Restoring Vision Using Stem Cells and Transplantation.

The replacement of retinal cells, or the support of surviving retinal neurons, in a degenerated retina presents a significant challenge in the fields of ophthalmology and regenerative medicine. Stem cell-based therapies are being explored as an approach for treating retinal dystrophies, such as retinitis pigmentosa (RP), Stargardt's disease, and age-related macular degeneration (AMD). This review provides an update on the recent progress made toward the restoration of vision lost to degenerative disease using stem cell-based transplantation strategies and the challenges that need to be overcome. Both retinal pigmented epithelium (RPE) and photoreceptor replacement therapies are discussed.

Engineering Extracellular Vesicles with the Tools of Enzyme Prodrug Therapy.

Extracellular vesicles (EVs) have recently gained significant attention as important mediators of intercellular communication, potential drug carriers, and disease biomarkers. These natural cell-derived nanoparticles are postulated to be biocompatible, stable under physiological conditions, and to show reduced immunogenicity as compared to other synthetic nanoparticles. Although initial clinical trials are ongoing, the use of EVs for therapeutic applications may be limited due to undesired off-target activity and potential "dilution effects" upon systemic administration which may affect their ability to reach their target tissues. To fully exploit their therapeutic potential, EVs are embedded into implantable biomaterials designed to achieve local delivery of therapeutics taking advantage of enzyme prodrug therapy (EPT). In this first application of EVs for an EPT approach, EVs are used as smart carriers for stabilizing enzymes in a hydrogel for local controlled conversion of benign prodrugs to active antiinflammatory compounds. It is shown that the natural EVs' antiinflammatory potential is comparable or superior to synthetic carriers, in particular upon repeated long-term incubations and in different macrophage models of inflammation. Moreover, density-dependent color scanning electron microscopy imaging of EVs in a hydrogel is presented herein, an impactful tool for further understanding EVs in biological settings.

Sequence-Dependent Self-Assembly and Structural Diversity of Islet Amyloid Polypeptide-Derived ß-Sheet Fibrils.

Determining the structural origins of amyloid fibrillation is essential for understanding both the pathology of amyloidosis and the rational design of inhibitors to prevent or reverse amyloid formation. In this work, the decisive roles of peptide structures on amyloid self-assembly and morphological diversity were investigated by the design of eight amyloidogenic peptides derived from islet amyloid polypeptide. Among the segments, two distinct morphologies were highlighted in the form of twisted and planar (untwisted) ribbons with varied diameters, thicknesses, and lengths. In particular, transformation of amyloid fibrils from twisted ribbons into untwisted structures was triggered by substitution of the C-terminal serine with threonine, where the side chain methyl group was responsible for the distinct morphological change. This effect was confirmed following serine substitution with alanine and valine and was ascribed to the restriction of intersheet torsional strain through the increased hydrophobic interactions and hydrogen bonding. We also studied the variation of fibril morphology (i.e., association and helicity) and peptide aggregation propensity by increasing the hydrophobicity of the peptide side group, capping the N-terminus, and extending sequence length. We anticipate that our insights into sequence-dependent fibrillation and morphological diversity will shed light on the structural interpretation of amyloidogenesis and development of structure-specific imaging agents and aggregation inhibitors.

Enhanced articular cartilage by human mesenchymal stem cells in enzymatically mediated transiently RGDS-functionalized collagen-mimetic hydrogels.

Recapitulation of the articular cartilage microenvironment for regenerative medicine applications faces significant challenges due to the complex and dynamic biochemical and biomechanical nature of native tissue. Towards the goal of biomaterial designs that enable the temporal presentation of bioactive sequences, recombinant bacterial collagens such as Streptococcal collagen-like 2 (Scl2) proteins can be employed to incorporate multiple specific bioactive and biodegradable peptide motifs into a single construct. Here, we first modified the backbone of Scl2 with glycosaminoglycan-binding peptides and cross-linked the modified Scl2 into hydrogels via matrix metalloproteinase 7 (MMP7)-cleavable or non-cleavable scrambled peptides. The cross-linkers were further functionalized with a tethered RGDS peptide creating a system whereby the release from an MMP7-cleavable hydrogel could be compared to a system where release is not possible. The release of the RGDS peptide from the degradable hydrogels led to significantly enhanced expression of collagen type II (3.9-fold increase), aggrecan (7.6-fold increase), and SOX9 (5.2-fold increase) by human mesenchymal stem cells (hMSCs) undergoing chondrogenesis, as well as greater extracellular matrix accumulation compared to non-degradable hydrogels (collagen type II; 3.2-fold increase, aggrecan; 4-fold increase, SOX9; 2.8-fold increase). Hydrogels containing a low concentration of the RGDS peptide displayed significantly decreased collagen type I and X gene expression profiles, suggesting a major advantage over either hydrogels functionalized with a higher RGDS peptide concentration, or non-degradable hydrogels, in promoting an articular cartilage phenotype. These highly versatile Scl2 hydrogels can be further manipulated to improve specific elements of the chondrogenic response by hMSCs, through the introduction of additional bioactive and/or biodegradable motifs. As such, these hydrogels have the possibility to be used for other applications in tissue engineering. STATEMENT OF SIGNIFICANCE: Recapitulating aspects of the native tissue biochemical microenvironment faces significant challenges in regenerative medicine and tissue engineering due to the complex and dynamic nature of the tissue. The ability to take advantage of, mimic, and modulate cell-mediated processes within novel naturally-derived hydrogels is of great interest in the field of biomaterials to generate constructs that more closely resemble the biochemical microenvironment and functions of native biological tissues such as articular cartilage. Towards this goal, the temporal presentation of bioactive sequences such as RGDS on the chondrogenic differentiation of human mesenchymal stem cells is considered important as it has been shown to influence the chondrogenic phenotype. Here, a novel and versatile platform to recreate a high degree of biological complexity is proposed, which could also be applicable to other tissue engineering and regenerative medicine applications.

Impact of an integrated medicines management service on preventable medicines-related readmission to hospital: a descriptive study.

BACKGROUND: Medication contributes to 5-20% of hospital admissions, of which half are considered preventable. An integrated medicines management service (IMMS) was developed at a large general hospital in London to identify and manage patients at risk of a preventable medicines-related readmission (PMRR) to reduce the risk of PMRR. OBJECTIVE: To investigate the effect of the pharmacy IMMS on the rate of PMRR within 30 days of the first discharge. METHOD: 744 patients were identified between October 2008 and October 2014, using the PREVENT tool. Patients at risk were managed by the IMMS with medication reconciliation, review, consultation and follow-up, as required. RESULTS: Of 744 patients, 119 were readmitted within 30 days of discharge, with a PMRR for 2 patients (1.7%). The main reason for referral to the service was to assess the need to start a compliance aid. Most interventions involved communication: 84% included patient consultations with 50% involving discussion with the patient's community pharmacist and 32% with their general practitioner surgery. CONCLUSIONS: An IMMS may be an effective method of reducing the rate of PMRR. Further work is needed to establish the cost-effectiveness of the service.

Pericyte Seeded Dual Peptide Scaffold with Improved Endothelialization for Vascular Graft Tissue Engineering.

The development of synthetic vascular grafts for coronary artery bypass is challenged by insufficient endothelialization, which increases the risk of thrombosis, and the lack of native cellular constituents, which favors pathological remodeling. Here, a bifunctional electrospun poly(ε-caprolactone) (PCL) scaffold with potential for synthetic vascular graft applications is presented. This scaffold incorporates two tethered peptides: the osteopontin-derived peptide (Adh) on the "luminal" side and a heparin-binding peptide (Hep) on the "abluminal" side. Additionally, the "abluminal" side of the scaffold is seeded with saphenous vein-derived pericytes (SVPs) as a source of proangiogenic growth factors. The Adh peptide significantly increases endothelial cell adhesion, while the Hep peptide promotes accumulation of vascular endothelial growth factor secreted by SVPs. SVPs increase endothelial migration both in a transwell assay and a modified scratch assay performed on the PCL scaffold. Seeding of SVPs on the "abluminal"/Hep side of the scaffold further increases endothelial cell density, indicating a combinatory effect of the peptides and pericytes. Finally, SVP-seeded scaffolds are preserved by freezing in a xeno-free medium, maintaining good cell viability and function. In conclusion, this engineered scaffold combines patient-derived pericytes and spatially organized functionalities, which synergistically increase endothelial cell density and growth factor retention.

Temporally degradable collagen-mimetic hydrogels tuned to chondrogenesis of human mesenchymal stem cells.

Tissue engineering strategies for repairing and regenerating articular cartilage face critical challenges to recapitulate the dynamic and complex biochemical microenvironment of native tissues. One approach to mimic the biochemical complexity of articular cartilage is through the use of recombinant bacterial collagens as they provide a well-defined biological 'blank template' that can be modified to incorporate bioactive and biodegradable peptide sequences within a precisely defined three-dimensional system. We customized the backbone of a Streptococcal collagen-like 2 (Scl2) protein with heparin-binding, integrin-binding, and hyaluronic acid-binding peptide sequences previously shown to modulate chondrogenesis and then cross-linked the recombinant Scl2 protein with a combination of matrix metalloproteinase 7 (MMP7)- and aggrecanase (ADAMTS4)-cleavable peptides at varying ratios to form biodegradable hydrogels with degradation characteristics matching the temporal expression pattern of these enzymes in human mesenchymal stem cells (hMSCs) during chondrogenesis. hMSCs encapsulated within the hydrogels cross-linked with both degradable peptides exhibited enhanced chondrogenic characteristics as demonstrated by gene expression and extracellular matrix deposition compared to the hydrogels cross-linked with a single peptide. Additionally, these combined peptide hydrogels displayed increased MMP7 and ADAMTS4 activities and yet increased compression moduli after 6 weeks, suggesting a positive correlation between the degradation of the hydrogels and the accumulation of matrix by hMSCs undergoing chondrogenesis. Our results suggest that including dual degradation motifs designed to respond to enzymatic activity of hMSCs going through chondrogenic differentiation led to improvements in chondrogenesis. Our hydrogel system demonstrates a bimodal enzymatically degradable biological platform that can mimic native cellular processes in a temporal manner. As such, this novel collagen-mimetic protein, cross-linked via multiple enzymatically degradable peptides, provides a highly adaptable and well defined platform to recapitulate a high degree of biological complexity, which could be applicable to numerous tissue engineering and regenerative medicine applications.

Harnessing the Versatility of Bacterial Collagen to Improve the Chondrogenic Potential of Porous Collagen Scaffolds.

Collagen I foams are used in the clinic as scaffolds to promote articular cartilage repair as they provide a bioactive environment for cells with chondrogenic potential. However, collagen I as a base material does not allow for precise control over bioactivity. Alternatively, recombinant bacterial collagens can be used as "blank slate" collagen molecules to offer a versatile platform for incorporation of selected bioactive sequences and fabrication into 3D scaffolds. Here, we show the potential of Streptococcal collagen-like 2 (Scl2) protein foams modified with peptides designed to specifically and noncovalently bind hyaluronic acid and chondroitin sulfate to improve chondrogenesis of human mesenchymal stem cells (hMSCs) compared to collagen I foams. Specific compositions of functionalized Scl2 foams lead to improved chondrogenesis compared to both nonfunctionalized Scl2 and collagen I foams, as indicated by gene expression, extracellular matrix accumulation, and compression moduli. hMSCs cultured in functionalized Scl2 foams exhibit decreased collagens I and X gene and protein expression, suggesting an advantage over collagen I foams in promoting a chondrocytic phenotype. These highly modular foams can be further modified to improve specific aspects chondrogenesis. As such, these scaffolds also have the potential to be tailored for other regenerative medicine applications.

Raman Spectroscopy Reveals New Insights into the Zonal Organization of Native and Tissue-Engineered Articular Cartilage.

Tissue architecture is intimately linked with its functions, and loss of tissue organization is often associated with pathologies. The intricate depth-dependent extracellular matrix (ECM) arrangement in articular cartilage is critical to its biomechanical functions. In this study, we developed a Raman spectroscopic imaging approach to gain new insight into the depth-dependent arrangement of native and tissue-engineered articular cartilage using bovine tissues and cells. Our results revealed previously unreported tissue complexity into at least six zones above the tidemark based on a principal component analysis and k-means clustering analysis of the distribution and orientation of the main ECM components. Correlation of nanoindentation and Raman spectroscopic data suggested that the biomechanics across the tissue depth are influenced by ECM microstructure rather than composition. Further, Raman spectroscopy together with multivariate analysis revealed changes in the collagen, glycosaminoglycan, and water distributions in tissue-engineered constructs over time. These changes were assessed using simple metrics that promise to instruct efforts toward the regeneration of a broad range of tissues with native zonal complexity and functional performance.

Collagen-mimetic peptide-modifiable hydrogels for articular cartilage regeneration.

Regenerative medicine strategies for restoring articular cartilage face significant challenges to recreate the complex and dynamic biochemical and biomechanical functions of native tissues. As an approach to recapitulate the complexity of the extracellular matrix, collagen-mimetic proteins offer a modular template to incorporate bioactive and biodegradable moieties into a single construct. We modified a Streptococcal collagen-like 2 protein with hyaluronic acid (HA) or chondroitin sulfate (CS)-binding peptides and then cross-linked with a matrix metalloproteinase 7 (MMP7)-sensitive peptide to form biodegradable hydrogels. Human mesenchymal stem cells (hMSCs) encapsulated in these hydrogels exhibited improved viability and significantly enhanced chondrogenic differentiation compared to controls that were not functionalized with glycosaminoglycan-binding peptides. Hydrogels functionalized with CS-binding peptides also led to significantly higher MMP7 gene expression and activity while the HA-binding peptides significantly increased chondrogenic differentiation of the hMSCs. Our results highlight the potential of this novel biomaterial to modulate cell-mediated processes and create functional tissue engineered constructs for regenerative medicine applications.

The use of N-methylpyrrolidone as a cosolvent and oxidant in pharmaceutical stress testing.

The use of N-methylpyrrolidone (NMP) as an oxidant and cosolvent in pharmaceutical stress testing (forced degradation) is examined. Various active pharmaceutical ingredients were heated in NMP-water solutions under nitrogen, air, and oxygen and then analyzed by high-performance liquid chromatography, usually with ultraviolet diode array detection and mass spectrometry detection. In some cases, degradation products were isolated and characterized by nuclear magnetic resonance. The NMP-water-air-heat system provided oxidative and hydrolytic degradation products. The observed oxidation products were consistent with products expected from free radical autoxidation, reactions with hydroperoxides, and possibly singlet oxygen. Oxidative and hydrolytic pathways could be distinguished by comparison of the reactions carried out under air/oxygen and nitrogen. In many cases, the oxidation products observed during stress testing were also observed during formal stability studies of drug products. The NMP-water-air-heat stress condition facilitates various oxidative degradation pathways, which are often relevant to drug product on stability. This approach facilitates stability-indicating method development and helps elucidate degradation pathways.

A model for selecting assessment methods for evaluating medical students in African medical schools.

Introduction of more effective and standardized assessment methods for testing students' performance in Africa's medical institutions has been hampered by severe financial and personnel shortages. Nevertheless, some African institutions have recognized the problem and are now revising their medical curricula, and, therefore, their assessment methods. These institutions, and those yet to come, need guidance on selecting assessment methods so as to adopt models that can be sustained locally. The authors provide a model for selecting assessment methods for testing medical students' performance in African medical institutions. The model systematically evaluates factors that influence implementation of an assessment method. Six commonly used methods (the essay examinations, short-answer questions, multiple-choice questions, patient-based clinical examination, problem-based oral examination [POE], and objective structured clinical examination) are evaluated by scoring and weighting against performance, cost, suitability, and safety factors. In the model, the highest score identifies the most appropriate method. Selection of an assessment method is illustrated using two institutional models, one depicting an ideal situation in which the objective structured clinical examination was preferred, and a second depicting the typical African scenario in which the essay and short-answer-question examinations were best. The POE method received the highest score and could be recommended as the most appropriate for Africa's medical institutions, but POE assessments require changing the medical curricula to a problem-based learning approach. The authors' model is easy to understand and promotes change in the medical curriculum and method of student assessment.

Melatonin protects against copper-mediated free radical damage.

Copper is an essential trace element which forms an integral component of many enzymes. While trace amounts of copper are needed to sustain life, excess copper is extremely toxic. Copper has been implicated in various neurodegenerative disorders, such as Wilson's and Alzheimer's diseases. Previous studies showed that melatonin, the principle secretory product of the pineal gland, binds Cupric chloride (Cu2+) and that this may have implications in copper-induced neurodegenerative diseases. In the present study, in vitro copper-mediated lipid peroxidation was induced. Melatonin (5 mM) protected against copper-mediated lipid peroxidation in liver homogenates. Electron micrographs of in vivo administered Cu2+ and melatonin show that melatonin affords some protection to rat hepatocytes in the presence of copper. Electrochemical studies performed show that melatonin, in addition to binding Cu2+, may provide protection against copper-mediated free radical damage by binding Cu1+. The findings of these studies provide further evidence for the neuroprotective role of melatonin.