Biocompatible 3D printed polymers <i>via</i> fused deposition modelling direct C₂C₁₂ cellular phenotype <i>in vitro</i>

The capability to 3D print bespoke biologically receptive parts within short time periods has driven the growing prevalence of additive manufacture (AM) technology within biological settings, however limited research concerning cellular interaction with 3D printed polymers has been undertaken. In this work, we used skeletal muscle C2C12 cell line in order to ascertain critical evidence of cellular behaviour in response to multiple bio-receptive candidate polymers; polylactic acid (PLA), acrylonitrile butadiene styrene (ABS), polyethylene terephthalate (PET) and polycarbonate (PC) 3D printed via fused deposition modelling (FDM). The extrusion based nature of FDM elicited polymer specific topographies, within which C2C12 cells exhibited reduced metabolic activity when compared to optimised surfaces of tissue culture plastic, however assay viability readings remained high across polymers outlining viable phenotypes. C2C12 cells exhibited consistently high levels of morphological alignment across polymers, however differential myotube widths and levels of transcriptional myogenin expression appeared to demonstrate response specific thresholds at which varying polymer selection potentiates cellular differentiation, elicits pre-mature early myotube formation and directs subsequent morphological phenotype. Here we observed biocompatible AM polymers manufactured via FDM, which also appear to hold the potential to simultaneously manipulate the desired biological phenotype and enhance the biomimicry of skeletal muscle cells in vitro via AM polymer choice and careful selection of machine processing parameters. When considered in combination with the associated design freedom of AM, this may provide the opportunity to not only enhance the efficiency of creating biomimetic models, but also to precisely control the biological output within such scaffolds

Some Pharmacological And Phytochemical Studies Of Clitoria Ternatea Extracts

In this thesis, detailed work was carried out to explore and determine the phytochemical screening, potential anti-infective, antioxidant and analgesic properties of C. ternatea plant parts using various bioassays techniques and appropriate experimental models. The initial qualitative phytochemical screening shows that C. ternatea plant parts contain tannin, phlobatannin, flavonoid, anthraquinone, alkaloid, saponin, cardiac glycosides, volatile oils, steroids and terpenoids. The flavonoids and phenolic content of the various plant parts were also determined in the plant parts. Analysis shows that C. ternatea flower extract has both high phenolic (104.77 ± 2.64 mg GAE/g) and flavonoid (28.10 ± 4.13 mg CE/g) content than other plant parts. This is evident by the fact that C. ternatea flower extract exhibited the highest antioxidant activity (FRAP: 6.80 ± 0.32 μM Fe(II)/g ; DPPH IC50 : 9.17 ± 0.003 μg/mL) when compared to other parts of the plant. One of the bioactives identified in the flower extract was kaempferol-3-glucoside (K3G) which is a flavonoid compound. Kaempferol-3-glucoside was also found in C. ternatea stem and leaf. This could be one of the identified bioactives responsible for the antioxidant activity. As for the in vitro anti-infective screening, both leaf and root crude extracts demonstrated marked antimicrobial activities. Interestingly, the leaf extract demonstrated a remarkable antifungal activity against Aspergillus niger with a MIC and MFC of 0.4 and 0.8 mg/mL, respectively. This was evident by the scanning electron microscope (SEM) analysis which showed a marked morphological alteration of the A. niger hyphal wall and conidiophores after exposure to C. ternatea leaf extract when compared to control

Materials 3D printed via fused deposition modelling elicit myofibrillar alignment and enhanced differentiation of skeletal muscle cells in-vitro

Materials 3D printed via fused deposition modelling elicit myofibrillar alignment and enhanced differentiation of skeletal muscle cells in-vitr

Solid-state additive manufacturing for metallized optical fiber integration

The formation of smart, Metal Matrix Composite (MMC) structures through the use of solid-state Ultrasonic Additive Manufacturing (UAM) is currently hindered by the fragility of uncoated optical fibers under the required processing conditions. In this work, optical fibers equipped with metallic coatings were fully integrated into solid Aluminum matrices using processing parameter levels not previously possible. The mechanical performance of the resulting manufactured composite structure, as well as the functionality of the integrated fibers, was tested. Optical microscopy, Scanning Electron Microscopy (SEM) and Focused Ion Beam (FIB) analysis were used to characterize the interlaminar and fiber/matrix interfaces whilst mechanical peel testing was used to quantify bond strength. Via the integration of metallized optical fibers it was possible to increase the bond density by 20–22%, increase the composite mechanical strength by 12–29% and create a solid state bond between the metal matrix and fiber coating; whilst maintaining full fiber functionality

Mechanical loading stimulates hypertrophy in tissue-engineered skeletal muscle: Molecular and phenotypic responses

Mechanical loading of skeletal muscle results in molecular and phenotypic adaptations typified by enhanced muscle size. Studies on humans are limited by the need for repeated sampling, and studies on animals have methodological and ethical limitations. In this investigation, three-dimensional skeletal muscle was tissue-engineered utilizing the murine cell line C2C12, which bears resemblance to native tissue and benefits from the advantages of conventional in vitro experiments. The work aimed to determine if mechanical loading induced an anabolic hypertrophic response, akin to that described in vivo after mechanical loading in the form of resistance exercise. Specifically, we temporally investigated candidate gene expression and Akt-mechanistic target of rapamycin 1 signalling along with myotube growth and tissue function. Mechanical loading (construct length increase of 15%) significantly increased insulin-like growth factor-1 and MMP-2 messenger RNA expression 21 hr after overload, and the levels of the atrophic gene MAFbx were significantly downregulated 45 hr after mechanical overload. In addition, p70S6 kinase and 4EBP-1 phosphorylation were upregulated immediately after mechanical overload. Maximal contractile force was augmented 45 hr after load with a 265% increase in force, alongside significant hypertrophy of the myotubes within the engineered muscle. Overall, mechanical loading of tissue-engineered skeletal muscle induced hypertrophy and improved force production

Functional regeneration of tissue engineered skeletal muscle in vitro is dependent on the inclusion of basement membrane proteins

Skeletal muscle has a high regenerative capacity, injuries trigger a regenerative program which restores tissue function to a level indistinguishable to the pre-injury state. However, in some cases where significant trauma occurs, such as injuries seen in military populations, the regenerative process is overwhelmed and cannot restore full function. Limited clinical interventions exist which can be used to promote regeneration and prevent the formation of non-regenerative defects following severe skeletal muscle trauma. Robust and reproducible techniques for modelling complex tissue responses are essential to promote the discovery of effective clinical interventions. Tissue engineering has been highlighted as an alternative method, allowing the generation of three-dimensional in vivo like tissues without laboratory animals. Reducing the requirement for animal models promotes rapid screening of potential clinical interventions, as these models are more easily manipulated genetically and pharmacologically and reduce the associated cost and complexity, whilst increasing access to models for laboratories without animal facilities. In this study an in vitro chemical injury using barium chloride is validated using the C2C12 myoblast cell line, and is shown to selectively remove multinucleated myotubes, whilst retaining a regenerative mononuclear cell population. Monolayer cultures showed limited regenerative capacity, with basement membrane supplementation or extended regenerative time incapable of improving the regenerative response. Conversely tissue engineered skeletal muscles, supplemented with basement membrane proteins, showed full functional regeneration, and a broader in vivo like inflammatory response. This work outlines a freely available and open access methodology to produce a cell line-based tissue engineered model of skeletal muscle regeneration

An assessment of myotube morphology, matrix deformation, and myogenic mRNA expression in custom-built and commercially available engineered muscle chamber configurations

There are several three-dimensional (3D) skeletal muscle (SkM) tissue engineered models reported in the literature. 3D SkM tissue engineering (TE) aims to recapitulate the structure and function of native (in vivo) tissue, within an in vitro environment. This requires the differentiation of myoblasts into aligned multinucleated myotubes surrounded by a biologically representative extracellular matrix (ECM). In the present work, a new commercially available 3D SkM TE culture chamber manufactured from polyether ether ketone (PEEK) that facilitates suitable development of these myotubes is presented. To assess the outcomes of the myotubes within these constructs, morphological, gene expression, and ECM remodeling parameters were compared against a previously published custom-built model. No significant differences were observed in the morphological and gene expression measures between the newly introduced and the established construct configuration, suggesting biological reproducibility irrespective of manufacturing process. However, TE SkM fabricated using the commercially available PEEK chambers displayed reduced variability in both construct attachment and matrix deformation, likely due to increased reproducibility within the manufacturing process. The mechanical differences between systems may also have contributed to such differences, however, investigation of these variables was beyond the scope of the investigation. Though more expensive than the custom-built models, these PEEK chambers are also suitable for multiple use after autoclaving. As such this would support its use over the previously published handmade culture chamber system, particularly when seeking to develop higher-throughput systems or when experimental cost is not a factor

Neural and aneural regions generated by the use of chemical surface coatings

The disordered environment found in conventional neural cultures impedes various applications where cell directionality is a key process for functionality. Neurons are highly specialized cells known to be greatly dependent on interactions with their surroundings. Therefore, when chemical cues are incorporated on the surface material, a precise control over neuronal behavior can be achieved. Here, the behavior of SH-SY5Y neurons on a variety of self-assembled monolayers (SAMs) and polymer brushes both in isolation and combination to promote cellular spatial control was determined. APTES and BIBB coatings promoted the highest cell viability, proliferation, metabolic activity, and neuronal maturation, while low cell survival was seen on PKSPMA and PMETAC surfaces. These cell-attractive and repulsive surfaces were combined to generate a binary BIBB-PKSPMA coating, whereby cellular growth was restricted to an exclusive neural region. The utility of these coatings when precisely combined could act as a bioactive/bioinert surface resulting in a biomimetic environment where control over neuronal growth and directionality can be achieved

Polydimethylsiloxane and poly(ether) ether ketone functionally graded composites for biomedical applications

Functionally graded materials (FGMs), with varying spatial, chemical and mechanical gradients (continuous or stepwise), have the potential to mimic heterogenous properties found across biological tissues. They can prevent stress concentrations and retain healthy cellular functions. Here, we show for the first time the fabrication of polydimethylsiloxane and poly(ether) ether ketone (PDMS-PEEK) composites. These were successfully manufactured as a bulk material and functionally graded (stepwise) without the use of hazardous solvents or the need of additives. Chemical, irreversible adhesion between layers (for the FGMs) was achieved without the formation of hard, boundary interfaces. The mechanical properties of PDMS-PEEK FGMs are proven to be further tailorable across the entirety of the build volume, mimicking the transition from soft to harder tissues. The introduction of 20 wt% PEEK particles into the PDMS matrix resulted in significant rises in the elastic modulus under tensile and compressive loading. Biological and thermal screenings suggested that these composites cause no adverse effects to human fibroblast cell lines and can retain physical state and mass at body temperature, which could make the composites suitable for a range of biomedical applications such as maxillofacial prosthetics, artificial blood vessels and articular cartilage replacement

Controlled arrangement of neuronal cells on surfaces functionalized with micro-patterned polymer brushes

Conventional in vitro cultures are useful to represent simplistic neuronal behaviour, however the lack of organisation results in random neurite spreading. To overcome this problem, control over the directionality of SH-SY5Y cells was attained, utilising photolithography to pattern the cell-repulsive anionic brush poly(potassium 3-sulfopropyl methacrylate) (PKSPMA) into tracks of 20, 40, 80 and 100 µm width. This data validates the use of PKSPMA brush coatings for long-term culture of SH-SY5Y cells, as well as providing a methodology by which the precise deposition of PKSPMA can be utilised to achieve targeted control over SH-SY5Y cells. Specifically, PKSPMA brush patterns prevented cell attachment, allowing SH-SY5Y cells to grow only on the non-coated glass (gaps of 20, 50, 75 and 100 µm width) at different cell densities (5000, 10000 and 15000 cells/cm2). This research demonstrates the importance of achieving cell directionality in vitro, whilst these simplistic models could provide new platforms to study complex neuron-neuron interactions