Validation of ERICVA Risk Score as a Predictor of One Year Amputation-Free Survival of Patients with Critical Limb Ischemia.

BACKGROUND: The ERICVA score was derived to predict amputation-free survival in patients with critical limb ischemia (CLI). It may be a useful tool to stratify patients in trials of novel interventions to treat CLI but, as yet, it has not been externally validated. METHODS: A prospective database of CLI patients was developed during prescreening of patients for a phase 1 stem cell therapy clinical trial. The primary outcome was amputation free survival (AFS) at 1 year. Both the full ERICVA scale (11 parameters) and simplified ERICVA scale (5 parameters) were validated. Data analysis was performed by calculation of the area under the receiver operating characteristic (ROC) curve examining the predictive value of the scores. The Chi-square test was used to examine the association between risk group and one-year AFS and the cumulative survival of the three risk groups was compared using Kaplan Meier survival curves. RESULTS: A series of 179 CLI patients were included in the analysis. The Chi-square test of independence showed a significant association between the risk group (high, medium and low) and one-year AFS outcome (P = 0.0007). Kaplan-Meier survival curve showed significant difference in one-year AFS between the three risk groups (log-rank P < 0.001). The area under the curve (AUC) was found to be 0.63 and 0.61 for the full and simplified score, respectively. The sensitivity of the full score was 0.44 with specificity of 0.84. The simplified score had a sensitivity of 0.28 and specificity of 0.92. CONCLUSION: The ERICVA risk score system was found to have a fair validity but cannot be considered reliable as a single predictor of one year AFS of CLI patients. The simplified score had an AUC almost identical to the full score and can accordingly replace the full score.

Anti-donor antibody induction following intramuscular injections of allogeneic mesenchymal stromal cells.

Allogeneic mesenchymal stromal cells (allo-MSC) are a promising "off-the-shelf" therapy with anti-inflammatory and pro-repair properties. This study investigated humoral immune responses to intramuscular (IM) injections of allo-MSC. Total and isotype-specific anti-donor IgG and donor-specific complement-mediated lysis were determined in sera from healthy mice 2 weeks after single or repeated IM injections of fully mismatched-MHC allo-MSC with comparison to mice receiving syngeneic MSC, allogeneic splenocytes or saline. In mice subjected to hind limb ischemia (HLI), anti-donor IgG was analyzed following IM allo-MSC injection with and without administration of the T-cell immunosuppressant tacrolimus. Recipients of single and repeated IM allo-MSC developed readily-detectable anti-donor IgG. Serum anti-donor IgG levels were similar to those of allo-splenocyte recipients but had higher IgG1/IgG2a ratio and variable capacity for complement-mediated lysis of donor cells. The induced anti-donor IgG bound readily to allo-MSC and this binding was increased following allo-MSC pretreatment with interferon gamma. In mice with HLI, IM injection of allo-MSC into the ischemic limb was also associated with induction of anti-donor IgG but this was abrogated by tacrolimus (FK-506). The results indicate that allo-MSC are inherently immunogenic when delivered intramuscularly to healthy and ischemic mouse hind limb, but induce an IgG1-skewed humoral response that is suppressed by tacrolimus.

Current good manufacturing practice considerations for mesenchymal stromal cells as therapeutic agents.

Producing human mesenchymal stromal cells (MSCs) for clinical use requires adherence to current good manufacturing practice (cGMP) standards. This is necessary for ensuring standardization and reproducibility through the manufacturing process, but also, for product quality and safety. However, the large-scale production of clinical-grade MSCs possesses unique regulatory challenges and hurdles related to the heterogeneous nature of MSC cultures as well as the complex manufacturing process. Following is a compilation of the major issues encountered in the manufacturing of MSCs for clinical use, and our views on the optimal characteristics of the final MSC product.

When Origin Matters: Properties of Mesenchymal Stromal Cells From Different Sources for Clinical Translation in Kidney Disease.

Advanced therapy medicinal products (ATMPs) offer new prospects to improve the treatment of conditions with unmet medical needs. Kidney diseases are a current major health concern with an increasing global prevalence. Chronic renal failure appears after many years of impairment, which opens a temporary window to apply novel therapeutic approaches to delay or halt disease progression. The immunomodulatory, anti-inflammatory, and pro-regenerative properties of mesenchymal stromal cells (MSCs) have sparked interest for their use in cell-based regenerative therapies. Currently, several early-phase clinical trials have been completed and many are ongoing to explore MSC safety and efficacy in a wide range of nephropathies. However, one of the current roadblocks to the clinical translation of MSC therapies relates to the lack of standardization and harmonization of MSC manufacturing protocols, which currently hinders inter-study comparability. Studies have shown that cell culture processing variables can have significant effects on MSC phenotype and functionality, and these are highly variable across laboratories. In addition, heterogeneity within MSC populations is another obstacle. Furthermore, MSCs may be isolated from several sources which adds another variable to the comparative assessment of outcomes. There is now a growing body of literature highlighting unique and distinctive properties of MSCs according to the tissue origin, and that characteristics such as donor, age, sex and underlying medical conditions may alter the therapeutic effect of MSCs. These variables must be taken into consideration when developing a cell therapy product. Having an optimal scale-up strategy for MSC manufacturing is critical for ensuring product quality while minimizing costs and time of production, as well as avoiding potential risks. Ideally, optimal scale-up strategies must be carefully considered and identified during the early stages of development, as making changes later in the bioprocess workflow will require re-optimization and validation, which may have a significant long-term impact on the cost of the therapy. This article provides a summary of important cell culture processing variables to consider in the scale-up of MSC manufacturing as well as giving a comprehensive review of tissue of origin-specific biological characteristics of MSCs and their use in current clinical trials in a range of renal pathologies.

Diabetes Mellitus and Gender Have a Negative Impact on the Outcome of Hip Fracture Surgery-A Pilot Study.

Diabetes mellitus (DM) is associated with an elevated risk of post-operative complications. The impact it has on patients living with DM following hip fracture surgery (HFS) is not completely understood and may represent a predictor of increased mortality. This study investigates the impact of DM, gender, American Society of Anaesthesiologists (ASA) grade, and fracture location, on the outcome of HFS in Ireland. The Hospital Inpatient Enquiry (HIPE) database records all fragility hip fractures within Galway University Hospital. Retrospective data collection was performed over a 3-year period. Data collected included patient age, gender, date of HFS, anatomical fracture location, type of operation, ASA grade, DM status, and mortality. A database of 650 individuals was created including 461 females and 189 males, with an average group age of 80.2 ± 9.3 years. Results showed a significantly higher incidence of hip fractures in males with DM (19.57%) than females with DM (12.36%) (χ2 test, p = 0.020). Cox regression survival analysis indicated that DM status and ASA grade were the two main independent predictors of patient survival following HFS. Nevertheless, when examining the combined impact of gender and DM status on survival after HFS, results showed that survival post HFS differed significantly with gender and presence of DM (log-rank test, p < 0.001), with males with DM performing worse than females with DM (p = 0.021) or males without DM (p = 0.001). This gender and disease-associated outcome should prompt an early multi-disciplinary team approach to the management of hip fractures in patients with DM. © 2019 Orthopaedic Research Society. Published by Wiley Periodicals, Inc. J Orthop Res 38:834-842, 2020.

Knowledge, Perceptions and Concerns of Diabetes -Associated Complications Among Individuals Living with Type 1 and Type 2 Diabetes Mellitus.

The purpose of this study was to investigate the knowledge, perceptions and concerns of individuals living with diabetes mellitus regarding the disorder and its associated long-term health complications. Individuals living with type 1 (N = 110) and type 2 (N = 100) diabetes were surveyed at the Diabetes Centre at University Hospital Galway (Ireland). A questionnaire was used to record respondent's perceptions and concerns about living with diabetes and developing associated long-term health complications, especially diabetes-induced osteopathy. Participants' responses revealed a variety of perspectives. Individuals with type 1 diabetes had a deeper understanding of the aetiology of diabetes and were more concerned about its complications than individuals with type 2 diabetes. The most recognized complications identified by the participants were retinopathy (92% type 1; 83% type 2), amputations (80% type 1; 70% type 2) and nephropathy (83% type 1; 63% type 2). Diabetes-related osteopathy was under-recognized, with 37% (type 1) and 23% (type 2) of respondents identifying bone fractures as a diabetes-related complication. Enhancing the patient awareness of this under-recognized diabetes-associated complication and ensuring that preventative measures are incorporated within health care programmes may offer methodologies to address this complication clinically.

In vitro models for assessing therapeutic angiogenesis.

Arterial obstruction leading to ischemia causes a reduction of oxygen and nutrient supply to distal tissues. The physiological response to tissue ischemia triggers a cascade of events that results in the development of accessory vasculature to increase local tissue perfusion and to salvage tissue. However, this adaptive mechanism of repair is suboptimal in some patients. Therapeutic angiogenesis aims to stimulate new blood vessel formation via the local administration of proangiogenic agents or cell therapy products (CTPs). In this review, we provide a summary of the current understanding of in vitro models for assessing the angiogenic potential of a product.

Macromolecular crowding meets oxygen tension in human mesenchymal stem cell culture - A step closer to physiologically relevant in vitro organogenesis.

Modular tissue engineering is based on the cells' innate ability to create bottom-up supramolecular assemblies with efficiency and efficacy still unmatched by man-made devices. Although the regenerative potential of such tissue substitutes has been documented in preclinical and clinical setting, the prolonged culture time required to develop an implantable device is associated with phenotypic drift and/or cell senescence. Herein, we demonstrate that macromolecular crowding significantly enhances extracellular matrix deposition in human bone marrow mesenchymal stem cell culture at both 20% and 2% oxygen tension. Although hypoxia inducible factor - 1α was activated at 2% oxygen tension, increased extracellular matrix synthesis was not observed. The expression of surface markers and transcription factors was not affected as a function of oxygen tension and macromolecular crowding. The multilineage potential was also maintained, albeit adipogenic differentiation was significantly reduced in low oxygen tension cultures, chondrogenic differentiation was significantly increased in macromolecularly crowded cultures and osteogenic differentiation was not affected as a function of oxygen tension and macromolecular crowding. Collectively, these data pave the way for the development of bottom-up tissue equivalents based on physiologically relevant developmental processes.

Inefficiency in macromolecular transport of SCS-based microcapsules affects viability of primary human mesenchymal stem cells but not of immortalized cells.

Microcapsules made of sodium cellulose sulphate (SCS) and poly-diallyl-dimethyl-ammonium chloride (pDADMAC) have been employed to encapsulate a wide range of established cell lines for several applications. However, little is known about the encapsulation of primary cells including human mesenchymal stem cells (hMSCs). Human MSCs are of interest in regenerative medicine applications due to pro-angiogenic, anti-inflammatory and immunomodulatory properties, which result from paracrine effects of this cell type. In the present work we have encapsulated primary hMSCs and hMSC-TERT immortalized cells and compared their behavior and in vitro angiogenic potential. We found that, although both cell types were able to secret angiogenic factors such as VEGF, there was a marked reduction of primary hMSC viability compared to hMSC-TERT cells when cultured in these microcapsules. Moreover, this applied to other primary cell cultures such as primary human fibroblasts but not to other cell lines such as human embryonic kidney 293 (HEK293) cells. We found that the microcapsule membrane had a molecular weight cut-off below a critical size, which caused impairment in the diffusion of essential nutrients and had a more detrimental effect on the viability of primary cell cultures compared to cell lines and immortalized cells.

A shape-controlled tuneable microgel platform to modulate angiogenic paracrine responses in stem cells.

Development of cell delivery platforms have been driven based on an empirical cytoprotective design. While cell-matrix and cell-cell interactions that influence biochemical effects beyond survival has been limited and overshadowed in an effort to incrementally improve biomimicking properties of the tissue-engineered constructs. Here we demonstrate fabrication of a shape controlled 3D type-I collagen-based microgel platform that can be tuned to modulate angiogenic paracrine- 'angiocrine' responses of human mesenchymal stem cells (hMSCs). Furthermore, these microgels were characterized as a 3D cell culture tool to assess optimal biological response as a function of cell-matrix and cell-cell interactions. Finally, optimised hMSC embedded microgels were shown to induce vascular repair and functional improvement in vivo in a mouse model of hind-limb ischemia. The approach described here in designing a tuneable cell delivery platform using naturally occurring extracellular matrix molecules highlights the need for highly customised matrices with an array of self-assembling proteins that dictate specific cell function resembling the native tissue of interest for repair.