Intradermal Delivery of an Immunomodulator for Basal Cell Carcinoma; Expanding the Mechanistic Insight into Solid Microneedle-Enhanced Delivery of Hydrophobic Molecules.

Basal cell carcinoma (BCC) is the most common cutaneous malignancy in humans. One of the most efficacious drugs used in the management of BCC is the immunomodulator, imiquimod. However, imiquimod has physiochemical properties that limit its permeation to reach deeper, nodular tumor lesions. The use of microneedles may overcome such limitations and promote intradermal drug delivery. The current work evaluates the effectiveness of using an oscillating microneedle device Dermapen either as a pre- or post-treatment with 5% w/w imiquimod cream application to deliver the drug into the dermis. The effectiveness of microneedles to enhance the permeation of imiquimod was evaluated ex vivo using a Franz cell setup. After a 24-h permeation experiment, sequential tape strips and vertical cross-sections of the porcine skin were collected and analyzed using time-of-flight secondary ion mass spectrometry (ToF-SIMS). In addition, respective Franz cell components were analyzed using high-performance liquid chromatography (HPLC). Analysis of porcine skin cross-sections demonstrated limited dermal permeation of 5% w/w imiquimod cream. Similarly, limited dermal permeation was also seen when 5% w/w imiquimod cream was applied to the skin that was pretreated with the Dermapen, this is known as poke-and-patch. In contrast, when the formulation was applied first to the skin prior to Dermapen application, this is known as patch-and-poke, we observed a significant increase in intradermal permeation of imiquimod. Such enhancement occurs immediately upon microneedle application, generating an intradermal depot that persists for up to 24 h. Intradermal colocalization of isostearic acid, an excipient in the cream, with imiquimod within microneedle channels was also demonstrated. However, such enhancement in intradermal delivery of imiquimod was not observed when the patch-and-poke strategy was used with a non-oscillating microneedle applicator, the Dermastamp. The current work highlights that using the patch-and-poke approach with an oscillating microneedle pen may be a viable approach to improve the current treatment in BCC patients who would prefer a less invasive intervention relative to surgery.

Self-Assembling Benzothiazole-Based Gelators: A Mechanistic Understanding of in Vitro Bioactivation and Gelation.

Low molecular weight gelators (LMWGs) of chemotherapeutic drugs represent a valid alternative to the existing polymer-based formulations used for targeted delivery of anticancer drugs. Herein we report the design and development of novel self-assembling gelators of the antitumor benzothiazole 5F 203 (1). Two different types of derivatives of 1 were synthesized, formed by an amide (2) and a carbamate (3a-3d) linker, respectively, which showed potent in vitro antitumor activity against MCF-7 mammary and IGROV-1 ovarian carcinoma cells. In contrast, MRC-5 fibroblasts were inherently resistant to the above derivatives (GI50 > 10 µM), thus revealing stark selectivity against the malignant cell lines over the nontransformed fibroblasts. Western blots assays demonstrated induction of CYP1A1 by 1 and its derivatives only in sensitive malignant cells (MCF-7), corroborating conservation of a CYP1A1-mediated mechanism of action. The ability to form stable gels under relatively high strains was supported by rheological tests; in addition, their inner morphology was characterized as possessing a crossed-linked nanostructure, with the formation of thick aggregates with variable widths between 1100 and 400 nm and lengths from 8 to 32 µm. Finally, in vitro dissolution studies proved the ability of hydrogel 2 to release 48% of 2 within 80 h, therefore demonstrating its ability to act as a platform for localized delivery.

Improving umbilical cord blood processing to increase total nucleated cell count yield and reduce cord input wastage by managing the consequences of input variation.

BACKGROUND AIMS: With the rising use of umbilical cord blood (UCB) as an alternative source of hematopoietic stem cells, storage inventories of UCB have grown, giving rise to genetically diverse inventories globally. In the absence of reliable markers such as CD34 or counts of colony-forming units, total nucleated cell (TNC) counts are often used as an indicator of potency, and transplant centers worldwide often select units with the largest counts of TNC. As a result, cord blood banks are driven to increase the quality of stored inventories by increasing the TNC count of products stored. However, these banks face challenges in recovering consistent levels of TNC with the use of the standard protocols of automated umbilical cord processing systems, particularly in the presence of input variation both of cord blood volume and TNC count, in which it is currently not possible to process larger but useable UCB units with consequent losses in TNC. METHODS: This report addresses the challenge of recovering consistently high TNC yields in volume reduction by proposing and validating an alternative protocol capable of processing a larger range of units more reliably. RESULTS: This work demonstrates improvements in plastic ware and tubing sets and in the recovery process protocol with consequent productivity gains in TNC yield and a reduction in standard deviation. CONCLUSIONS: This work could pave the way for cord blood banks to improve UCB processing and increase efficiency through higher yields and lower costs.

Multi-material 3D bioprinting of porous constructs for cartilage regeneration.

The current gold standard for nasal reconstruction after rhinectomy or severe trauma includes transposition of autologous cartilage grafts in conjunction with coverage using an autologous skin flap. Harvesting autologous cartilage requires a major additional procedure that may create donor site morbidity. Major nasal reconstruction also requires sculpting autologous cartilages to form a cartilage framework, which is complex, highly skill-demanding and very time consuming. These limitations have prompted facial reconstructive surgeons to explore different techniques such as tissue engineered cartilage. This work explores the use of multi-material 3D bioprinting with chondrocyte-laden gelatin methacrylate (GelMA) and polycaprolactone (PCL) to fabricate constructs that can potentially be used for nasal reconstruction. In this study, we have investigated the effect of 3D manufacturing parameters including temperature, needle gauge, UV exposure time, and cell carrier formulation (GelMA) on the viability and functionality of chondrocytes in bioprinted constructs. Furthermore, we printed chondrocyte-laden GelMA and PCL into composite constructs to combine biological and mechanical properties. It was found that 20% w/v GelMA was the best concentration for the 3D bioprinting of the chondrocytes without comprising the scaffold's porous structure and cell functionality. In addition, the 3D bioprinted constructs showed neocartilage formation and similar mechanical properties to nasal alar cartilage after a 50-day culture period. Neocartilage formation was also observed in the composite constructs evidenced by the presence of glycosaminoglycans and collagen type II. This study shows the feasibility of manufacturing neocartilage using chondrocytes/GelMA/PCL 3D bioprinted porous constructs which could be applied as a method for fabricating implants for nose reconstruction.

Improving children's and their visitors' hand hygiene compliance.

BACKGROUND: Numerous interventions have tried to improve healthcare workers' hand hygiene compliance. However, little attention has been paid to children's and their visitors' compliance. AIM: To test whether interactive educational interventions increase children's and visitors' compliance with hand hygiene. METHODS: This was a cluster randomised study of hand hygiene compliance before and after the introduction of educational interventions. Observations were compared for different moments of hygiene and times of the day. Qualitative data in the form of questionnaire-based structured interviews were obtained. FINDINGS: Hand hygiene compliance increased by 24.4% (P < 0.001) following the educational interventions, with children's compliance reaching 40.8% and visitors' being 50.8%. Compliance varied depending on which of the five moments of hygiene was observed (P < 0.001), with the highest compliance being 'after body fluid exposure' (72.7%). Responses from questionnaires showed educational interventions raised awareness of the importance of hand hygiene (69%, 57%) compared to those who had not experienced the educational intervention (50%). CONCLUSION: Educational interventions may result in a significant increase in children's and visitors' hand hygiene (P < 0.001).

Intradermal delivery of imiquimod using polymeric microneedles for basal cell carcinoma.

Despite being one of the most efficacious drugs used in the treatment of basal cell carcinoma (BCC), imiquimod has limited cutaneous permeation. The current work presents the development of polyvinylpyrrolidone-co-vinyl acetate (PVPVA) microneedles loaded with imiquimod for improving intradermal delivery of imiquimod for the treatment of nodular BCC. In vitro permeation studies, using full thickness ex vivo porcine skin were used to evaluate the effectiveness of these imiquimod loaded polymeric microneedles in comparison to the topical application of commercial Aldara™ cream. HPLC analysis demonstrated similar intradermal permeation of imiquimod from Aldara™ cream and imiquimod-loaded microneedles despite the microneedle having a six-fold lower drug loading than the clinical dose of Aldara™ used for BCC management. In addition, ToF-SIMS analysis of skin cross sections demonstrated intradermal localisation of imiquimod following microneedle-based delivery while the Aldara™ treated skin showed the drug localised predominantly within the stratum corneum. ToF-SIMS analysis also demonstrated intradermal co-localisation of the PVPVA polymer, used in fabricating the microneedle, with imiquimod within the microneedle channels in a label-free manner. This study demonstrates that a polymeric microneedle system may be a viable approach to improving the intradermal delivery of imiquimod for the treatment of nodular BCC with lower drug loading.

Intradermal and transdermal drug delivery using microneedles - Fabrication, performance evaluation and application to lymphatic delivery.

The progress in microneedle research is evidenced by the transition from simple 'poke and patch' solid microneedles fabricated from silicon and stainless steel to the development of bioresponsive systems such as hydrogel-forming and dissolving microneedles. In this review, we provide an outline on various microneedle fabrication techniques which are currently employed. As a range of factors, including materials, geometry and design of the microneedles, affect the performance, it is important to understand the relationships between them and the resulting delivery of therapeutics. Accordingly, there is a need for appropriate methodologies and techniques for characterization and evaluation of microneedle performance, which will also be discussed. As the research expands, it has been observed that therapeutics delivered via microneedles has gained expedited access to the lymphatics, which makes them a favorable delivery method for targeting the lymphatic system. Such opportunity is valuable in the area of vaccination and treatment of lymphatic disorders, which is the final focus of the review.

Expanding the applications of microneedles in dermatology.

Since the first patent for microneedles was filed in the 1970s, research on utilising microneedles as a drug delivery system has progressed significantly. In addition to the extensive research on microneedles for improving transdermal drug delivery, there is a growing interest in using these devices to manage dermatological conditions. This review aims to provide the background on microneedles, the clinical benefits, and challenges of the device along with the potential dermatological conditions that may benefit from the application of such a drug delivery system. The first part of the review provides an outline on benefits and challenges of translating microneedle-based drug delivery systems into clinical practice. The second part of the review covers the application of microneedles in treating dermatological conditions. The efficacy of microneedles along with the limitations of such a strategy to treat diseased skin shall be addressed.

Review of additive manufactured tissue engineering scaffolds: relationship between geometry and performance.

Material extrusion additive manufacturing has rapidly grown in use for tissue engineering research since its adoption in the year 2000. It has enabled researchers to produce scaffolds with intricate porous geometries that were not feasible with traditional manufacturing processes. Researchers can control the structural geometry through a wide range of customisable printing parameters and design choices including material, print path, temperature, and many other process parameters. Currently, the impact of these choices is not fully understood. This review focuses on how the position and orientation of extruded filaments, which sometimes referred to as the print path, lay-down pattern, or simply "scaffold design", affect scaffold properties and biological performance. By analysing trends across multiple studies, new understanding was developed on how filament position affects mechanical properties. Biological performance was also found to be affected by filament position, but a lack of consensus between studies indicates a need for further research and understanding. In most research studies, scaffold design was dictated by capabilities of additive manufacturing software rather than free-form design of structural geometry optimised for biological requirements. There is scope for much greater application of engineering innovation to additive manufacture novel geometries. To achieve this, better understanding of biological requirements is needed to enable the effective specification of ideal scaffold geometries.

Characterisation of the surface structure of 3D printed scaffolds for cell infiltration and surgical suturing.

3D printing is of great interest for tissue engineering scaffolds due to the ability to form complex geometries and control internal structures, including porosity and pore size. The porous structure of scaffolds plays an important role in cell ingrowth and nutrition infusion. Although the internal porosity and pore size of 3D printed scaffolds have been frequently studied, the surface porosity and pore size, which are critical for cell infiltration and mass transport, have not been investigated. The surface geometry can differ considerably from the internal scaffold structure depending on the 3D printing process. It is vital to be able to control the surface geometry of scaffolds as well as the internal structure to fabricate optimal architectures. This work presents a method to control the surface porosity and pore size of 3D printed scaffolds. Six scaffold designs have been printed with surface porosities ranging from 3% to 21%. We have characterised the overall scaffold porosity and surface porosity using optical microscopy and microCT. It has been found that surface porosity has a significant impact on cell infiltration and proliferation. In addition, the porosity of the surface has been found to have an effect on mechanical properties and on the forces required to penetrate the scaffold with a surgical suturing needle. To the authors' knowledge, this study is the first to investigate the surface geometry of extrusion-based 3D printed scaffolds and demonstrates the importance of surface geometry in cell infiltration and clinical manipulation.

A 3D bioprinting exemplar of the consequences of the regulatory requirements on customized processes.

Computer-aided 3D printing approaches to the industrial production of customized 3D functional living constructs for restoration of tissue and organ function face significant regulatory challenges. Using the manufacture of a customized, 3D-bioprinted nasal implant as a well-informed but hypothetical exemplar, we examine how these products might be regulated. Existing EU and USA regulatory frameworks do not account for the differences between 3D printing and conventional manufacturing methods or the ability to create individual customized products using mechanized rather than craft approaches. Already subject to extensive regulatory control, issues related to control of the computer-aided design to manufacture process and the associated software system chain present additional scientific and regulatory challenges for manufacturers of these complex 3D-bioprinted advanced combination products.

Degradation and Characterization of Resorbable Phosphate-Based Glass Thin-Film Coatings Applied by Radio-Frequency Magnetron Sputtering.

Quinternary phosphate-based glasses of up to 2.67 µm, deposited by radio-frequency magnetron sputtering, were degraded in distilled water and phosphate-buffered saline (PBS) to investigate their degradation characteristics. Magnetron-sputtered coatings have been structurally compared to their compositionally equivalent melt-quenched bulk glass counterparts. The coatings were found to have structurally variable surfaces to melt-quenched glass such that the respective bridging oxygen to nonbridging oxygen bonds were 34.2% to 65.8% versus 20.5% to 79.5%, forming metaphosphate (PO3)(-) (Q(2)) versus less soluble (P2O7)(4-) (Q(1)) and (PO4)(3-) (Q(0)), respectively. This factor led to highly soluble coatings, exhibiting a t(1/2) degradation dependence in the first 2 h in distilled water, followed by a more characteristic linear profile because the subsequent layers were less soluble. Degradation was observed to preferentially occur, forming voids characteristic of pitting corrosion, which was confirmed by the use of a focused ion beam. Coating degradation in PBS precipitated a (PO3)(-) metaphosphate, an X-ray amorphous layer, which remained adherent to the substrate and seemingly formed a protective diffusion barrier, which inhibited further coating degradation. The implications are that while compositionally similar, sputter-deposited coatings and melt-quenched glasses are structurally dissimilar, most notably, with regard to the surface layer. This factor has been attributed to surface etching of the as-deposited coating layer during deposition and variation in the thermal history between the processes of magnetron sputtering and melt quenching.

A novel technique for the production of electrospun scaffolds with tailored three-dimensional micro-patterns employing additive manufacturing.

Electrospinning is a common technique used to fabricate fibrous scaffolds for tissue engineering applications. There is now growing interest in assessing the ability of collector plate design to influence the patterning of the fibres during the electrospinning process. In this study, we investigate a novel method to generate hybrid electrospun scaffolds consisting of both random fibres and a defined three-dimensional (3D) micro-topography at the surface, using patterned resin formers produced by rapid prototyping (RP). Poly(D,L-lactide-co-glycolide) was electrospun onto the engineered RP surfaces and the ability of these formers to influence microfibre patterning in the resulting scaffolds visualized by scanning electron microscopy. Electrospun scaffolds with patterns mirroring the microstructures of the formers were successfully fabricated. The effect of the resulting fibre patterns and 3D geometries on mammalian cell adhesion and proliferation was investigated by seeding enhanced green fluorescent protein labelled 3T3 fibroblasts onto the scaffolds. Following 24 h and four days of culture, the seeded scaffolds were visually assessed by confocal macro- and microscopy. The patterning of the fibres guided initial cell adhesion to the scaffold with subsequent proliferation over the geometry resulting in the cells being held in a 3D micro-topography. Such patterning could be designed to replicate a specific in vivo structure; we use the dermal papillae as an exemplar here. In conclusion, a novel, versatile and scalable method to produce hybrid electrospun scaffolds has been developed. The 3D directional cues of the patterned fibres have been shown to influence cell behaviour and could be used to culture cells within a similar 3D micro-topography as experienced in vivo.

Growth of the chorioallantoic membrane into a rapid-prototyped model pore system: experiments and mathematical model.

This paper presents a mathematical model to describe the growth of tissue into a rapid-prototyped porous scaffold when it is implanted onto the chorioallantoic membrane (CAM). The scaffold was designed to study the effects of the size and shape of pores on tissue growth into conventional tissue engineering scaffolds, and consists of an array of pores each having a pre-specified shape. The experimental observations revealed that the CAM grows through each pore as an intact layer of tissue, provided the width of the pore exceeds a threshold value. Based on these results a mathematical model is described to simulate the growth of the membrane, assuming that the growth is a function of the local isotropic membrane tension. The model predictions are compared against measurements of the extent of membrane growth through the pores as a function of time for pores with different dimensions.