of suitable coatings is desired to improve biocompatibility and corrosion resistance of metallic medical devices to be implanted in human body. In the present work, a thin hydrogel layer of chitosan was deposited on 304SS by a galvanic displacement reaction, which is advantageous first as it does not require external power supply. 304SS was immersed into an aqueous solution of chitosan/lactic acid and electrochemically coupled with magnesium acting as a sacrificial anode. SEM images showed the formation of a uniform layer of chitosan with a thickness controlled by deposition time. Corrosion tests in simulating body fluid showed that chitosan coatings shift the corrosion potential of 304 substrates toward nobler values. Finally, the cytotoxicity of the coating was investigated through cell viability assays with osteoblastic cell MC3T3-E1. The results revealed highly satisfying biocompatibility of the coating.