Improvement of Osteogenic Differentiation of Mouse Pre-Osteoblastic MC3T3-E1 Cells on Core-Shell Polylactic Acid/Chitosan Electrospun Scaffolds for Bone Defect Repair.

Electrospun hybrid scaffolds composed of synthetic and natural polymers have gained increasing interest in tissue engineering applications over the last decade. In this work, scaffolds composed of polylactic acid electrospun fibers, either treated (P-PLA) or non-treated (PLA) with air-plasma, were coated with high molecular weight chitosan to create a core-shell microfibrous structure. The effective thickness control of the chitosan layer was confirmed by gravimetric, spectroscopic (FTIR-ATR) and morphological (SEM) investigations. The chitosan coating increased the fiber diameter of the microfibrous scaffolds while the tensile mechanical tests, conducted in dry and wet environments, showed a reinforcing action of the coating layer on the scaffolds, in particular when deposited on P-PLA samples. The stability of the Chi coating on both PLA and P-PLA substrates was confirmed by gravimetric analysis, while their mineralization capacity was evaluated though scanning electron microscopy (SEM) and energy-dispersive spectroscopy (EDS) after immersing the scaffolds in simulated body fluids (SBF) at 37 °C for 1 week. Sample biocompatibility was investigated through cell viability assay and SEM analysis on mouse pre-osteoblastic MC3T3-E1 cells grown on scaffolds at different times (1, 7, 14 and 21 days). Finally, Alizarin Red assay and qPCR analysis suggested that the combination of plasma treatment and chitosan coating on PLA electrospun scaffolds influences the osteoblastic differentiation of MC3T3-E1 cells, thus demonstrating the great potential of P-PLA/chitosan hybrid scaffolds for bone tissue engineering applications.

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.

Green and Integrated Wearable Electrochemical Sensor for Chloride Detection in Sweat.

Wearable sensors for sweat biomarkers can provide facile analyte capability and monitoring for several diseases. In this work, a green wearable sensor for sweat absorption and chloride sensing is presented. In order to produce a sustainable device, polylactic acid (PLA) was used for both the substrate and the sweat absorption pad fabrication. The sensor material for chloride detection consisted of silver-based reference, working, and counter electrodes obtained from upcycled compact discs. The PLA substrates were prepared by thermal bonding of PLA sheets obtained via a flat die extruder, prototyped in single functional layers via CO2 laser cutting, and bonded via hot-press. The effect of cold plasma treatment on the transparency and bonding strength of PLA sheets was investigated. The PLA membrane, to act as a sweat absorption pad, was directly deposited onto the membrane holder layer by means of an electrolyte-assisted electrospinning technique. The membrane adhesion capacity was investigated by indentation tests in both dry and wet modes. The integrated device made of PLA and silver-based electrodes was used to quantify chloride ions. The calibration tests revealed that the proposed sensor platform could quantify chloride ions in a sensitive and reproducible way. The chloride ions were also quantified in a real sweat sample collected from a healthy volunteer. Therefore, we demonstrated the feasibility of a green and integrated sweat sensor that can be applied directly on human skin to quantify chloride ions.

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.

Occurrence of Microplastics in Waste Sludge of Wastewater Treatment Plants: Comparison between Membrane Bioreactor (MBR) and Conventional Activated Sludge (CAS) Technologies.

In this study, the presence of microplastics in the sludge of three wastewater treatment plants (WWTPs) was examined. The investigated WWTPs operated based on a conventional activated sludge (CAS) process, with (W1) or without (W2) primary clarification, and a membrane bioreactor process (MBR) (W3). The microplastics (MPs) concentration in the samples of W3 was approximately 81.1 ± 4.2 × 103 particles/kg dry sludge, whereas MPs concentrations in W1 and W2 were 46.0 ± 14.8 × 103 particles/kg dry sludge and 36.0 ± 5.2 × 103 particles/kg dry sludge, respectively. Moreover, MPs mainly consisted of fragments (66-68%) in the CAS plants, whereas the fractions of MPs shapes in the MBR sludge were more evenly distributed, although fiber (47%) was the most abundant fraction. Furthermore, samples from the MBR showed a greater diversity in MPs composition. Indeed, all the main polyesters (i.e., textile fibers and polyethylene terephthalate), polyolefins (i.e., polyethylene and polypropylene) and rubber (i.e., polybutadiene) were observed, whereas only polybutadiene, cellulose acetate and polyester were detected in the CAS plants. These findings confirmed that MPs from wastewater are transferred and concentrated in the waste sludge. This is a critical finding since sludge disposal could become a new pathway for microplastic release into the environment and because MPs might affect the fouling behavior of the membrane.

Poly-l-Lactic Acid (PLLA)-Based Biomaterials for Regenerative Medicine: A Review on Processing and Applications.

Synthetic biopolymers are effective cues to replace damaged tissue in the tissue engineering (TE) field, both for in vitro and in vivo application. Among them, poly-l-lactic acid (PLLA) has been highlighted as a biomaterial with tunable mechanical properties and biodegradability that allows for the fabrication of porous scaffolds with different micro/nanostructures via various approaches. In this review, we discuss the structure of PLLA, its main properties, and the most recent advances in overcoming its hydrophobic, synthetic nature, which limits biological signaling and protein absorption. With this aim, PLLA-based scaffolds can be exposed to surface modification or combined with other biomaterials, such as natural or synthetic polymers and bioceramics. Further, various fabrication technologies, such as phase separation, electrospinning, and 3D printing, of PLLA-based scaffolds are scrutinized along with the in vitro and in vivo applications employed in various tissue repair strategies. Overall, this review focuses on the properties and applications of PLLA in the TE field, finally affording an insight into future directions and challenges to address an effective improvement of scaffold properties.

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.

Combining carvacrol and nisin in biodegradable films for antibacterial packaging applications.

In this work, the feasibility of antibacterial biopolymeric films containing carvacrol (CRV) and a nisin commercial formulation (Nis) for potential food packaging applications was investigated. As polymer matrix, a commercial biodegradable polymer formulation of Mater-Bi (MB) was chosen due to its significant food packaging applications. CRV and Nis were chosen due to their well-established antibacterial properties and their potential synergistic effect. MB/CRV, MB/Nis, and MB/CRV/Nis systems were produced by melt mixing and compression molding. The mechanical properties of the films were evaluated by tensile tests. Differential scanning calorimetry was assessed aiming at investigating the effect of the two compounds and their mixture on the thermal properties of MB. The release profile of CRV and Nis from the MB-based films was evaluated in water at 4 °C by UV-Vis measurements and it was fitted with a power-law model. The antibacterial activity of MB-based films was tested in vitro against Listeria monocytogenes, Salmonella enteritidis, Escherichia coli, and Staphylococcus aureus. The combination of CRV and Nis strongly affected the properties of the MB-based films and ensured higher antibacterial activity if compared to MB/CRV and MB/Nis systems.

Engineered Membranes for Residual Cell Trapping on Microfluidic Blood Plasma Separation Systems: A Comparison between Porous and Nanofibrous Membranes.

Blood-based clinical diagnostics require challenging limit-of-detection for low abundance, circulating molecules in plasma. Micro-scale blood plasma separation (BPS) has achieved remarkable results in terms of plasma yield or purity, but rarely achieving both at the same time. Here, we proposed the first use of electrospun polylactic-acid (PLA) membranes as filters to remove residual cell population from continuous hydrodynamic-BPS devices. The membranes hydrophilicity was improved by adopting a wet chemistry approach via surface aminolysis as demonstrated through Fourier Transform Infrared Spectroscopy and Water Contact Angle analysis. The usability of PLA-membranes was assessed through degradation measurements at extreme pH values. Plasma purity and hemolysis were evaluated on plasma samples with residual red blood cell content (1, 3, 5% hematocrit) corresponding to output from existing hydrodynamic BPS systems. Commercially available membranes for BPS were used as benchmark. Results highlighted that the electrospun membranes are suitable for downstream residual cell removal from blood, permitting the collection of up to 2 mL of pure and low-hemolyzed plasma. Fluorometric DNA quantification revealed that electrospun membranes did not significantly affect the concentration of circulating DNA. PLA-based electrospun membranes can be combined with hydrodynamic BPS in order to achieve high volume plasma separation at over 99% plasma purity.

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.

Polylactic is a Sustainable, Low Absorption, Low Autofluorescence Alternative to Other Plastics for Microfluidic and Organ-on-Chip Applications.

Organ-on-chip (OOC) devices are miniaturized devices replacing animal models in drug discovery and toxicology studies. The majority of OOC devices are made from polydimethylsiloxane (PDMS), an elastomer widely used in microfluidic prototyping, but posing a number of challenges to experimentalists, including leaching of uncured oligomers and uncontrolled absorption of small compounds. Here we assess the suitability of polylactic acid (PLA) as a replacement material to PDMS for microfluidic cell culture and OOC applications. We changed the wettability of PLA substrates and demonstrated the functionalization method to be stable over a time period of at least 9 months. We successfully cultured human cells on PLA substrates and devices, without coating. We demonstrated that PLA does not absorb small molecules, is transparent (92% transparency), and has low autofluorescence. As a proof of concept of its manufacturability, biocompatibility, and transparency, we performed a cell tracking experiment of prostate cancer cells in a PLA device for advanced cell culture.

Development of injectable and durable kefiran hydro-alcoholic gels.

Injectable, in-situ forming kefiran gels have been developed for potential applications as implantable drug delivery devices or scaffolds for tissue regeneration. Concentrated solutions (4, 5 and 6%w) of kefiran, extracted from kefir grains, have been assessed in term of viscosity and injectability through G26 syringe needles, and for their ability to undergo gelation upon mixing with different alcohols. Propylene glycol (PG) has been selected as gelling agent because it ensures homogenous gelation in relatively short times (from few minutes up to 6 h). The investigation of the rheological behavior of kefiran/PG gels varying polymer concentration and temperature (25 °C and 37 °C) has provided interesting hints to support a possible gelation mechanism that accounts also for the observed influence of the alcohol type. Finally, the study of kefiran/PG gels has been complemented with the investigation on selected formulations of the swelling/degradation behavior upon immersion in isotonic buffer solution for up to 40 days at 37 °C; of the ability of the gels to retain and/or release two model molecules; and within vitro cell viability and cytotoxicity tests, to support the absence of toxic effects on cells induced by direct contact with the gels or by leached components from these gels.

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.