Hyaluronic acid-ibuprofen conjugation: a novel ototherapeutic approach protecting inner ear cells from inflammation-mediated damage.

There is a substantial need of effective drugs for the treatment of hearing loss, which affects nearly 500 million individuals globally. Hearing loss can be the result of intense or prolonged noise exposure, ototoxic drugs, infections, and trauma, which trigger inflammatory signaling cascades that lead to irreversible damage to cochlear structures. To address this, we developed and characterized a series of covalent conjugates of anti-inflammatory drugs to hyaluronic acid (HA), for potential use as topical ototherapeutics. These conjugates were tested in in vitro assays designed to mirror physiological processes typically observed with acoustic trauma. Intense noise exposure leads to macrophage recruitment to the cochlea and subsequent inflammatory damage to sensory cells. We therefore first tested our conjugates' ability to reduce the release of inflammatory cytokines in macrophages. This anti-inflammatory effect on macrophages also translated to increased cochlear cell viability. In our initial screening, one conjugate, ibuprofen-HA, demonstrated significantly higher anti-inflammatory potential than its counterparts. Subsequent cytokine release profiling of ibuprofen-HA further confirmed its ability to reduce a wider range of inflammatory markers, to a greater extent than its equivalent unconjugated drug. The conjugate's potential as a topical therapeutic was then assessed in previously developed tympanic and round window membrane tissue permeation models. As expected, our data indicate that the conjugate has limited tympanic membrane model permeability; however, it readily permeated the round window membrane model and to a greater extent than the unconjugated drug. Interestingly, our data also revealed that ibuprofen-HA was well tolerated in cellular and tissue cytocompatibility assays, whereas the unconjugated drug displayed significant cytotoxicity at equivalent concentrations. Moreover, our data highlighted the importance of chemical conjugation of ibuprofen to HA; the conjugate had improved anti-inflammatory effects, significantly reduced cytotoxicity, and is more suitable for therapeutic formulation. Overall, this work suggests that ibuprofen-HA could be a promising safe and effective topical ototherapeutic for inflammation-mediated cochlear damage.

In vitro characterization of novel hyaluronan-antioxidant conjugates as potential topical therapeutics against hearing loss.

Noise-induced hearing loss affects roughly 430 million people worldwide. Current treatment options often require invasive medical procedures, and to date, there are no FDA-approved drug therapies. While the causes can be diverse, noise induced hearing loss is unequivocally associated with oxidative stress and inflammation, and subsequent damage to the inner ear structures. Several studies have shown that various antioxidants such as glutathione, cysteine, and methionine can be used to mitigate oxidative damage from reactive oxygen species; however, these studies relied on invasive or systemic drug delivery methods. This study focused on the development and characterization of a novel series of antioxidant compounds that would be suitable for non or minimally invasive topical inner ear delivery and could mitigate reactive oxygen species associated cellular damage. Specifically, a series of covalent conjugates were synthesized by using hyaluronan as a drug carrier, and methionine, cysteine or glutathione as antioxidant drugs. The conjugates were tested for their ability to readily permeate though in vitro round window membrane and tympanic membrane permeation models, as well as their in vitro internalization into cochlear cells. Our data revealed interdependence between the molecular weight of the hyaluronan carrier, and the tissue and cellular membrane permeation capacity. Subsequent screening of the adequately sized conjugates in in vitro acellular assays revealed the strongest antioxidant activity for the cysteine and glutathione conjugates. These oxidative stress protective effects were further confirmed in cellular in vitro assays. Collectively, the data herein showcase the potential value of these conjugates as therapeutics against oxidative-stress-mediated cellular damage specific to noise-induced hearing loss.

An Evaluation of the Drug Permeability Properties of Human Cadaveric In Situ Tympanic and Round Window Membranes.

It is estimated that hearing loss currently affects more than 1.5 billion people, or approximately 20% of the global population; however, presently, there are no Food and Drug Administration-approved therapeutics or prophylactics for this condition. While continued research on the development of otoprotective drugs to target this clear unmet need is an obvious path, there are numerous challenges to translating promising therapeutic candidates into human clinical testing. The screening of promising drug candidates relies exclusively on preclinical models. Current models do not permit the rapid high-throughput screening of promising drug candidates, and their relevance to clinical scenarios is often ambiguous. With the current study, we seek to understand the drug permeability properties of the cadaveric tympanic and round window membranes with the goal of generating knowledge that could inform the design and/or evaluation of in vitro organotypic models. The development of such models could enable the early high-throughput screening of topical therapeutic candidates and should address some of the limitations of currently used animal models.

Development and Characterization of an In Vitro Round Window Membrane Model for Drug Permeability Evaluations.

Hearing loss and balance disorders are highly common disorders, and the development of effective oto-therapeutics remains an area of intense research. Drug development and screening in the hearing research field heavily rely on the use of preclinical models with often ambiguous translational relevance. This often leads to failed advancement in the market of effective therapeutics. In this context, especially for inner ear-specific pathologies, the availability of an in vitro, physiologically relevant, round window membrane (RWM) model could enable rapid, high-throughput screening of potential topical drugs for inner ear and cochlear dysfunctions and could help accelerate the advancement to clinic and market of more viable drug candidates. In this study, we report the development and evaluation of an in vitro model that mimics the native RWM tissue morphology and microenvironment as shown via immunostaining and histological analyses. The developed three-dimensional (3D) in vitro model was additionally assessed for barrier integrity by transepithelial electrical resistance, and the permeability of lipophilic and hydrophilic drugs was determined. Our collective findings suggest that this in vitro model could serve as a tool for rapid development and screening of topically deliverable oto-therapeutics.

An In Vitro Model for Characterization of Drug Permeability across the Tympanic Membrane.

Otic disorders, such as otitis media and hearing loss, affect a substantial portion of the global population. Despite this, oto-therapeutics, in particular those intended to treat hearing loss, have seen limited development and innovation. A significant factor to this is likely a result of the inherent costs and complexities of drug discovery and development. With in vitro 3D tissue models seeing increased utility for the rapid, high-throughput screening of drug candidates, it stands to reason that the field of otology could greatly benefit from such innovations. In this study, we propose and describe an in vitro 3D model, designed using a physiologically based approach, which we suggest can be used to estimate drug permeability across human tympanic membranes (TM). We characterize the permeability properties of several template drugs in this model under various growth and storage conditions. The availability of such cost-effective, rapid, high-throughput screening tools should allow for increased innovation and the discovery of novel drug candidates over the currently used animal models. In the context of this TM permeation model, it may promote the development of topical drugs and formulations that can non-invasively traverse the TM and provide tissue-targeted drug delivery as an alternative to systemic treatment, an objective which has seen limited study until present.

Stem Cells in Tumour Microenvironment Aid in Prolonged Survival Rate of Cancer Cells and Developed Drug Resistance: Major Challenge in Osteosarcoma Treatment.

Osteosarcoma is an aggressive bone cancer found in children and adolescents. The combined treatment strategy includes the surgical removal of tumour and subsequent chemotherapy to prevent the reoccurrence has been a widely accepted approach. However, the drug resistance developed by tumour cells causes recurrence of cancer. It is imperative to understand the molecular mechanism involved in the development of drug resistance and tumour progression for developing potential therapy. Tumour microenvironment and cellular cross-talk via activation of various signalling pathways are responsible for tumour progression and metastasis. The comprehensive reviews are already available on the tumour microenvironment, signalling cascades responsible for tumour progression, and cellular crosstalk between malignant cells and immune cells. Therefore, we intend to provide comprehend review postulating the importance of mesenchymal stem cells (MSCs) in osteosarcoma progression and metastasis. This paper is aimed to provide information sequentially includes: tumour microenvironment, MSCs role in osteosarcoma progression, the hypoxic environment in MSCs recruitment at the tumour site and the importance of exosomes in tumorigenesis, progression and metastasis. Overall, this review may enlighten the research on the role of MSCs and MSCs derived exosome in osteosarcoma progression and drug resistance. This possibly may result in developing novel therapeutic approaches to combat the osteosarcoma effectively and contributes for the development of prognosis tools for early diagnosis.

Mechanistic role of perfusion culture on bone regeneration.

Bone tissue engineering (BTE) aims to develop engineered bone tissue to substitute conventional bone grafts. To achieve this, culturing the cells on the biocompatible three-dimensional (3D) scaffold is one alternative approach. The new functional bone tissue regeneration could be feasible by the synergetic combinations of cells, biomaterials and bioreactors. Although the field of biomaterial design/development for BTE applications attained reasonable success, development of suitable bioreactor remains still a major challenge. Tissue engineering bioreactors provide the microenvironment required for neo-tissue regeneration, and also can be used to study the physio-chemical cues effect on cell proliferation and differentiation in order to produce functional tissue. In this direction, various bioreactors have been developed and evaluated for the successful development of engineered bone tissue. Continues assessment of tissue development and limitations of the bioreactors lead to the progression of perfusion flow bioreactor system. Improvements in perfusion reactor system were able to yield multiple tissue engineered constructs with uniform cell distribution, easy to operate protocols and also effectively handled for the functional tissue development to meet the adequate supply of engineered graft for clinical application.

Improved osteogenic differentiation of umbilical cord blood MSCs using custom made perfusion bioreactor.

BACKGROUND: 3D cell culture is an appropriate method to develop engineered bone tissue, where different bioreactors have been designed to mitigate the challenges in 3D culture. Currently, we tailored a perfusion reactor to witness human mesenchymal stem cells (MSCs) proliferation and differentiation over polylactic acid-polyethylene glycol (PLA/PEG) composite scaffolds. METHODS: The composite scaffolds with different weight ratios of PLA and PEG were prepared using solvent casting-particulate leaching technique. Human umbilcal card blood MSCs were cultured under dynamic and static conditions to elucidate the role of dynamic fluid flow in osteogenesis of MSCs. RESULTS: The human MSCs distribution over the scaffolds was confirmed with fluorescent microscopy. Alkaline phosphatase (ALP), calcium mineralization, and collagen formation were found to be higher in PLA90 scaffolds than PLA100 and PLA75. PLA90 scaffolds with better cell adhesion/proliferartion were considered for bioreactor studies and they exhibited enhanced ALP, Ca+2 mineralization and collagen formation under dynamic perfusion than static culture. We further confirmed our observation by looking at expression levels of osteogenic marker (Runx2 and osteonectin) in differentiated MSCs subjected to perfusion culture compared to static culture. CONCLUSION: The results of the current investigation once again proves that dynamic perfusion cultures improve the osteogenic differentiation of MSCs over hybrid polymer scaffolds (PLA90) for effective bone regeneration.

PCL-Based Composite Scaffold Matrices for Tissue Engineering Applications.

Biomaterial-based scaffolds are important cues in tissue engineering (TE) applications. Recent advances in TE have led to the development of suitable scaffold architecture for various tissue defects. In this narrative review on polycaprolactone (PCL), we have discussed in detail about the synthesis of PCL, various properties and most recent advances of using PCL and PCL blended with either natural or synthetic polymers and ceramic materials for TE applications. Further, various forms of PCL scaffolds such as porous, films and fibrous have been discussed along with the stem cells and their sources employed in various tissue repair strategies. Overall, the present review affords an insight into the properties and applications of PCL in various tissue engineering applications.