A novel therapeutic management for diabetes patients with chronic limb-threatening ischemia: comparison of autologous bone marrow mononuclear cells versus allogenic Wharton jelly-derived mesenchymal stem cells.

BACKGROUND: Chronic limb-threatening ischemia (CLTI) represents the final stage of peripheral arterial disease. Approximately one-third of patients with CLTI are not eligible for conventional surgical treatments. Furthermore, patients with advanced stage of CLTI are prone to amputation and death. Thus, an effective therapeutic strategy is urgently needed. In this context, autologous bone marrow mononuclear cell (auto-BM-MNC) and allogeneic mesenchymal stem cells represent a promising therapeutic approach for treating CLTI. In this study, we compared the safety and beneficial therapeutic effect of auto-BM-MNC versus allogeneic Wharton jelly-derived mesenchymal stem cells (allo-WJ-MSCs) in diabetic patients with CLTI. METHODS: We performed a randomized, prospective, double-blind and controlled pilot study. Twenty-four diabetic patients in the advanced stage of CLTI (4 or 5 in Rutherford's classification) and a transcutaneous oxygen pressure (TcPO2) below 30 mmHg were randomized to receive 15 injections of (i) auto-BM-MNC (7.197 × 106 ± 2.984 × 106 cells/mL) (n = 7), (ii) allo-WJ-MSCs (1.333 × 106 cells/mL) (n = 7) or (iii) placebo solution (1 mL) (n = 10), which were administered into the periadventitial layer of the arterial walls under eco-Doppler guidance. The follow-up visits were at months 1, 3, 6, and 12 to evaluate the following parameters: (i) Rutherford's classification, (ii) TcPO2, (iii) percentage of wound closure, (iv) pain, (v) pain-free walking distance, (vi) revascularization and limb-survival proportion, and (vii) life quality (EQ-5D questionnaire). RESULTS: No adverse events were reported. Patients with CLTI who received auto-BM-MNC and allo-WJ-MSCs presented an improvement in Rutherford's classification, a significant increase in TcPO2 values‬, a reduction in the lesion size in a shorter time, a decrease in the pain score and an increase in the pain-free walking distance, in comparison with the placebo group. In addition, the participants treated with auto-BM-MNC and allo-WJ-MSCs kept their limbs during the follow-up period, unlike the placebo group, which had a marked increase in amputation. CONCLUSIONS: Our results showed that patients with CLTI treated with auto-BM-MNC and allo-WJ-MSCs conserved 100% of their limb during 12 months of the follow-up compared to the placebo group, where 60% of participants underwent limb amputation in different times. Furthermore, we observed a faster improvement in the allo-WJ-MSC group, unlike the auto-BM-MNC group. Trial registration This study was retrospectively registered at ClinicalTrials.gov (NCT05631444).

Role of mesenchymal stromal cells derivatives in diabetic foot ulcers: a controlled randomized phase 1/2 clinical trial.

BACKGROUND: Diabetes-related foot complications have been identified as the most common isolated cause of morbidity among patients with diabetes and the leading cause of amputation. Therefore, new strategies to stimulate skin regeneration may provide a novel therapeutic approach to reduce non-healing ulcer disease. Recently, we demonstrated in proof-of-concept in humans that administration of allogeneic bone marrow mesenchymal stromal cellss derivatives (allo-hBM-MSCDs) is effective in a similar way to the use of allogeneic bone marrow mesenchymal stromal cellss (allo-hBM-MSCs) in grade 2 diabetic foot ulcers (DFUs). AIM: To assess the safety and efficacy profile of the allo-hBM-MSCDs relative to the conventional approach (PolyMen® dressing) in 1/2 clinical trial phases in patients with grade 1 and 2 DFUs. METHODS: In the present study, we used 2 doses of allo-hBM-MSCDs (1 mL) or 1 dose of allo-hBM-MSCs (1 × 106 cells) intradermally injected around wounds and assessed their safety and effectiveness, relative to the conventional approach (PolyMem dressing). Allo-hBM-MSCDs and allo-hBM-MSCs were produced in a certified Good Manufacturing Practice-type Laboratory. Patients with grade 1 and 2 DFUs were randomized to receive allo-hBM-MSCDs (n=12), allo-hBM-MSCs (n=6) or conventional treatment (PolyMem dressing) (n=10). The wound-healing process was macroscopically evaluated until the complete closure of the ulcers. RESULTS: No adverse events were reported. Patients with grade 1 and 2 DFUs treated with either allo-hBM-MSCDs or allo-hBM-MSCs, achieved greater percentages of wound closure, enhanced skin regeneration in shorter times and a greater ulcer-free survival relative to the patients who received conventional treatment. Finally, through proteomic analysis, we elucidated the proteins and growth factors that are secreted by allo-hBM-MSCs and relevant to the wound-healing process. In addition, by combining proteomics with Gene Ontology analysis, we comprehensively classified secreted proteins on both biological process and molecular function. CONCLUSIONS: In this phase 1/2 trial, our cumulative results suggest that 2 doses of allo-hBM-MSCDs combined with a wound dressing are a safe and effective treatment for grade 1 and 2 DFUs.

Current Advances in the Development of Hydrogel-Based Wound Dressings for Diabetic Foot Ulcer Treatment.

Diabetic foot ulcers (DFUs) are one of the most prevalent complications associated with diabetes mellitus. DFUs are chronic injuries that often lead to non-traumatic lower extremity amputations, due to persistent infection and other ulcer-related side effects. Moreover, these complications represent a significant economic burden for the healthcare system, as expensive medical interventions are required. In addition to this, the clinical treatments that are currently available have only proven moderately effective, evidencing a great need to develop novel strategies for the improved treatment of DFUs. Hydrogels are three-dimensional systems that can be fabricated from natural and/or synthetic polymers. Due to their unique versatility, tunability, and hydrophilic properties, these materials have been extensively studied for different types of biomedical applications, including drug delivery and tissue engineering applications. Therefore, this review paper addresses the most recent advances in hydrogel wound dressings for effective DFU treatment, providing an overview of current perspectives and challenges in this research field.

Decellularized Tissues for Wound Healing: Towards Closing the Gap Between Scaffold Design and Effective Extracellular Matrix Remodeling.

The absence or damage of a tissue is the main cause of most acute or chronic diseases and are one of the appealing challenges that novel therapeutic alternatives have, in order to recover lost functions through tissue regeneration. Chronic cutaneous lesions are the most frequent cause of wounds, being a massive area of regenerative medicine and tissue engineering to have efforts to develop new bioactive medical products that not only allow an appropriate and rapid healing, but also avoid severe complications such as bacterial infections. In tissue repair and regeneration processes, there are several overlapping stages that involve the synergy of cells, the extracellular matrix (ECM) and biomolecules, which coordinate processes of ECM remodeling as well as cell proliferation and differentiation. Although these three components play a crucial role in the wound healing process, the ECM has the function of acting as a biological platform to permit the correct interaction between them. In particular, ECM is a mixture of crosslinked proteins that contain bioactive domains that cells recognize in order to promote migration, proliferation and differentiation. Currently, tissue engineering has employed several synthetic polymers to design bioactive scaffolds to mimic the native ECM, by combining biopolymers with growth factors including collagen and fibrinogen. Among these, decellularized tissues have been proposed as an alternative for reconstructing cutaneous lesions since they maintain the complex protein conformation, providing the required functional domains for cell differentiation. In this review, we present an in-depth discussion of different natural matrixes recently employed for designing novel therapeutic alternatives for treating cutaneous injuries, and overview some future perspectives in this area.

Effect of biomolecules derived from human platelet-rich plasma on the ex vivo expansion of human adipose-derived mesenchymal stem cells for clinical applications.

Mesenchymal stem cells are a tool in cell therapies but demand a large cell number per treatment, for that, suitable culture media is required which contains fetal bovine serum (FBS). However, for cell-based therapy applications, the use of FBS is problematic. Several alternatives to FBS have been explored, including human derivatives from platelet-rich plasma (hD-PRP). Although various studies have evaluated the impact of hD-PRP on MSC proliferation and differentiation, few of them have assessed their influence on processes, such as metabolism and gene expression. Here, we cultured human adipose-derived MSCs (hAD-MSCs) in media supplemented with either 10% hD-PRP (hD-PRP-SM) or 10% FBS (FBS-SM) in order to characterize them and evaluate the effect of hD-PRP on cell metabolism, gene expression of associated regenerative factors, as well as chromosome stability during cell expansion. We found that hAD-MSCs cultured in hD-PRP-SM have a greater cell elongation but express similar surface markers; in addition, hD-PRP-SM promoted a significant osteogenic differentiation in the absence of differentiation medium and increased the growth rate, maintaining chromosomal stability. In terms of cell metabolic profile, hAD-MSC behavior did not reveal any differences between both culture conditions. Conversely, significant differences in collagen I and angiopoietin 2 expression were observed between both conditions. The present results suggest that hD-PRP may influence hAD-MSC behavior.

Mesenchymal stem cells derivatives as a novel and potential therapeutic approach to treat diabetic foot ulcers.

SUMMARY: Diabetic foot ulcer morbidity and mortality are dramatically increasing worldwide, reinforcing the urgency to propose more effective interventions to treat such a devastating condition. Previously, using a diabetic mouse model, we demonstrated that administration of bone marrow mesenchymal stem cells derivatives is more effective than the use of bone marrow mesenchymal stem cells alone. Here, we used the aforementioned treatments on three patients with grade 2 diabetic foot ulcers and assessed their beneficial effects, relative to the conventional approach. In the present study, two doses of cell derivatives, one dose of mesenchymal stem cells or one dose of vehicle (saline solution with 5% of human albumin), were intradermally injected around wounds. Wound healing process and changes on re-epithelialization were macroscopically evaluated until complete closure of the ulcers. All ulcers were simultaneously treated with conventional treatment (PolyMen® dressing). Patients treated with either cell derivatives or mesenchymal stem cells achieved higher percentages of wound closure in shorter times, relative to the patient treated with the conventional treatment. The cell derivative and mesenchymal stem cells approaches resulted in complete wound closure and enhanced skin regeneration at some point between days 35 and 42, although no differences between these two treatments were observed. Moreover, wounds treated with the conventional treatment healed after 161 days. Intradermal administration of cell derivatives improved wound healing to a similar extent as mesenchymal stem cells. Thus, our results suggest that mesenchymal stem cell derivatives may serve as a novel and potential therapeutic approach to treat diabetic foot ulcers. LEARNING POINTS: In diabetic mouse models, the administration of mesenchymal stem cells derivatives have been demonstrated to be more effective than the use of marrow mesenchymal stem cells alone. Mesenchymal stem cells have been explored as an attractive therapeutic option to treat non-healing ulcers. Mesenchymal stem cells derivatives accelerate the re-epithelialization on diabetic foot ulcers.

C-reactive protein, interleukin-6 and pre-eclampsia: large-scale evidence from the GenPE case-control study.

Multiple small studies have suggested that women with pre-eclampsia present elevated levels of C-reactive protein (CRP) and interleukin-6 (IL-6). However, little is known regarding the source of this CRP and IL-6 increase. Therefore, the aim of this study was to evaluate the relationship between CRP and IL-6 levels with pre-eclampsia considering different confounding factors. Using data from a large Colombian case-control study (3,590 cases of pre-eclampsia and 4,564 normotensive controls), CRP and IL-6 levels were measured in 914 cases and 1297 controls. The association between maternal serum levels of CRP and IL-6 with pre-eclampsia risk was evaluated using adjusted logistic regression models. Pre-eclampsia was defined as presence of blood pressure ≥140/90 mmHg and proteinuria ≥300mg/24 h (or ≥1 + dipstick). There was no evidence of association between high levels of CRP and IL-6 with pre-eclampsia after adjusting for the following factors: maternal and gestational age, ethnicity, place and year of recruitment, multiple-pregnancy, socio-economic position, smoking, and presence of infections during pregnancy. The adjusted OR for 1SD increase in log-CRP and log-IL-6 was 0.96 (95%CI 0.85, 1.08) and 1.09 (95%CI 0.97, 1.22), respectively. Although previous reports have suggested an association between high CRP and IL-6 levels with pre-eclampsia, sample size may lack the sufficient power to draw robust conclusions, and this association is likely to be explained by unaccounted biases. Our results, the largest case-control study reported up to date, demonstrate that there is not a causal association between elevated levels of CRP and IL-6 and the presence of pre-eclampsia.

Influence of collagen-based integrin α1 and α2 mediated signaling on human mesenchymal stem cell osteogenesis in three dimensional contexts.

Collagen I interactions with integrins α1 and α2 are known to support human mesenchymal stem cell (hMSC) osteogenesis. Nonetheless, elucidating the relative impact of specific integrin interactions has proven challenging, in part due to the complexity of native collagen. In the present work, we employed two collagen-mimetic proteins-Scl2-2 and Scl2-3- to compare the osteogenic effects of integrin α1 versus α2 signaling. Scl2-2 and Scl2-3 were both derived from Scl2-1, a triple helical protein lacking known cell adhesion, cytokine binding, and matrix metalloproteinase sites. However, Scl2-2 and Scl2-3 were each engineered to display distinct collagen-based cell adhesion motifs: GFPGER (binding integrins α1 and α2 ) or GFPGEN (binding only integrin α1 ), respectively. hMSCs were cultured within poly(ethylene glycol) (PEG) hydrogels containing either Scl2-2 or Scl2-3 for 2 weeks. PEG-Scl2-2 gels were associated with increased hMSC osterix expression, osteopontin production, and calcium deposition relative to PEG-Scl2-3 gels. These data indicate that integrin α2 signaling may have an increased osteogenic effect relative to integrin α1 . Since p38 is activated by integrin α2 but not by integrin α1 , hMSCs were further cultured in PEG-Scl2-2 hydrogels in the presence of a p38 inhibitor. Results suggest that p38 activity may play a key role in collagen-supported hMSC osteogenesis. This knowledge can be used toward the rational design of scaffolds which intrinsically promote hMSC osteogenesis. © 2018 Wiley Periodicals, Inc. J Biomed Mater Res Part A: 106A: 2594-2604, 2018.

Evaluation of late outgrowth endothelial progenitor cell and umbilical vein endothelial cell responses to thromboresistant collagen-mimetic hydrogels.

Bioactive coatings which support the adhesion of late-outgrowth peripheral blood endothelial progenitor cells (EOCs) are actively being investigated as a means to promote rapid endothelialization of "off-the-shelf," small-caliber arterial graft prostheses following implantation. In the present work, we evaluated the behavior of EOCs on thromboresistant graft coatings based on the collagen-mimetic protein Scl2-2 and poly(ethylene glycol) (PEG) diacrylate. Specifically, the attachment, proliferation, migration, and phenotype of EOCs on PEG-Scl2-2 hydrogels were evaluated as a function of Scl2-2 concentration (4, 8, and 12 mg/mL) relative to human umbilical vein endothelial cells (HUVECs). Results demonstrate the ability of each PEG-Scl2-2 hydrogel formulation to support EOC and HUVEC adhesion, proliferation, and spreading. However, only the 8 and 12 mg/mL PEG-Scl2-2 hydrogels were able to support stable EOC and HUVEC confluence. These PEG-Scl2-2 formulations were, therefore, selected for evaluation of their impact on EOC and HUVEC phenotype relative to PEG-collagen hydrogels. Cumulatively, both gene and protein level data indicated that 8 mg/mL PEG-Scl2-2 hydrogels supported similar or improved levels of EOC maturation relative to PEG-collagen controls based on evaluation of CD34, VEGFR2, PECAM-1, and VE-Cadherin. The 8 mg/mL PEG-Scl2-2 hydrogels also appeared to support similar or improved levels of EOC homeostatic marker expression relative to PEG-collagen hydrogels based on von Willebrand factor, collagen IV, NOS3, thrombomodulin, and E-selectin assessment. Combined, the present results indicate that PEG-Scl2-2 hydrogels warrant further investigation as "off-the-shelf" graft coatings. © 2017 Wiley Periodicals, Inc. J Biomed Mater Res Part A: 105A: 1712-1724, 2017.

Collagen-mimetic hydrogels promote human endothelial cell adhesion, migration and phenotypic maturation.

This work evaluates the response of human aortic endothelial cells (HAECs) to thromboresistant collagen-mimetic hydrogel coatings toward improving the biocompatibility of existing "off-the-shelf" small-caliber vascular grafts. Specifically, bioactive hydrogels - previously shown to support α1/α2 integrin-mediated cell adhesion but to resist platelet activation - were fabricated by combining poly(ethylene glycol) (PEG) with a 120 kDa, triple-helical collagen-mimetic protein(Scl2-2) containing the GFPGER adhesion sequence. Analysis of HAECs seeded onto the resulting PEG-Scl2-2 hydrogels demonstrated that HAEC adhesion increased with increasing Scl2-2 concentration, while HAEC migration rate decreased over this same concentration range. In addition, evaluation of HAEC phenotype at confluence indicated significant differences in the gene expression of NOS3, thrombomodulin, and E-selectin on the PEG-Scl2-2 hydrogels relative to PEG-collagen controls. At the protein level, however, only NOS3 was significantly different between the PEG-Scl2-2 and PEG-collagen surfaces. Furthermore, PECAM-1 and VE-cadherin expression on PEG-Scl2-2 hydrogels versus PEG-collagen controls could not be distinguished at either the gene or protein level. Cumulatively, these data indicate the PEG-Scl2-2 hydrogels warrant further investigation as "off-the-shelf" graft coatings. In future studies, the Scl2-2 protein can potentially be modified to include additional extracellular matrix or cytokine binding sites to further improve endothelial cell responses.