Employment of Melt Electrowriting for the design of regenerative grafts

BACKGROUND Cell-sized structures such as electrospun mats have been shown to tailor cell growth in a variety of ways and thus have great potential in the development of regenerative implants. Due to their thinness of several hundreds of micrometers, these mats mostly act as coatings on larger matrices, but the use of cytotoxic solvents complicates the translational process. A relatively new technique, melt electrowriting (MEW), offers similar properties but relinquishes cytotoxic solvents. Instead, thermoplastics such as polycaprolactone (PCL) are melted and processed under high voltage to form fibers with a precise fiber diameter that can be deposited in highly ordered meshes using a three-axis system. Outcome of the MEW processes are fiber architectures with defined fiber diameter, fiber spacing and tailored porosity within the cellular dimensions. This contrasts with previous electrospun mats, which mostly exhibit chaotic fiber architectures. RESEARCH QUESTIONS Due to the novelty of MEW, all studies so far used highly customized laboratory printers to produce MEW membranes, complicating translation to the clinic. Therefore, one of the first commercial MEW printer had to be established and the printing characteristics needed to be found to maintain homogeneous fiber diameters throughout the printing process and to investigate the compatibility with other printing techniques. Large tympanic membrane defects, such as those caused by chronic otitis media and other conditions, are currently closed with autologous materials in an elaborate procedure that may result in side effects such as hearing loss. Customized MEW meshes could improve this situation if they demonstrate similar mechanical and vibrational properties as the TM or the respective autologous materials. To this end, the variety of different design parameters such as fiber diameter, fiber spacing, layer-to-layer orientation, and number of layers should be investigated. Ideally, the collagen fiber structure and the curvature of the TM could be mimicked, too. Furthermore, the behavior of TM-typical cells such as keratinocytes and fibroblasts on the artificial scaffolds should be analyzed. This should simulate a potential regrowth of the TM collagen structure in vivo by migrating cells from the surrounding tissue onto the artificial membranes. Larger regenerative implant materials produced by other (additive) manufacturing processes do not offer geometries within cell dimension, therefore a combination with MEW could facilitate the scaffold-cell interaction. Hence, the objective of the present work was to investigate to what extent MEW can be combined with other additive manufacturing processes and what effects combined implant materials have on cell adhesion, alignment, and migration. MATERIALS & METHODS A GeSiM Printer 3.1. equipped with a MEW module was used for printing. Medical grade PCL was utilized to produce artificial membranes. Medical grade collagen type I was applied to create airtight membranes and improve cell compatibility. All scaffolds were mechanically tested using a uniaxial testing machine to characterize either the bending stiffness of the artificial TM grafts or the Young's modulus of the bone and tendon grafts. Compression tests were performed on the bone grafts, whereas tensile tests were used to evaluate the tendon grafts. Vibration analysis of the TM grafts was performed in collaboration at the Ear Clinic of Dresden University Hospital. For in vitro analyses of the TM grafts, immortalized and primary keratinocytes (HaCaT, HEKn) and primary fibroblasts (NHDF) were seeded on the grafts and analyzed. Murine calvarial osteoblast progenitor cells (MC3T3-E1) and immortalized human mesenchymal stem cells (hTERT-MSC) were seeded on the calcium phosphate cement and PCL composites. Adipose-derived mesenchymal stem cells (AT-MSC) were seeded onto the PLA-PCL tendon grafts. Standardized biochemical assays and fluorescence microscopy were used to analyze cell behavior. RESULTS Specific scaffold designs were developed to create a TM graft with comparable mechanical and similar vibrational properties to the native TM. In addition, the constructs showed high cell compatibility. Circular and radial fibers were integrated to mimic the native collagen structure closer. The combination of MEW with extrusion printing of sacrificial pyramids allowed for resembling the curvature of the TM. Overall, these adjustments minimized the gap between implant and native TM. By increasing the number of layers, the yield strength of the TM could be increased, but with a (small) decrease in vibration properties. A co-culture of primary fibroblasts and keratinocytes mimicked the in vivo migration of these cell types on the scaffolds so that the native collagen architecture could be restored after implantation in vivo (Publication I + II). For the first time, the bone graft material calcium phosphate cement (CPC) has been combined with PCL microfibers in a single fabrication process by combining MEW and extrusion printing (Publication III). Geometries in clinically relevant defect sizes of up to 3 cm with a variety of different pore structures were realized. The microporosity thus created within the macroporosity of the CPC structures had no significant effect on the cell growth closing these pores compared to CPC scaffolds without microfibers. From a mechanical point of view, the microfibers did not affect the adhesion between the CPC layers but fixed the CPC fragments during and after mechanical loading, so that the PCL-reinforced CPC scaffolds did not splinter in contrast to the pure CPC structures. When the PCL mats were printed wider than the CPC scaffolds, the protruding mesh provided an additional fixation option for the composite scaffolds in a potential defect area during surgery

Passive and active middle ear implants

Besides eradication of chronic middle ear disease, the reconstruction of the sound conduction apparatus is a major goal of modern ear microsurgery. The material of choice in cases of partial ossicular replacement prosthesis is the autogenous ossicle. In the event of more extensive destruction of the ossicular chain diverse alloplastic materials, e.g. metals, ceramics, plastics or composits are used for total reconstruction. Their specialised role in conducting sound energy within a half-open implant bed sets high demands on the biocompatibility as well as the acoustic-mechanic properties of the prosthesis. Recently, sophisticated titanium middle ear implants allowing individual adaptation to anatomical variations are widely used for this procedure. However, despite modern developments, hearing restoration with passive implants often faces its limitations due to tubal-middle-ear dysfunction. Here, implantable hearing aids, successfully used in cases of sensorineural hearing loss, offer a promising alternative. This article reviews the actual state of affairs of passive and active middle ear implants

Finite Element Dynamics of Human Auditory System Comprising Middle Ear and Cochlea in Inner Ear

Ehime University (愛媛大学)博士(工学)doctoral thesi

Regenerative therapies for tympanic membrane

It is estimated that by 2050 one in every ten people will be suffering from disabling hearing loss. Perforated tympanic membranes (TMs) are the most common injury to the human ear, resulting in a partial or complete hearing loss due to inept sound conduction. Commonly known as the eardrum, the TM is a thin, concave tissue of the middle ear that captures sound pressure waves from the environment and transmits them as mechanical vibrations to the inner ear. Microsurgical placement of autologous tissue graft has been the “gold standard” for treating damaged TMs; however, the incongruent structural and mechanical properties of these autografts often impair an optimal hearing restoration following recovery. Moreover, given the lack of available tissues for transplantations, regenerative medicine has emerged as a promising alternative. Several tissue engineered approaches applying bio-instructive scaffolds and stimuli have been reported for the TM regeneration, which can be broadly classified into TM repair and TM reconstruction. This review evaluates the current advantages and challenges of both strategies with a special focus on the use of recent biofabrication technologies for advancing TM tissue engineering

Tympanic Membrane Collagen Expression by Dynamically Cultured Human Mesenchymal Stromal Cell/Star-Branched Poly(ε-Caprolactone) Nonwoven Constructs

The tympanic membrane (TM) primes the sound transmission mechanism due to special fibrous layers mainly of collagens II, III, and IV as a product of TM fibroblasts, while type I is less represented. In this study, human mesenchymal stromal cells (hMSCs) were cultured on star-branched poly("-caprolactone) (*PCL)-based nonwovens using a TM bioreactor and proper dierentiating factors to induce the expression of the TM collagen types. The cell cultures were carried out for one week under static and dynamic conditions. Reverse transcriptase-polymerase chain reaction (RT-PCR) and immunohistochemistry (IHC) were used to assess collagen expression. A Finite Element Model was applied to calculate the stress distribution on the scaolds under dynamic culture. Nanohydroxyapatite (HA) was used as a filler to change density and tensile strength of *PCL scaolds. In dynamically cultured *PCL constructs, fibroblast surface marker was overexpressed, and collagen type II was revealed via IHC. Collagen types I, III and IV were also detected. Von Mises stress maps showed that during the bioreactor motion, the maximum stress in *PCL was double that in HA/*PCL scaolds. By using a *PCL nonwoven scaold, with suitable physico-mechanical properties, an oscillatory culture, and proper dierentiative factors, hMSCs were committed into fibroblast lineage-producing TM-like collagens

Nanomechanics of Electrospun Nanofibres for Tissue Engineering of the Tympanic Membrane

The Tympanic Membrane (TM), also known as the eardrum, includes layers of organized collagen nanofibres which play an essential role in sound transmission. Perforations that are caused by infection or accident must be repaired in order to restore hearing. Tympanoplasty is performed using grafts that are prepared from bladder, cartilage, temporal fascia and cadaveric skin. However, since mechanical properties of these grafts do not match those of the original TM, normal hearing is not fully restored. The goal of this study is to develop nanofibrous scaffolds for tissue engineering of the TM in order to circumvent the complications addressed with the conventional grafts. Mechanical properties of scaffolds greatly influence cellular behaviour, since cells can sense and respond to the stiffness of their substrate. In this study we investigated the Young’s modulus of single poly(caprolactone) (PCL) nanofibres as well as the moduli of as-spun and genipin-cross-linked collagen type I nanofibres using multi-point bending test with atomic force microscope (AFM). The effect of shear and tension on bending behaviour of fibres was investigated using four different analytical models. The Young’s modulus of electrospun PCL fibres (100 d 400 nm) was obtained with a mean value of 0.48 0.03 GPa. For as-spun and genipin-cross-linked collagen nanofibres a range of 1.66 – 13.9 GPa and 8.22 – 40.1 GPa were found for their Young’s moduli, respectively. The results indicate that there is a great potential for electrospun PCL and collagen nanofibres to be successfully applied in tissue engineering scaffolds because of their promising mechanical properties and biocompatibility

A Comprehensive study on Cartilage Tympanoplasty in Adhesive Otitis Media

INTRODUCTION: The management of the atelectatic ear continues to be one of the most controversial issues facing the otolaryngologist. Much of the confusion associated with this disorder stems from a poor understanding of the underlying pathophysiologic conditions that ultimately lead to changes in the tympanic membrane, resulting in atrophy, diffuse or local retractions, and cholesteatoma formation. Likewise, the lack of an accepted classification or grading scheme for the atelectatic ear has made it difficult to elucidate and predict the natural history of this disease and effectively predict those cases that will ultimately develop complications, such as cholesteatoma. The controversy is augmented by the fact that, early in the course of the disease, and even in the presence of incus erosion, hearing loss is frequently minimal and the patient, for the most part, asymptomatic. OBJECTIVE: The surgical management of adhesive otitis media is debatable. Adhesive otitis media progressing to cholesteatoma cannot be predicted, and hearing remains normal until later in the disease course. Hence surgery is done only when there is a hearing loss or frank cholesteatoma develops, where an extensive surgery may be needed. Earlier intervention is often avoided due to near normal hearing levels at this stage in some cases. Hearing results who have undergone cartilage tympanoplasty with or without ossicular reconstruction are reported for patients with adhesive otitis media. Study design: This is a prospective study. Setting: Study was done at Madras Medical College and Rajiv Gandhi Govt General Hospital, Chennai-3. Patients: A total of 30 patients (31 ears) aged 13-48 years underwent cartilage tympanoplasty with or without ossicular reconstruction. Interventions: Tympanotomy followed by cartilage reconstruction of the tympanic membrane, with ossicular reconstruction if there is any ossicular discontinuity. Main Outcome Measure(s): Post-operative pure tone average, air-bone gap for 3 frequencies (500, 1000, 2000 Hz) compared to pre-operative levels. RESULTS: There was a statistically significant improvement in hearing. CONCLUSIONS: Management of adhesive otitis media with cartilage perichondrium tympanoplasty with or without ossiculoplasty is a proven modality of treatment with successful results. Cartilage gives a tensile strength to the tympanic membrane which prevents further retractions inspite of the continuing Eustachian tube dysfunction and thus prevents cholesteatoma formation without compromising on hearing

Multimodal additive manufacturing of biomimetic tympanic membrane replacements with near tissue-like acousto-mechanical and biological properties

The three additive manufacturing techniques fused deposition modeling, gel plotting and melt electrowriting were combined to develop a mimicry of the tympanic membrane (TM) to tackle large TM perforations caused by chronic otitis media. The mimicry of the collagen fiber orientation of the TM was accompanied by a study of multiple funnel-shaped mimics of the TM morphology, resulting in mechanical and acoustic properties similar to those of the eardrum. For the different 3D printing techniques used, the process parameters were optimized to allow reasonable microfiber arrangements within the melt electrowriting setup. Interestingly, the fiber pattern was less important for the acousto-mechanical properties than the overall morphology. Furthermore, the behavior of keratinocytes and fibroblasts is crucial for the repair of the TM, and an in vitro study showed a high biocompatibility of both primary cell types while mimicking the respective cell layers of the TM. A simulation of the in vivo ingrowth of both cell types resulted in a cell growth orientation similar to the original collagen fiber orientation of the TM. Overall, the combined approach showed all the necessary parameters to support the growth of a neo-epithelial layer with a similar structure and morphology to the original membrane. It therefore offers a suitable alternative to autologous materials for the treatment of chronic otitis media

The chicken eggshell membrane: a versatile, sustainable, biological material for translational biomedical applications

Naturally derived materials are often preferred to than synthetic materials for biomedical applications due to their innate biological characteristics, relative availability, sustainability, and agreement with conscientious end-users. The chicken eggshell membrane (ESM) is an abundant resource with a defined structural profile, chemical composition, and validated morphological and mechanical characteristics. These unique properties have not only allowed the ESM to be exploited within the food industry, but has also led to it be considered for other novel translational applications such as tissue regeneration and replacement, filtration aids and barrier devices, and environmental health engagement. However, challenges still exist in order to enhance the native ESM: the need to improve its mechanical properties, the ability to combine/join fragments of ESM together, and the addition or incorporation of drugs/growth factors to advance its therapeutic capacity. This review article provides a succinct background to the native ESM, its extraction, isolation, and consequent physical, mechanical and biological characterisation including possible approaches to enhancement. Moreover, it also highlights current applications of the ESM in regenerative medicine and hints at future novel applications in which this novel biomaterial could be exploited to beneficial use