Nanocomposite Fibrous Scaffolds Research Articles

Nanocomposite pectin fibers incorporating folic acid-decorated carbon quantum dots

Pectin has recently attracted increasing attention as an alternative biomaterial commonly used in biomedical and pharmaceutical fields. It shows several promising properties, including good biocompatibility, health benefits, nontoxicity, and biodegradation. In this research, novel nanocomposite fibers composed of folic acid-decorated carbon dots (CDs) in pectin/PEO matrix were fabricated using the electrospinning technique, which was never reported previously. Nitrogen-doped and nitrogen, sulfur-doped CDs were synthesized with average diameters of 2.74 nm and 2.17 nm using the one-step hydrothermal method, studied regarding their physicochemical, optical, and biocompatibility properties. The relative Quantum yields of N-CDs and N, S doped CDs were measured to be 54.7 % and 30.2 %, respectively. Nanocomposite fibers containing CDs were prepared, and their morphology, physicochemical properties, conductivity, drug release behavior, and cell viability were characterized. The results indicated that CDs improve fibrous scaffolds' tensile strength from 13.74 to 35.22 MPa while maintaining comparable extensibility. Furthermore, by incorporation of CDs in the prepared fibers conductivity enhanced from 8.69 × 10−9 S·m−1 to 1.36 × 10−4 S·m−1. The nanocomposite fibrous scaffold was also biocompatible with controlled drug release over 212 h, potentially promising tissue regeneration.

Electroconductive Polythiophene Nanocomposite Fibrous Scaffolds for Enhanced Osteogenic Differentiation via Electrical Stimulation.

Biophysical cues are key distinguishing characteristics that influence tissue development and regeneration, and significant efforts have been made to alter the cellular behavior by means of cell-substrate interactions and external stimuli. Electrically conductive nanofibers are capable of treating bone defects since they closely mimic the fibrillar architecture of the bone matrix and deliver the endogenous and exogenous electric fields required to direct cell activities. Nevertheless, previous studies on conductive polymer-based scaffolds have been limited to polypyrrole, polyaniline, and poly(3,4-ethylenedioxythiophene) (PEDOT). In the present study, chemically synthesized polythiophene nanoparticles (PTh NPs) are incorporated into polycaprolactone (PCL) nanofibers, and subsequent changes in physicochemical, mechanical, and electrical properties are observed in a concentration-dependent manner. In murine preosteoblasts (MC3T3-E1), we examine how substrate properties modified by adding PTh NPs contribute to changes in the cellular behavior, including viability, proliferation, differentiation, and mineralization. Additionally, we determine that external electrical stimulation (ES) mediated by PTh NPs positively affects such osteogenic responses. Together, our results provide insights into polythiophene's potential as an electroconductive composite scaffold material.

Nanocomposite fibrous scaffold mediated mandible reconstruction and dental rehabilitation: An experimental study in pig model

Mandible reconstruction and dental rehabilitation after trauma or tumor resection represent a serious challenge for maxillofacial surgeons. This study aimed to investigate the bone formation potential of nanocomposite fibrous scaffold (silica-nanohydroxyapatite-gelatin reinforced with poly L-lactic acid yarns - CSF) for delayed Titanium (Ti) implantation, which was compared to autograft (AG) taken from the iliac crest. The grafts were placed in critical-sized mandibular defects in an adult pig model for 6 months followed by dental implant placement for another 3 months. There was complete union and vascularised lamellar bone formation within 6 months. Moreover, the biological processes associated with angiogenesis, bone maturation and remodelling were seen in CSF, which was comparable to AG. Later, when Ti dental implant was placed on newly formed bone, CSF group demonstrated better osseointegration. In short, nanocomposite fibrous scaffold promoted quality bone formation in mandible defect that leads to successful osseointegration, suggesting as a potential candidate for implant-based rehabilitation in clinics in future.

Silver nanowire loaded poly(ε-caprolactone) nanocomposite fibers as electroactive scaffolds for skeletal muscle regeneration

Volumetric muscle loss (VML) due to trauma and tumor removal operations affects millions of people every year. Although skeletal muscle has a natural repair mechanism, it cannot provide self-healing above a critical level of VML. In this study, nanocomposite aligned fiber scaffolds as support materials were developed for volumetric skeletal muscle regeneration. For this purpose, silver nanowire (Ag NW) loaded poly(ε-caprolactone) (PCL) nanocomposite fiber scaffolds (PCL-Ag NW) were prepared to mimic the aligned electroactive structure of skeletal muscle and provide topographic and conductive environment to modulate cellular behavior and orientation. A computer-aided rotational wet spinning (RWS) system was designed to produce high-yield fiber scaffolds. Nanocomposite fiber bundles with lengths of 50 cm were fabricated via this computer-aided RWS system. The morphological, chemical, thermal properties and biodegradation profiles of PCL and PCL-Ag NW nanocomposite fibers were characterized in detail. The proliferation behavior and morphology of C2C12 mouse myoblasts were investigated on PCL and PCL-Ag NW nanocomposite fibrous scaffolds with and without electrical stimulation. Significantly enhanced cell proliferation was observed on PCL-Ag NW nanocomposite fibers compared to neat PCL fibers with electrical stimulations of 1.5 V, 3 V and without electrical stimulation.

Ca2+-supplying black phosphorus-based scaffolds fabricated with microfluidic technology for osteogenesis

Effective osteogenesis remains a challenge in the treatment of bone defects. The emergence of artificial bone scaffolds provides an attractive solution. In this work, a new biomineralization strategy is proposed to facilitate osteogenesis through sustaining supply of nutrients including phosphorus (P), calcium (Ca), and silicon (Si). We developed black phosphorus (BP)-based, three-dimensional nanocomposite fibrous scaffolds via microfluidic technology to provide a wealth of essential ions for bone defect treatment. The fibrous scaffolds were fabricated from 3D poly (l-lactic acid) (PLLA) nanofibers (3D NFs), BP nanosheets, and hydroxyapatite (HA)-porous SiO2 nanoparticles. The 3D BP@HA NFs possess three advantages: i) stably connected pores allow the easy entrance of bone marrow-derived mesenchymal stem cells (BMSCs) into the interior of the 3D fibrous scaffolds for bone repair and osteogenesis; ii) plentiful nutrients in the NFs strongly improve osteogenic differentiation in the bone repair area; iii) the photothermal effect of fibrous scaffolds promotes the release of elements necessary for bone formation, thus achieving accelerated osteogenesis. Both in vitro and in vivo results demonstrated that the 3D BP@HA NFs, with the assistance of NIR laser, exhibited good performance in promoting bone regeneration. Furthermore, microfluidic technology makes it possible to obtain high-quality 3D BP@HA NFs with low costs, rapid processing, high throughput and mass production, greatly improving the prospects for clinical application. This is also the first BP-based bone scaffold platform that can self-supply Ca2+, which may be the blessedness for older patients with bone defects or patients with damaged bones as a result of calcium loss.

Statistical optimization of novel acemannan polysaccharides assisted TiO2 nanorods based nanofibers for skin cancer application

In our work, we report the fabrication of novel aloe vera acemannan polysaccharides mediated green synthesized titanium dioxide nanorods loaded resveratrol drug incorporated poly vinyl alcohol nanofibers against A431 skin cancer cells. An innovative approach using RSM Box-Behnken design showed an R2 value of 97.23, 90.69 and 90.1 for fiber diameter, porosity and pore size demonstrating a good fitting for the experimental values observed. The composite nanofibers were characterized through SEM, XRD and FT-IR analysis. Cell viability percentage of about 37.2% at 500 µg/ml and a good zone of inhibition around 7.5 ± 0.5 mm and 5.4 ± 0.4 mm for S. aureus and P. aeruginosa respectively was observed for our PVA+TNR+RSV nanofibers. An effective scavenging of reactive oxygen species was confirmed from our DCFH-DA assay through real time fluorescence spectroscopy study. Thus, the acemannan carbohydrate polysaccharides adhered nano-composite fibrous scaffold proves to be a potent transdermal patch for melanoma applications.

A ternary nanocomposite fibrous scaffold composed of poly(ε‐caprolactone)/Gelatin/Gehlenite (Ca2Al2SiO7): Physical, chemical, and biological properties in vitro

Scaffolds are regarded as 3D substrates providing an appropriate environment through which the cells attachment, proliferation, and differentiation rate can be accelerated; based on their application, these scaffolds must have certain characteristics like suitable mechanical properties and porosity, desired degradation rate, and cell compatibility. In the present study, a novel nanocomposite fibrous scaffold composed of poly(ε‐caprolactone) (PCL)/Gelatin (GT)/Gehlenite (GLN) nanoparticles was fabricated through electrospinning method. Different weight ratios of GLN nanoparticles in the fibrous scaffolds were added and optimized and a series of samples including PCL, PCL/GT, and PCL/GT/GLN scaffolds were constructed in order to reach a better comparison between the scaffolds. It turned out that 7% was the optimized GLN weight ratio to be included into the scaffolds without destroying the fibers structure. Different characterization techniques were applied to assess the physical and chemical properties of scaffolds. Moreover, the scaffolds' degradation rate, bioactivity potential, cell viability, attachment, DAPI and Alizarin staining, and ALP activity were assessed in vitro as well. The overall results indicate that the ternary scaffold (PCL/GT/GLN7%) has a promising potential for bone tissue regeneration.

Biomimetically Mineralized Alginate Nanocomposite Fibers for Bone Tissue Engineering: Mechanical Properties and in Vitro Cellular Interactions.

We report herein the structural and mechanical properties and in vitro cellular response of hydroxyapatite (HAp)/alginate nanocomposite fibrous scaffolds mimicking the mineralized collagen fibrils of bone tissue. The biomimetically "engineered" nanocomposites, fabricated by electrospinning and in situ synthesis strategy, were compared with pure alginate nanofibers and micrometer-level HAp/alginate composite fibers. The tensile strength and elastic modulus of the nanocomposites increased by 79.3 and 158.4%, respectively, compared to those of alginate. The uniform nucleation and HAp nanocrystal growth on the alginate nanofibers resulted in such enhancement of the mechanical properties via a stress-transfer effect. Rat calvarial osteoblasts were stably attached and stretched more extensively on the nanocomposites' surface than on the pristine alginate. The controlled deposition of the HAp nanophase contributed to a much faster cell proliferation rate on the nanocomposites than on the others. The improved structural stability and osteoblast interactions suggest the fibrous nanocomposite scaffold's potential advantages for bone tissue regeneration.

Biodegradable nanocomposite fibrous scaffold mediated local delivery of vancomycin for the treatment of MRSA infected experimental osteomyelitis.

There is an increased demand for an ideal biodegradable biomaterial that eradicates infection, while concurrently promoting tissue regeneration in osteomyelitic bone, which eliminates the need for revision surgery. In this study, our objective was to evaluate the efficacy of a nanocomposite fibrous scaffold (silica coated nanohydroxyapatite-gelatin reinforced with poly-l-lactic acid yarns) containing vancomycin for treating methicillin-resistant Staphylococcus aureus (MRSA) induced osteomyelitis in rat models. The antibiotic was either incorporated during scaffold synthesis (SE-V) or loaded directly after the development of the scaffold (SA-V) at 5 wt% and 15 wt%. There was a sustained release of vancomycin from both the groups of scaffolds for 30 days and the released drug demonstrated antibacterial activity against MRSA. Furthermore, implantation of the composite scaffold into osteomyelitic rat femur resulted in significant bacterial reduction, mainly with 15 wt% drug and its efficacy was comparable to that of commercial graft Stimulan. Both drug entrapped and absorbed composite scaffolds promoted bone regeneration in 3 months, with no distinguishable difference between them. However, Stimulan resorbed fast and there were bone voids at the defect site after 3 months. Hence, the nanocomposite fibrous scaffold containing vancomycin can be proposed as a bi-functional graft that can reduce bacterial infection, while subsequently engineer new bone in osteomyelitis.

Bioinspired nanocomposite fibrous scaffold mediated delivery of ONO-1301 and BMP2 enhance bone regeneration in critical sized defect.

Treatment aiming to enhance bone tissue regeneration can benefit from multiple growth factor or small molecule delivery. In the present study, the objective was to evaluate the feasibility of using nanocomposite fibrous scaffold to deliver prostacyclin I2 agonist ONO-1301 in combination with BMP2 for treating critical sized bone defect. For this, ONO-1301 at three different concentrations (1.67μg, 5μg, 15μg) and a fixed dose of BMP2 (5μg) was loaded on the scaffold via physical adsorption. The results showed fast release of ONO-1301 for two weeks, whereas BMP2 exhibited slow and sustained release for four weeks. The scaffold with dual factors promoted the migration and osteogenic differentiation of mesenchymal stem cells (MSCs) in vitro when compared to the scaffold with BMP2 alone. It also augmented bone tissue regeneration in critical sized rat calvarial defect at 12weeks; mainly with lower dose of ONO-1301. However, synergistic effect on osteogenic differentiation and bone regeneration were not obtained through the concurrent release of BMP-2 and ONO-1301.

Evaluation of osseointegration of staged or simultaneously placed dental implants with nanocomposite fibrous scaffolds in rabbit mandibular defect.

Implant-supported dental prosthesis in patients with edentulism or those with reconstructed bone have long survival rate, but the success depends largely on the quality and quantity of the available bone at the recipient site. The usage of autograft is the gold standard treatment for vertical bone augmentation, but it has many limitations. In this study, we have developed a nanocomposite fibrous scaffold [silica coated nanoHA-gelatin reinforced with electrospun poly(L-lactic acid) (PLLA) nanoyarns] and evaluated its efficacy to promote osseointegration in rabbit mandibular defect in comparison to the scaffold without fibers and commercial nanoHA-collagen graft. For this, critical sized bilateral defect (10 mm length, 3 mm depth and 3 mm width) was created in rabbit mandible and dental implantation was done in two manners. In strategy 1, Ti dental implant was placed along with the scaffold and in strategy 2, the scaffold was implanted for 3 months to facilitate new bone formation followed by Ti dental implantation. In strategy 2, the fibrous scaffold could promote new bone formation and osseointegration in rabbit mandibular defect when compared to the scaffolds without fibers and commercial graft, but strategy 1 was not successful. These findings demonstrated that nanocomposite fibrous scaffold is a promising biomaterial to promote new bone formation and osseointegration in mandibular defect.

Dual release of growth factor from nanocomposite fibrous scaffold promotes vascularisation and bone regeneration in rat critical sized calvarial defect

A promising strategy for augmenting bone formation involves the local delivery of multiple osteoinductive and vasculogenic growth factors. However, success depends on sustained growth factor release and its appropriate combination to induce stem cells and osteogenic cells at the bony site. Herein, we have developed a nanocomposite fibrous scaffold loaded with fibroblast growth factor 2 (FGF2), vascular endothelial growth factor (VEGF) and bone morphogenetic protein 2 (BMP2) and its ability to promote vascularisation and bone regeneration in critical sized calvarial defect was compared to the scaffold with VEGF + BMP2 and FGF2 + BMP2. Simple loading of growth factors on the scaffold could provide a differential release pattern, both in vitro and in vivo (VEGF release for 1 week where as BMP2 and FGF2 release for 3 weeks). Among all the groups, dual growth factor loaded scaffold (VEGF + BMP2 & FGF2 + BMP2) enhanced vascularisation and new bone formation, but there was no difference between FGF2 and VEGF loaded scaffolds although its release pattern was different. FGF2 mainly promoted stem cell migration, whereas VEGF augmented new blood vessel formation at the defect site. This study suggests that biomimetic nanocomposite scaffold is a promising growth factor delivery vehicle to improve bone regeneration in critical sized bone defects. Statement of SignificanceMany studies have shown the effect of growth factors like VEGF-BMP2 or FGF2-BMP2 in enhancing bone formation in critical sized defects, but there are no reports that demonstrate the direct comparison of VEGF-BMP2 and FGF2-BMP2. In this study, we have developed a nanocomposite fibrous scaffold that could differentially release growth factors like VEGF, BMP2 and FGF2 (VEGF release for 1 week where as BMP2 and FGF2 release for 3 weeks), which in turn promoted neovascularisation and new bone formation in critical sized defects. There was no difference in vascularisation and bone formation induced by VEGF + BMP2 or FGF2 + BMP2. The growth factor was loaded in a simple manner, which would ensure ease of use for the end-user, especially for the surgeons treating a patient in an operating room.

Incorporation of nanofibrillated chitosan into electrospun PCL nanofibers makes scaffolds with enhanced mechanical and biological properties

Fabricating polycaprolactone (PCL) composite can be a facile approach to improve wettability, mechanical properties and cellular compatibility of PCL-based scaffolds. In this study, nanofibrillated chitosan (NC) was utilized as dispersing phase in PCL matrix to acquire electrospun nanocomposite fibrous scaffolds. Various amounts of NC were added to PCL solutions and the solutions were electrospun under constant electrospinning parameters. Adding NC to PCL solutions was accompanied with notable changes in the solutions viscosity, conductivity and electrospinnability. Whiles the pure PCL solutions with concentration of 8 wt. % and 10 wt. % were not electrospinnable, adding 5–10 % NC made them electrospinnable. The mechanical properties, wettability and cellular compatibility of electrospun PCL/NC composites were improved as well. PCL/NC scaffolds showed remarkable enhancement in both tensile strength and Young’s modulus compared to neat PCL scaffold. Contact angle measurements revealed improvement in wettability of scaffolds after adding NC. In addition, proliferation and adhesion of cells was enhanced when NC was incorporated to nanofibers. The results suggest PCL/NC as a proper scaffold for tissue engineering applications.

Novel polycaprolactone/hydroxyapatite nanocomposite fibrous scaffolds by direct melt-electrospinning writing

Melt electrospinning writing (MEW) using an automated stage has recently been developed as a direct additive manufacturing method for the fabrication of orderly, precise and complex porous 3D fibrous structures that can promote cell infiltration and growth. The further incorporation of inorganic particles within fibrous scaffolds is desirable in order to enhance bioactivity, however this remains challenging with the MEW fabrication process. To address this challenge, flexible, osteoconductive, medical grade polycaprolactone (m-PCL) - hydroxyapatite (HAp) composite 3D fibrous structures with high porosity (96–98%) and fully interconnected pore architectures were fabricated using MEW under precisely controlled parameters. The physical properties of these 3D fibrous composite scaffolds including fibre size, mechanical characteristics, and in vitro degradation rate were investigated. The results showed that the composite m-PCL/HAp fibrous scaffolds degraded in an alkaline environment at 37 °C faster than plain m-PCL and provided a favourable platform for the infiltration and growth of human osteoblasts. Moreover, confocal imaging confirmed that the scaffolds contained HAp nano-particles (NPs) which induced a more homogeneous distribution of cells within the scaffold particularly after 7 days of culture. Osteoblast activity and viability in the m-PCL/HAp composite scaffolds indicated a favourable cell/material interaction, suggesting great potential for use in mineralised tissue reconstruction / regeneration applications.

Self-supporting electrospun PHB and PHBV/organoclay nanocomposite fibrous scaffolds

Here we report the preparation of novel electrospun poly (β-hydroxybutyrate) (PHB) and poly(3-hydroxybutyrate-co-3-hydroxyvalerate) (PHBV)/organo-modified montmorillonite (OMMT) nanocomposite fibrous scaffolds. The incorporation of OMMT particles leads to significant amorphization of the deposited fibrous scaffolds and greatly improves the mechanical parameters. The addition of 5 wt% OMMT in the 12 wt% PHBV solution leads to predominant electrospinning and self-supporting 3D network fibrous scaffold formation. In the case of electrospun PHB mats, the addition of OMMT particles leads to significant reduction of average fiber diameter and formation of surface clay filled pore structure.

Nanobead-on-string composites for tendon tissue engineering.

Tissue engineering holds great potential in the production of functional substitutes to restore, maintain or improve the functionality in defective or lost tissues. So far, a great variety of techniques and approaches for fabrication of scaffolds have been developed and evaluated, allowing researchers to tailor precisely the morphological, chemical and mechanical features of the final constructs. Electrospinning of biocompatible and biodegradable polymers is a popular method for producing homogeneous nanofibrous structures, which might reproduce the nanosized organization of the tendons. Moreover, composite scaffolds obtained by incorporating nanoparticles within electrospun fibers have been lately explored in order to enhance the properties and the functionalities of the pristine polymeric constructs. The present study is focused on the design and fabrication of biocompatible electrospun nanocomposite fibrous scaffolds for tendon regeneration. A mixture of poly(amide 6) and poly(caprolactone) is electrospun to generate constructs with mechanical properties comparable to that of native tendons. To improve the biological activity of the constructs and modify their topography, wettability, stiffness and degradation rate, we incorporated silica particles into the electrospun substrates. The use of nanosize silica particles enables us to form bead-on-fiber topography, allowing the better exposure of ceramic particles to better profit their beneficial characteristics. In vitro biocompatibility studies using L929 fibroblasts demonstrated that the presence of 20 wt% of silica nanoparticles in the engineered scaffolds enhanced cell spreading and proliferation as well as extracellular matrix deposition. The results reveal that the electrospun nanocomposite scaffold represents an interesting candidate for tendon tissue engineering.

Strontium eluting nanofibers augment stem cell osteogenesis for bone tissue regeneration

Strontium is known to offer a therapeutic benefit to osteoporotic patients by promoting bone formation. Thus, toward engineering scaffolds for bone tissue regeneration we have prepared polymer nanocomposite scaffolds by electrospinning. Strontium carbonate nanoparticles (nSrCO3) were added to poly(ε-caprolactone) (PCL) at 10 and 20wt% to develop nanocomposite fibrous scaffolds (PCL/SrC10 and PCL/SrC20) with fiber diameter in the range of 300⿿500nm. Incorporation of nSrCO3 decreased crystallinity and the elastic modulus of PCL. The composite scaffolds released Sr2+ ions with up to 65ppm in 4days from the PCL/SrC20 scaffolds. Cell studies confirmed that the composite scaffold with 20% nSrCO3 enhanced proliferation of human mesenchymal stem cells in vitro. There was marked increase in mineral deposition up to four folds in PCL/SrC20 suggesting enhanced osteogenesis. This was corroborated by increased mRNA and protein expression of various osteogenic markers such as BMP-2, Osterix and Runx2 in the PCL/SrC20 fibers. Thus, incorporation of nSrCO3 in polymer scaffolds is a promising strategy for bone tissue engineering as an alternative to the use of labile growth factors to impart bioactivity to polymer scaffolds.

Enhancing the Biomechanical Performance of Anisotropic Nanofibrous Scaffolds in Tendon Tissue Engineering: Reinforcement with Cellulose Nanocrystals.

Anisotropically aligned electrospun nanofibrous scaffolds based on natural/synthetic polymer blends have been established as a reasonable compromise between biological and biomechanical performance for tendon tissue engineering (TE) strategies. However, the limited tensile properties of these biomaterials restrict their application in this field due to the load-bearing nature of tendon/ligament tissues. Herein, the use of cellulose nanocrystals (CNCs) as reinforcing nanofillers in aligned electrospun scaffolds based on a natural/synthetic polymer blend matrix, poly-ε-caprolactone/chitosan (PCL/CHT) is reported. The incorporation of small amounts of CNCs (up to 3 wt%) into tendon mimetic nanofiber bundles has a remarkable biomaterial-toughing effect (85% ± 5%, p < 0.0002) and raises the scaffolds mechanical properties to tendon/ligament relevant range (σ = 39.3 ± 1.9 MPa and E = 540.5 ± 83.7 MPa, p < 0.0001). Aligned PCL/CHT/CNC nanocomposite fibrous scaffolds meet not only the mechanical requirements for tendon TE applications but also provide tendon mimetic extracellular matrix (ECM) topographic cues, a key feature for maintaining tendon cell's morphology and behavior. The strategy proposed here may be extended to other anisotropic aligned nanofibrous scaffolds based on natural/synthetic polymer blends and enable the full exploitation of the advantages provided by their tendon mimetic fibrous structures in tendon TE.

Fabrication of anisotropically aligned nanofibrous scaffolds based on natural/synthetic polymer blends reinforced with cellulose nanocrystals for tendon tissue engineering

Event Abstract Back to Event Fabrication of anisotropically aligned nanofibrous scaffolds based on natural/synthetic polymer blends reinforced with cellulose nanocrystals for tendon tissue engineering Rui M. Domingues1, 2*, Silvia Chiera3, 4*, Pavel Gershovich1, 2, Antonella Motta3, 4*, Rui L. Reis1, 2* and Manuela E. Gomes1, 2* 1 University of Minho, 3B’s Research Group, Portugal 2 ICVS/3B's-PT Associate Laboratory, Portugal 3 University of Trento, Department of Industrial Engineering and Biotech Research Centre, Italy 4 European Institute of Excellence on Tissue Engineering and Regenerative Medicine, Italy Introduction: Tendon disorders and injuries are among the most common musculoskeletal problems and their regeneration after injury remains a significant challenge. Given the prevalent fibrous nature of tendons’ ECM, which exhibits an aligned and hierarchical organization in structures from the nano to the macro scale, uniaxial aligned electrospun nanofibers produced from natural/synthetic polymer blends are among the most successful tendon scaffolds in tissue engineering (TE) strategies[1]. These biomaterials can provide the topographical cues to direct cell adhesion and proliferation, as well as positively affecting cell’s differentiation, phenotype maintenance and matrix deposition. However, the limited mechanical properties of electrospun biomaterials restrict their potential application in this field. In the present study, we propose the use of cellulose nanocrystals (CNCs), the “nature carbon nanotubes”, as a strategy for the reinforcement of electrospun poly-ɛ-caprolactone-chitosan (PCL-C) nanofiber scaffolds without compromising their biological performance and thus expand their potential range of application in tendon TE strategies. Materials and Methods: Random (reference materials) and anisotropically aligned nanocomposite nanofiber scaffolds were fabricated by electrospinning using a static plate or rotating wheel as collecting targets (Fig. 1A), respectively, and their morphology, thermodynamic, and mechanical properties were investigated. The biological performance of human derived tendon cells (TCs) on aligned PCL-C and PCL-C/CNC nanofibers was compared with those of corresponding random nanofibrous materials as composition and topographical controls, assessing their biocompatibility and influence over cell morphology and orientation (Fig. 1B). Figure1. A) Schematic representation of the nanocomposite scaffolds fabrication process. B) SEM and confocal microscopy images of TCs seeded on PCL-C scaffolds filled with 3 wt% CNCs. Red: Phalloidin-rhodamine for F-actin; Blue: DAPI for nuclei. Results and discussion: CNCs were incorporated in PCL-C nanofiber bundles, resembling the typical aligned structure of tendon EMC while raising their mechanical properties up to a tendon meaningful level. In the optimized conditions, the incorporation of 3 wt.% CNCs in the PCL-C nanofiber bundles increased the Young’s modulus to 534 MPa and the tensile strength to 39 MPa, representing an increase of 132% and 83%, respectively, compared to reference PCL-C nanofiber scaffolds, without significantly affecting the biomaterial’s ductility. The incorporation of CNCs in PCL-C nanofibers didn’t affect the cytocompatibility of the biomaterials. Moreover, the aligned nanotopography of the material surface noticeably promoted TCs to orient along the axis of the scaffold through contact guidance mechanisms (Fig. 1B). The observed actin stress fibers demonstrated longitudinal arrangement along the elongated cell shape axis, and their nuclei were evidently more elongated and elliptically shaped as well. These findings are particularly important, as actin cytoskeleton has been shown to play important role on the stage of connective tissue development[2] and elongated cell shape has been demonstrated to be essential for tenocyte function and phenotype maintenance[3]. Conclusions: Our results demonstrate that aligned PCL-C-CNC nanocomposite fibrous scaffolds meet not only the mechanical requirements for tendon TE applications but also provide mimetic ECM topographic cues which are key features for maintaining tendon cell’s morphology and behavior. The authors also acknowledge the financial support from the Project RL1 - ABMR - NORTE-01-0124-FEDER-000016 cofinanced by North Portugal Regional Operational Programme (ON.2 – O Novo Norte), under the National Strategic Reference Framework (NSRF), through the European Regional Development Fund (ERDF).

Novel biomimetic hydroxyapatite/alginate nanocomposite fibrous scaffolds for bone tissue regeneration

Hydroxyapatite/alginate nanocomposite fibrous scaffolds were fabricated via electrospinning and a novel in situ synthesis of hydroxyapatite (HAp) that mimics mineralized collagen fibrils in bone tissue. Poorly crystalline HAp nanocrystals, as confirmed by X-ray diffractometer peak approximately at 2θ = 32° and Fourier transform infrared spectroscopy spectrum with double split bands of PO4(v 4) at 564 and 602 cm(-1), were induced to nucleate and grow at the [-COO(-)]-Ca(2+)-[-COO(-)] linkage sites on electrospun alginate nanofibers impregnated with PO4 (3-) ions. This novel process resulted in a uniform deposition of HAp nanocrystals on the nanofibers, overcoming the severe agglomeration of HAp nanoparticles processed by the conventional mechanical blending/electrospinning method. Preliminary in vitro cell study showed that rat calvarial osteoblasts attached more stably on the surface of the HAp/alginate scaffolds than on the pure alginate scaffold. In general, the osteoblasts were stretched and elongated into a spindle-shape on the HAp/alginate scaffolds, whereas the cells had a round-shaped morphology on the alginate scaffold. The unique nanofibrous topography combined with the hybridization of HAp and alginate can be advantageous in bone tissue regenerative medicine applications.