Development and optimisation of hydroxyapatite-polyethylene glycol diacrylate hydrogel inks for 3D printing of bone tissue engineered scaffolds.

In the event of excessive damage to bone tissue, the self-healing process alone is not sufficient to restore bone integrity. Three-dimensional (3D) printing, as an advanced additive manufacturing technology, can create implantable bone scaffolds with accurate geometry and internal architecture, facilitating bone regeneration. This study aims to develop and optimise hydroxyapatite-polyethylene glycol diacrylate (HA-PEGDA) hydrogel inks for extrusion 3D printing of bone tissue scaffolds. Different concentrations of HA were mixed with PEGDA, and further incorporated with pluronic F127 (PF127) as a sacrificial carrier. PF127 provided good distribution of HA nanoparticle within the scaffolds and improved the rheological requirements of HA-PEGDA inks for extrusion 3D printing without significant reduction in the HA content after its removal. Higher printing pressures and printing rates were needed to generate the same strand diameter when using a higher HA content compared to a lower HA content. Scaffolds with excellent shape fidelity up to 75-layers and high resolution (∼200 µm) with uniform strands were fabricated. Increasing the HA content enhanced the compression strength and decreased the swelling degree and degradation rate of 3D printed HA-PEGDA scaffolds. In addition, the incorporation of HA improved the adhesion and proliferation of human bone mesenchymal stem cells (hBMSCs) onto the scaffolds. 3D printed scaffolds with 2 wt% HA promoted osteogenic differentiation of hBMSCs as confirmed by the expression of alkaline phosphatase activity and calcium deposition. Altogether, the developed HA-PEGDA hydrogel ink has promising potential as a scaffold material for bone tissue regeneration, with excellent shape fidelity and the ability to promote osteogenic differentiation of hBMSCs.

3D printing of chitooligosaccharide-polyethylene glycol diacrylate hydrogel inks for bone tissue regeneration.

To date, lack of functional hydrogel inks has limited 3D printing applications in tissue engineering. This study developed a series of photocurable hydrogel inks based on chitooligosaccharide (COS)-polyethylene glycol diacrylate (PEGDA) for extrusion-based 3D printing of bone tissue scaffolds. The scaffolds were prepared by aza-Michael addition of COS and PEGDA followed by photopolymerisation of unreacted PEGDA. The hydrogel inks showed sufficient shear thinning properties required for extrusion 3D printing. The printed scaffolds exhibited excellent shape fidelity and fine microstructure with a resolution of 250 µm. By increasing the COS content, the swelling ratio of the scaffolds decreased, while the compressive strength increased. 3D printed COS-PEGDA scaffolds showed high viability of human bone mesenchymal stem cells in vitro. In addition, scaffolds containing 2 wt% COS showed significantly higher alkaline phosphatase activity, calcium deposition, and bioactivity in simulated body fluid compared to the control (PEGDA). Altogether, 3D printed COS-PEGDA scaffolds represent promising candidates for bone tissue regeneration.

Optimization of thermoresponsive chitosan/ß-glycerophosphate hydrogels for injectable neural tissue engineering application.

Regeneration of neural tissue and recovery of lost functions following an accident or disease to the central nervous system remains a major challenge worldwide, with limited treatment options available. The main reason for the failure of conventional therapeutic techniques to regenerate neural tissue is the presence of blood-brain barrier separating nervous system from systemic circulation and the limited capacity of self-regeneration of the nervous system. Injectable hydrogels have shown great promise for neural tissue engineering given their suitability for minimally invasive in situ delivery and tunable mechanical and biological properties. Chitosan (CS)/ß-glycerophosphate (ß-GP) hydrogels have been extensively investigated and shown regenerative potential in a wide variety of tissues such as bone and cartilage tissue engineering. However, the potential of CS/ß-GP hydrogels has never been tested for injectable neural tissue engineering applications. In the present study, CS/ß-GP hydrogels, consisting of 0.5-2% CS and 2-3% ß-GP, were prepared and characterized to investigate their suitability for injectable neural tissue engineering applications. The resulting CS/ß-GP-hydrogels showed a varying range of properties depending on the CS/ß-GP blend ratio. In particular, the 0.5%:3% and 0.75%:3% CS/ß-GP hydrogels underwent rapid gelation (3 min and 5 min, respectively) at physiological temperature (37 °C) and pH (7.4). They also had suitable porosity, osmolality, swelling behavior and biodegradation for tissue engineering. The biocompatibility of hydrogels was determined in vitro using PC12 cells, an immortalized cell line with neuronal cell-like properties, revealing that these hydrogels supported cell growth and proliferation. In conclusion, the thermoresponsive 0.5%:3% and 0.75%:3% CS/ß-GP hydrogels had the greatest potential for neural tissue engineering.

Green synthesis of chitooligosaccharide-PEGDA derivatives through aza-Michael reaction for biomedical applications.

Chitooligosaccharide (COS) as an emerging material carbohydrate polymer with huge potential in biomedical applications was prepared using a microwave-assisted process. The obtained COS exhibited reduced molecular weight (Mw) and higher water solubility in comparison to chitosan while preserving the main saccharide structure and same degree of deacetylation (DD). The optimized COS (13 kDa) was then used to synthesize a new family of COS-poly(ethylene glycol) diacrylate (PEGDA) derivatives based on aza-Michael addition of acrylate groups of PEGDA to the amine groups of COS in the absence of any exterior agents. The modulation of the reaction time, temperature, pH and NH2:acrylate molar ratio, had a strong influence on the Michael reaction progress. At higher degrees of conversion of acrylate groups, COS-PEGDA derivative formed gel with high biocompatibility towards human bone mesenchymal stem cells (hBMSCs). These COS-PEGDA hydrogels synthesized at mild conditions through a green chemistry are, therefore, an innovative system combining adequate biological performance, ease of preparation, and an environmentally friendly concept of production.

A review of current advancements for wound healing: Biomaterial applications and medical devices.

Wound healing is a complex process that is critical in restoring the skin's barrier function. This process can be interrupted by numerous diseases resulting in chronic wounds that represent a major medical burden. Such wounds fail to follow the stages of healing and are often complicated by a pro-inflammatory milieu attributed to increased proteinases, hypoxia, and bacterial accumulation. The comprehensive treatment of chronic wounds is still regarded as a significant unmet medical need due to the complex symptoms caused by the metabolic disorder of the wound microenvironment. As a result, several advanced medical devices, such as wound dressings, wearable wound monitors, negative pressure wound therapy devices, and surgical sutures, have been developed to correct the chronic wound environment and achieve skin tissue regeneration. Most medical devices encompass a wide range of products containing natural (e.g., chitosan, keratin, casein, collagen, hyaluronic acid, alginate, and silk fibroin) and synthetic (e.g., polyvinyl alcohol, polyethylene glycol, poly[lactic-co-glycolic acid], polycaprolactone, polylactic acid) polymers, as well as bioactive molecules (e.g., chemical drugs, silver, growth factors, stem cells, and plant compounds). This review addresses these medical devices with a focus on biomaterials and applications, aiming to deliver a critical theoretical reference for further research on chronic wound healing.

Fabrication and characterization of 3-dimensional electrospun poly(vinyl alcohol)/keratin/chitosan nanofibrous scaffold.

Layer-by-layer three-dimensional nanofibrous scaffolds (3DENS) were produced using the electrospinning technique. Interest in using biopolymers and application of electrospinning fabrication techniques to construct nanofibers for biomedical application has led to the development of scaffolds composed of PVA, keratin, and chitosan. To date, PVA/keratin blended nanofibers and PVA/chitosan blended nanofibers have been fabricated and studied for biomedical applications. Electrospun scaffolds comprised of keratin and chitosan have not yet been reported in published literature, thus a novel nanofibrous PVA/keratin/chitosan scaffold was fabricated by electrospinning. The resulting 3DENS were characterized using fourier transform infrared (FT-IR) spectroscopy, scanning electron microscopy (SEM), differential scanning colorimetry (DSC), and thermogravimetric analysis (TGA). Physiochemical properties of the polymer solutions such as viscosity (rheology) and conductivity were also investigated. The 3DENS possess a relatively uniform fibrous structure, suitable porosity, swelling properties, and degradation which are affected by the mass ratio of keratin, and chitosan to PVA. These results demonstrate that PVA/keratin/chitosan 3DENS have the potential for biomedical applications.

Types of biomaterials useful in brain repair.

Biomaterials is an emerging field in the study of brain tissue engineering and repair or neurogenesis. The fabrication of biomaterials that can replicate the mechanical and viscoelastic features required by the brain, including the poroviscoelastic responses, force dissipation, and solute diffusivity are essential to be mapped from the macro to the nanoscale level under physiological conditions in order for us to gain an effective treatment for neurodegenerative diseases. This research topic has identified a critical study gap that must be addressed, and that is to source suitable biomaterials and/or create reliable brain-tissue-like biomaterials. This chapter will define and discuss the various types of biomaterials, their structures, and their function-properties features which would enable the development of next-generation biomaterials useful in brain repair.

Chitooligosaccharides and their structural-functional effect on hydrogels: A review.

Chitosan's lack of solubility in physiological pH and high molecular weight (MW) limits its use in hydrogel scaffolds. Conversion of chitosan to low MW chitooligosaccharides (COS) not only imparts water solubility, it also enhances several other biological properties. When used in hydrogels, the low MW improves the performance of the hydrogels, e.g., the absorptive property, biocompatibility and cell proliferation capability. Most importantly, properties of COS, namely the degree of polymerization (DP) and degree of deacetylation (DD), can be altered to support specific functions in hydrogels used in regenerative medicine. Methods of preparation of COS must therefore be simple and convenient, leading to COS that can be readily used in biomedical applications without requiring extensive post-purification. This review compares these various methods of production of COS and discusses critically the specific advantages that COS can lend to hydrogels, which make COS better alternatives to chitosan in cell-related applications.

Chitosan hydrogels in 3D printing for biomedical applications.

Tissue engineering and regenerative medicine have entered a new stage of development by the recent progress in biology, material sciences, and particularly an emerging additive manufacturing technique, three-dimensional (3D) printing. 3D printing is an advanced biofabrication technique which can generate patient-specific scaffolds with highly complex geometries while hosting cells and bioactive agents to accelerate tissue regeneration. Chitosan hydrogels themselves have been widely used for various biomedical applications due to its abundant availability, structural features and favorable biological properties; however, the 3D printing of chitosan-based hydrogels is still under early exploration. Therefore, 3D printing technologies represent a new avenue to explore the potential application of chitosan as an ink for 3D printing, or as a coating on other 3D printed scaffolds. The combination of chitosan-based hydrogels and 3D printing holds much promise in the development of next generation biomedical implants.

Fabrication and characterisation of melt-extruded chitosan/keratin/PCL/PEG drug-eluting sutures designed for wound healing.

Diclofenac potassium loaded sutures based upon PEG/PCL/chitosan-keratin blends were fabricated using the hot-melt extrusion technique. Polymer sutures were evaluated based on their physical, thermal and mechanical properties, while the drug-eluting sutures were evaluated for drug release properties. Lastly, the performance of the drug-loaded sutures in the contact with the human keratinocyte cell line HaCat were assessed. Results showed that the sutures extruded homogeneously at a temperature of 63 ± 1 °C providing a uniform thickness of fibres. Analysis by Differential Scanning Calorimetry (DSC) and Thermogravimetric Analysis (TGA) showed that completely amorphous and miscible solid dispersions were created. Fourier transform infrared (FTIR) spectroscopy indicated that the presence of hydrogen bonds between the polymers improved material miscibility. Tensile properties of the sutures were clearly affected by the PEG, chitosan and keratin additions. The optimal formulation of tensile strength was obtained when PCL/PEG/chitosan-keratin were combined at a ratio of 80/19/1 w/w. Rapid and sustained drug release rates were achieved with the PEG/PCL/chitosan/keratin blends at various combinations. The composite of PCL/PEG/chitosan-keratin with 30 wt% of diclofenac potassium also exhibited high cell viability and wound healing rates in vitro cytotoxicity testing. The anti-inflammatory properties imparted by the PCL/PEG/chitosan/keratin/drug sutures may further the use of composite sutures for wound healing in clinical settings.

Biodegradable methacrylated casein for cardiac tissue engineering applications.

Casein is a naturally derived amino group (-NH2) rich protein, that enables surface functionalization leading to hydrophilicity, which in turn facilitates better cell adhesion. Casein obtained from either commercial ß-casein rich skim milk (A2 milk) or dissolved air flotation (DAF) technology was tested for its potential for tissue engineering applications in a comparative study. A novel biodegradable biomaterial was synthesized from casein by chemically modifying with methacrylic anhydride (MA) and combined with polyvinyl alcohol (PVA) and polyvinyl pyrrolidone (PVP) blend. The resulting methacrylated casein (CasMA) with the two polymers was processed into porous scaffolds with low and high MA concentrations to demonstrate CasMA's ease of modification and reproducibility. Fourier Transform Infrared Microscopy (FTIR) and Proton Nuclear Magnetic Resonance (1H NMR) revealed the presence of all the components and the successful modification of casein. The rheological and morphological analysis presented viscous behaviour and columnar hollow tube-like microstructures in agreement with the biomaterials' swelling and biodegradation behaviour. The live/dead in vitro assay showed high cell viability that agreed with the cell proliferation (MTT) assay in vitro, which indicated increased proliferation upon casein modification at appropriate biomaterial concentrations and volumes. This study not only showed a possible mechanism of casein methacrylation but also presented the potential use of waste materials like DAF-casein as a value-added product for tissue engineering applications.

Untapped potentials of hazardous nanoarchitectural biopolymers.

The First Industrial Revolution began when manual labour transitioned to machines. Fossil fuels and steam eventually replaced wood and water as an energy source used predominantly for the mechanized production of textiles and iron. The emergence of the required numerous enormous factories gave rise to smoke pollution due to the immense growth in coal consumption. The manufactured gas industry produced highly toxic effluent that was released into sewers and rivers polluting the water. Many pieces of legislation were introduced to overcome this issue, but with varying degrees of effectiveness. Alongside our growth in world population, the problems that we had with waste remained, but together with our increase in number the waste produced has also increased additionally. The immense volume of waste materials generated from human activity and the potentially detrimental effects on the environment and on public health have awakened in ourselves a critical need to embrace current scientific methods for the safe disposal of wastes. We are informed daily that our food waste must be better utilized to ensure enough food is available to feed the world's growing population in a sustainable way (Thyberg and Tonjes, 2016). Some things are easy, like waste food and cellulose products can be turned into compost, but how do we recycle sheep's wool? Or shrimp shells? Despite the fact that both these substances are hazardous, and have caused environmental and economic impact from being incinerated; but we anticipate that those substances may have the potential to convert into added value applications.We have been working in this area for over 15 years, working towards managing them and seeking their added value applications. We take the biological products, process (reconstitute) and engineer them into added value products such as functional and nanostructure materials including edible films, foams and composites including medical devices useful in the human body. Anything that we can ingest, should not cause an immune response in the human system. Natural biomacromolecules display the inherent ability to perform very specific chemical, mechanical or structural roles. Specifically, protein- and polysaccharide-based biomaterials have come to light as the most promising candidates for many biomedical applications due their biomimetic and nanostructured arrangements, their multi-functional features, and their capability to function as matrices that are capable of facilitating cell-cell and cell-matrix interactions.

Three-Dimensional Melt-Electrowritten Polycaprolactone/Chitosan Scaffolds Enhance Mesenchymal Stem Cell Behavior.

Melt electrowriting (MEW) is an emerging technique that precisely fabricates microfibrous scaffolds, ideal for tissue engineering, where biomimetic microarchitectural detail is required. Polycaprolactone (PCL), a synthetic polymer, was selected as the scaffold material due to its biocompatibility, biodegradability, mechanical strength, and melt processability. To increase PCL bioactivity, a natural polymer, chitosan, was added to construct MEW fibrous composite scaffolds. To date, this is the first study of its kind detailing the effects of stem cell behavior on PCL containing chitosan MEW scaffolds. The aim of this study was to melt electrowrite a range of PCL/chitosan tissue-engineered constructs (TECs) and assess their suitability to promote the growth of human bone-marrow-derived mesenchymal stem cells (hBMSCs). In vitro physical and biological characterizations of melt-electrowritten TECs were performed. Physical characterization showed that reproducible, layered micron-range scaffolds could be successfully fabricated. As well, cell migration and proliferation were assessed via an assay to monitor cell infiltration throughout the three-dimensional (3D) melt-electrowritten scaffold structure. A statistically significant increase (∼140%) in hBMSC proliferation in 1 wt % chitosan PCL blends in comparison to PCL-only scaffolds was found when monitored over two weeks. Overall, our study demonstrates the fabrication of melt-electrowritten PCL/chitosan composite scaffolds with controlled microarchitecture and their potential use for regenerative, tissue engineering applications.

Highly Stretchable and Flexible Melt Spun Thermoplastic Conductive Yarns for Smart Textiles.

This study demonstrates a scalable fabrication process for producing biodegradable, highly stretchable and wearable melt spun thermoplastic polypropylene (PP), poly(lactic) acid (PLA), and composite (PP:PLA = 50:50) conductive yarns through a dip coating process. Polydopamine (PDA) treated and poly(3,4-ethylenedioxythiophene):poly(styrenesulfonate) (PEDOT:PSS) coated conductive PP, PLA, and PP/PLA yarns generated electric conductivity of 0.75 S/cm, 0.36 S/cm and 0.67 S/cm respectively. Fourier Transform Infrared Spectroscopy (FTIR) confirmed the interactions among the functional groups of PP, PLA, PP/PLA, PDA, and PEDOT:PSS. The surface morphology of thermoplastic yarns was characterized by optical microscope and Scanning Electron Microscope (SEM). The mechanical properties of yarns were also assessed, which include tensile strength (TS), Young's modulus and elongation at break (%). These highly stretchable and flexible conductive PP, PLA, and PP/PLA yarns showed elasticity of 667%, 121% and 315% respectively. The thermal behavior of yarns was evaluated by differential scanning calorimetry (DSC) and thermo-gravimetric analysis (TGA). Wash stability of conductive yarns was also measured. Furthermore, ageing effect was determined to predict the shelf life of the conductive yarns. We believe that these highly stretchable and flexible PEDOT:PSS coated conductive PP, PLA, and PP/PLA composite yarns fabricated by this process can be integrated into textiles for strain sensing to monitor the tiny movement of human motion.

Melt Electrowritten Sandwich Scaffold Technique Using Sulforhodamine B to Monitor Stem Cell Behavior.

Background: Three-dimensional (3D) printing using melt electrowriting (MEW) technology is a recently developed technique to produce biocompatible micron-level mesh scaffolds layer-by-layer that can be seeded with cells for tissue engineering. Examining cell behavior, such as growth rate and migration, can be problematic in these opaque 3D scaffolds. A straightforward and quantitative method was developed to examine these cellular parameters on poly-ɛ-caprolactone (PCL) multilayered MEW scaffolds developed as components of the annulus fibrosus region of bioengineered intervertebral discs. Experiment: The anti-adhesion protein, bovine serum albumin (BSA), was used to coat plasticware to improve mesenchymal stem cell (T0523) adhesion to MEW scaffolds. Cells were seeded on circular MEW (cMEW) discs as defined growth starting points sandwiched between two test template scaffolds investigated at varying pore sizes. Cell expansion, growth, and migration were quantitated utilizing the protein-specific dye sulforhodamine B (SRB). Live cell imaging combined with image analysis were used to examine cell motility and expansion on 3D scaffolds. Results: After one coating of BSA, cells remained nonadherent for the duration of the study with cell spheroids formed and enlarging over 21 days and becoming entangled in MEW scaffold pores. Cells grown on the 250 µm pore size scaffolds exhibited a doubling time of 7 days, whereas the 400 µm pore size scaffolds time was 11.5 days. Conclusions: BSA coating of tissue culture dishes prevented surface adhesion of cells to vessel surfaces and promoted spheroid formation that encouraged attachment to the PCL scaffolds. Batch-printed cMEW scaffolds were useful as a defined starting point for quantitative assays that successfully measured cell migration, expansion and proliferation on test scaffolds. The SRB assay was shown to be a useful and straightforward way to quantitate cell numbers in multilayered MEW scaffolds. A pore size of 250 µm exhibited the fastest cell growth, spread, and expansion. Impact statement In this article, a new, useful, and straightforward method to quantitate cell numbers on three-dimensional (3D) melt electrowritten (MEW) scaffolds is presented. By using the sulforhodamine B assay on bovine serum albumin-coated dishes cell migration, expansion and proliferation in 3D printed MEW test scaffolds were quantitatively measured. Printed circular MEW (cMEW) scaffolds sandwiched between two MEW test scaffolds (Fig. 3) were used as defined cellular growth starting points with a particular pore size of 250 µm displaying the fastest cell growth and migration. This MEW sandwich technique could potentially be used to quantitate cell numbers and migration in other 3D multilayered MEW scaffold systems.

Bio-scaffolds produced from irradiated squid pen and crab chitosan with hydroxyapatite/ß-tricalcium phosphate for bone-tissue engineering.

In this study, bio-scaffolds have been developed using irradiated chitosan from different sources - squid pen (RS) and crab shell (RC) - with hydroxyapatite/ß-tricalcium phosphate (HA/ß-TCP) at a chitosan/HA/ß-TCP ratio of 50/30/20. The bio-scaffolds were prepared at two different freezing temperature (-20°C and -80°C) followed by lyophilisation. To enhance the mechanical properties, the bio-scaffolds were cross-linked using sodium tripolyphosphate (TPP) followed by lyophilisation. The composition and morphology of the bio-scaffolds were characterized using XRD, SEM, TEM and µ-CT. The pore size of the porous scaffolds ranged from 90 to 220µm and the scaffolds had 70-80% porosity. The scaffolds had a water uptake ratio of more than 10, and a controlled biodegradation in the range of 30-40%. These results suggest that the physical and biological properties of chitosan-based bio-scaffolds can be a promising biomaterial for bone-tissue regeneration.

Development and characterization of hydroxyapatite/ß-TCP/chitosan composites for tissue engineering applications.

Calcium phosphate ceramics that mimic bone composition provide interesting possibilities for the advancement in bone tissue engineering. The present study reports on a chitosan composite reinforced by hydroxyapatite (HA) and ß-tricalcium phosphate (ß-TCP) obtained from waste mussel shells and cross-linked using tripolyphosphate (TPP). The ratios of the ceramic components in composites were 20/10/70, 30/20/50 and 40/30/30 (HA/ß-TCP/CH, w/w %). Biodegradation rate, structural properties and in-vitro degradation of the bone-like composite scaffolds were investigated. The optimum amount of TPP required for composite was 2.5% and glycerol was used as plasticizer at an optimized concentration of 1%. Tripolyphosphate cross-linked chitosan composites were developed by freezing and lyophilisation. The Young's modulus of the scaffolds was increased from 4kPa to 17kPa and the porosity of composites dropped from 85 to 68% by increasing the HA/ß-TCP ratio. After 28days in physiological solution, bone-like composite scaffolds with a higher ratio of HA/ß-TCP (e.g. 40/30/30) showed about 2% lower biodegradation in comparison to scaffolds with a lower ratio of HA/ß-TCP (i.e. 20/10/70). The obtained data suggest that the chitosan based bone-like composites could be potential candidates for biomedical applications.

Bio-mimetic composite scaffold from mussel shells, squid pen and crab chitosan for bone tissue engineering.

In the present study, chitosan/hydroxyapatite (HA)/ß-tircalcium phosphate (ß-TCP) composites were produced using squid pen derived chitosan (CHS) and commercial crab derived chitosan (CHC). CHS was prepared from squid pens by alkaline N-deacetylation. HA and ß-TCP were extracted from mussel shells using a microwave irradiation method. Two different composites were prepared by incorporating 50% (w/w) HA/(ß-TCP) in CHS or CHC followed by lyophilization and cross-linking of composites by tripolyphosphate (TPP). The effect of different freezing temperatures of -20, -80 and -196 °C on the physicochemical characteristics of composites was investigated. A simulated body fluid (SBF) solution was used for preliminary in vitro study for 1, 7, 14 and 28 days and the composites were characterized by XRD, FTIR, TGA, SEM, µ-CT and ICP-MS. Porosity, pore size, water uptake; water retention abilities and in vitro degradations of the prepared composites were evaluated. The CHS composites were found to have higher porosity (62%) compared to the CHC composites (porosity 42%) and better mechanical properties. The results of this study indicated that composites produced at -20 °C had higher mechanical properties and lower degradation rate compared with -80 °C. Chitosan from the squid pen is an excellent biomaterial candidate for bone tissue engineering applications.

Aberrantly expressed long noncoding RNAs in human intervertebral disc degeneration: a microarray related study.

INTRODUCTION: In addition to the well-known short noncoding RNAs such as microRNAs (miRNAs), increasing evidence suggests that long noncoding RNAs (lncRNAs) act as key regulators in a wide aspect of biologic processes. Dysregulated expression of lncRNAs has been demonstrated being implicated in a variety of human diseases. However, little is known regarding the role of lncRNAs with regards to intervertebral disc degeneration (IDD). In the present study we aimed to determine whether lncRNAs are differentially expressed in IDD. METHODS: An lncRNA-mRNA microarray analysis of human nucleus pulposus (NP) was employed. Bioinformatics prediction was also applied to delineate the functional roles of the differentially expressed lncRNAs. Several lncRNAs and mRNAs were chosen for quantitative real-time PCR (qRT-PCR) validation. RESULTS: Microarray data profiling indicated that 116 lncRNAs (67 up and 49 down) and 260 mRNAs were highly differentially expressed with an absolute fold change greater than ten. Moreover, 1,052 lncRNAs and 1,314 mRNAs were differentially expressed in the same direction in at least four of the five degenerative samples with fold change greater than two. Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway analysis for the differentially expressed mRNAs indicated a number of pathways, such as extracellular matrix (ECM)-receptor interaction. A coding-noncoding gene co-expression (CNC) network was constructed for the ten most significantly changed lncRNAs. Annotation terms of the coexpressed mRNAs were related to several known degenerative alterations, such as chondrocyte differentiation. Moreover, lncRNAs belonging to a particular subgroup were identified. Functional annotation for the corresponding nearby coding genes showed that these lncRNAs were mainly associated with cell migration and phosphorylation. Interestingly, we found that Fas-associated protein factor-1 (FAF1), which potentiates the Fas-mediated apoptosis and its nearby enhancer-like lncRNA RP11-296A18.3, were highly expressed in the degenerative discs. Subsequent qRT-PCR results confirmed the changes. CONCLUSIONS: This is the first study to demonstrate that aberrantly expressed lncRNAs play a role in the development of IDD. Our study noted that up-regulated RP11-296A18.3 highly likely induced the over-expression of FAF1, which eventually promoted the aberrant apoptosis of disc cells. Such findings further broaden the understanding of the etiology of IDD.

Structure-function characteristics of the biomaterials based on milk-derived proteins.

There is an impetus on development of implantable biomaterials with the characteristics of improved biodegradability, bio-absorbability and wound healing activities. The milk proteins have valuable nutritional and biological properties, which lead to the promotion of quality health. In this study, whey protein isolate or WPI (highly aggregated) and its component lactalbumin (less aggregated) were melt blended with polycaprolactone (PCL) and then compression moulded into thin sheets ( approximately 1mm thickness). The effects of structural morphologies of the proteins on the mechanical, morphological, in vitro enzymatic degradation, and cytotoxicity and cell proliferation characteristics of the biomaterials were examined. In general, the tensile strength and modulus of the biomaterials decreased with increasing protein content. Compared to WPI, lactalbumin showed a better compatibility with the PCL matrix as observed in the mechanical properties and scanning electron microscopic morphology. The biomaterials exhibited a good retention of the mechanical characteristics after digestion in a physiologically simulated fluid containing trypsin enzyme. However, lactalbumin containing biomaterials showed a better retention of the tensile properties compared to WPI containing biomaterials. The cell culture studies indicated that the biomaterials have no cytotoxic effects, moreover they enhanced the proliferation of L929 cells compared to the pure PCL. Finally, this study indicated that the PCL based biomaterials with a protein content of 20wt% may be applied in fabrication of implantable devices for soft tissue engineering, where it requires a reasonably low to moderate mechanical strength (e.g., approximately 10MPa tensile strength), and improved biodegradability, biocompatibility and tissue healing activities.