Anandhan Dhanasingh

Method to Estimate the Complete and Two-turn Cochlear Duct Length

Hypothesis Using a linear measurement of the cochlea on a single radiographic image can reliably estimate the complete and two-turn cochlear duct length (CDL) in a normal human temporal bone. Background CDL is measured from the middle of the round window to the helicotrema. Histologic studies have shown the length of the organ of Corti (OC) to range from 25 to 35 mm. CDL measurements, performed either radiographically or histologically, are quite tedious and time-consuming. We propose equations that can reliably estimate both two-turn and complete CDL using a single computed tomography (CT) image. Methods Prior studies of CDL, measured either histologically or radiographically, were reviewed, which yielded distributions of CDL measured at the OC and the lateral wall of the cochlea. Using Escudé’s third equation as a basis, we were able to extrapolate complete and two-turn CDL based on a CT scan measurement of the diameter of the basal turn (A). Results Using measurement A, the relationship of two-turn CDL measured at the OC is 2TL(oc) = 3.65(A-1) and for 2TL(i) = 3.65(A-0.7). The equation for estimation of complete CDL is CDL(oc) = 4.16A − 4 and for CDL(i) = 4.16A − 2.7. Conclusion Using a single linear measurement from a CT scan image can reliably estimate the two-turn and complete CDLs in human temporal bones. The two-turn length represents the best compromise of cochlear coverage while minimizing intracochlear trauma for electrode insertions.

Tailored hyaluronic acid hydrogels through hydrophilic prepolymer cross-linkers

Despite its enormous biological importance, a lack of mechanical stability and fast degradability often hampers more intense application of hyaluronic acid (HA) for biomedical applications. We present a method to prepare hyaluronic acid based hydrogels with tailored stability, degradability and porosity by cross-linking with functional 18 kDa six arm star shaped poly(ethylene oxide-stat-propylene oxide) (sP(EO-stat-PO)) prepoylmers in aqueous solutions. In the case of NCO groups at the distal ends of the arms, cross-linking occurs through reaction with OH groups of the HA resulting in urethane bridges between star molecules and HA. In parallel, slow hydrolysis of isocyanates to amines that subsequently rapidly aminolyze other isocyanate groups and lead to urea bridges between prepolymers occurs as a side reaction. Overall, this process leads to cross-linking within 5 to 15 min depending on the molar ratios of NCO-sP(EO-stat-PO) to HA. A chemically orthogonal way of cross-linking can be achieved with acrylate functional prepolymers and thiolated HA where cross-linking exclusively results from Michael-type addition of the thiol to the acrylate groups. This process also results in gelation times of 5 to 15 min. The two mechanisms are compared and the resulting gels are analyzed regarding chemical composition, equilibrium water content, degree of sol-content, porosity, mechanical stability and resistance towards enzymatic degradation by hyaluronidase.

In Vitro and In Vivo Evaluation of a Hydrogel Reservoir as a Continuous Drug Delivery System for Inner Ear Treatment

Fibrous tissue growth and loss of residual hearing after cochlear implantation can be reduced by application of the glucocorticoid dexamethasone-21-phosphate-disodium-salt (DEX). To date, sustained delivery of this agent to the cochlea using a number of pharmaceutical technologies has not been entirely successful. In this study we examine a novel way of continuous local drug application into the inner ear using a refillable hydrogel functionalized silicone reservoir. A PEG-based hydrogel made of reactive NCO-sP(EO-stat-PO) prepolymers was evaluated as a drug conveying and delivery system in vitro and in vivo. Encapsulating the free form hydrogel into a silicone tube with a small opening for the drug diffusion resulted in delayed drug release but unaffected diffusion of DEX through the gel compared to the free form hydrogel. Additionally, controlled DEX release over several weeks could be demonstrated using the hydrogel filled reservoir. Using a guinea-pig cochlear trauma model the reservoir delivery of DEX significantly protected residual hearing and reduced fibrosis. As well as being used as a device in its own right or in combination with cochlear implants, the hydrogel-filled reservoir represents a new drug delivery system that feasibly could be replenished with therapeutic agents to provide sustained treatment of the inner ear.

Polysaccharide based covalently linked multi-membrane hydrogels

Layer-by-layer techniques bear great potential for biomaterials as they allow sequential construction of hierarchical constructs. This especially accounts for hydrogels which can be tuned for biochemically, morphologically and mechanically different microenvironments and find numerous applications for encapsulation and release of drugs and cells. Here we present a novel radiation-free preparation method for multi-membrane hydrogels based on polysaccharides using prepolymer cross-linkers. Isocyanate functional prepolymers were used either alone or for cross-linking unmodified hyaluronic acid to form hydrolytically stable hydrogel membranes, whereas acrylate-bearing prepolymers were used to cross-link thiofunctional hyaluronic acid resulting in fully degradable hydrogel layers. Covalently linked multi-membrane systems can be formed through initial formation of a central hydrogel core followed by sequential embedding of the preformed hydrogel in an aqueous precursor solution for the following membrane. As a proof of principle, sequential drug release from a three layer system is shown in which ampicillin and dexamethasone were embedded in the outer and inner membranes, respectively, with a hydrolytically stable middle membrane as separation layer and diffusion barrier.

Hydrogel-fibre composites with independent control over cell adhesion to gel and fibres as an integral approach towards a biomimetic artificial ECM

In the body, cells are surrounded by an interconnected mesh of insoluble, bioactive protein fibres to which they adhere in a well-controlled manner, embedded in a hydrogel-like highly hydrated matrix. True morphological and biochemical mimicry of this so-called extracellular matrix (ECM) remains a challenge but appears decisive for a successful design of biomimetic three-dimensional in vitro cell culture systems. Herein, an approach is presented which describes the fabrication and in vitro assessment of an artificial ECM which contains two major components, i.e. specifically biofunctionalized fibres and a semi-synthetic hyaluronic acid-based hydrogel, which allows control over cell adhesion towards both components. As proof of principle for the control of cell adhesion, RGD as well-known cell adhesive cue and the control sequence RGE are immobilized in the system. In vitro studies with primary human dermal fibroblasts were conducted to evaluate the specificity of cell adhesion and the potential of the composite system to support cell growth. Finally, one possible application example for guided cell growth is shown by the use of oriented fibres in a hydrogel matrix.

Imaging cochlear implantation with round window insertion in human temporal bones and cochlear morphological variation using high-resolution cone beam CT.

Abstract Conclusions: The present experimental set-up of high spatial resolution cone-beam computed tomography (CBCT) showed advantages of demonstrating the critical landmarks of the cochlea in identifying the position of intracochlear electrode contacts and has the potential for clinical application in cochlear implant (CI) surgery. Objective: To evaluate a newly developed CBCT system in defining CI electrode array in human temporal bone and cochlear morphological variation. Methods: Standard electrode, flexible tip electrode (Flex28), and an experimental electrode array with 36 contacts from MED-EL were implanted into the cochleae of six human temporal bones through an atraumatic round window membrane insertion. The cochleae were imaged with 900 frames using an experimental set-up based on a CBCT scanner installed with Superior SXR 130-15-0.5 X-ray tube in combination with filtration of copper and aluminum. Results: In all temporal bones, the landmarks of the cochlea, modiolus, osseous spiral lamina, round window niche, and stapes were demonstrated at an average level of 3.4–4.5. The contacts of electrode arrays were clearly shown to locate in the scala tympani. There was a linear correlation between the ‘A’ value and cochlea height, and between the A value and actual electrode insertion length for the first 360° insertion depth.

Cochlear Dummy Electrodes for Insertion Training and Research Purposes: Fabrication, Mechanical Characterization, and Experimental Validation

To develop skills sufficient for hearing preservation cochlear implant surgery, surgeons need to perform several electrode insertion trials in ex vivo temporal bones, thereby consuming relatively expensive electrode carriers. The objectives of this study were to evaluate the insertion characteristics of cochlear electrodes in a plastic scala tympani model and to fabricate radio opaque polymer filament dummy electrodes of equivalent mechanical properties. In addition, this study should aid the design and development of new cochlear electrodes. Automated insertion force measurement is a new technique to reproducibly analyze and evaluate the insertion dynamics and mechanical characteristics of an electrode. Mechanical properties of MED-ELs FLEX28, FLEX24, and FLEX20 electrodes were assessed with the help of an automated insertion tool. Statistical analysis of the overall mechanical behavior of the electrodes and factors influencing the insertion force are discussed. Radio opaque dummy electrodes of comparable characteristics were fabricated based on insertion force measurements. The platinum-iridium wires were replaced by polymer filament to provide sufficient stiffness to the electrodes and to eradicate the metallic artifacts in X-ray and computed tomography (CT) images. These low-cost dummy electrodes are cheap alternatives for surgical training and for in vitro, ex vivo, and in vivo research purposes.

Integrin α4 impacts on differential adhesion of preadipocytes and stem cells on synthetic polymers

Stem cells represent an ideal cell source for tissue engineering and regenerative medicine, because they can be readily isolated, expanded, differentiated and transplanted. For stem cell‐based therapies, biomaterials are required to allow for a spatial distribution of the stem cells within a defined area in the body. In our recent studies, we analysed the interaction of a large panel of stem cell types with an array of biomaterials and demonstrated that a rational prediction of stem cell behaviour on a specific biomaterial is so far not possible. Interestingly, even ontogenetically related stem cell types, such as mesenchymal stem cells (MSCs), preadipocytes and dental pulp stem cells (DPSCs), exhibit distinct adhesion properties on the very same biomaterial surface. Therefore, we investigated integrin and extracellular matrix (ECM) protein expression of stem cells to relate gene expression to adhesion behaviour. MSCs, preadipocytes and DPSCs were cultured on selected synthetic polymers, such as Texin, a thermoplastic polyurethane, poly(dimethyl siloxane) (PDMS), poly‐d,l‐lactic acid (PDLLA) and l‐lactic acid‐trimehylene carbonate (Resomer® LT706). Integrins and ECM proteins were analysed by RT–PCR, real‐time PCR and immunohistochemistry. Analysis of several adhesion molecules yielded that only one molecule, integrin α4, might play a significant role in differential adhesion on polymers for preadipocytes compared to DPSCs and MSCs. Thus, our studies on the molecular interactions of stem cells and polymers are expected to lead to a more profound understanding of the stem cell–biomaterial interactions to eventually allow for a rational biomaterial design. Copyright

Differential mineralization of human dental pulp stem cells on diverse polymers.

Abstract In tissue engineering, biomaterials are used as scaffolds for spatial distribution of specific cell types. Biomaterials can potentially influence cell proliferation and extracellular matrix formation, both in positive and negative ways. The aim of the present study was to investigate and compare mineralized matrix production of human dental pulp stem cells (DPSC), cultured on 17 different well-characterized polymers. Osteogenic differentiation of DPSC was induced for 21 days on biomaterials using dexamethasone, L-ascorbic-acid-2-phosphate, and sodium β-glycerophosphate. Success of differentiation was analyzed by quantitative RealTime PCR, alkaline phosphatase (ALP) activity, and visualization of calcium accumulations by alizarin red staining with subsequent quantification by colorimetric method. All of the tested biomaterials of an established biomaterial bank enabled a mineralized matrix formation of the DPSC after osteoinductive stimulation. Mineralization on poly(tetrafluoro ethylene) (PTFE), poly(dimethyl siloxane) (PDMS), Texin, LT706, poly(epsilon-caprolactone) (PCL), polyesteramide type-C (PEA-C), hyaluronic acid, and fibrin was significantly enhanced (p<0.05) compared to standard tissue culture polystyrene (TCPS) as control. In particular, PEA-C, hyaluronic acid, and fibrin promoted superior mineralization values. These results were confirmed by ALP activity on the same materials. Different biomaterials differentially influence the differentiation and mineralized matrix formation of human DPSC. Based on the present results, promising biomaterial candidates for bone-related tissue engineering applications in combination with DPSC can be selected.