Xenogeneic mesenchymal stem cell biocurative improves skin wounds healing in diabetic mice by increasing mast cells and the regenerative profile.

Introduction: Diabetes mellitus (DM) is a chronic disease and a major cause of mortality and morbidity worldwide. The hyperglycemia caused by DM induces micro and macrovascular complications that lead, among other consequences, to chronic wounds and amputations. Cell therapy and tissue engineering constitute recent therapeutic alternatives to improve wound healing in diabetic patients. The current study aimed to analyze the effectiveness of biocuratives containing human mesenchymal stem cells (MSCs) associated with a hydrogel matrix in the wound healing process and related inflammatory cell profile in diabetic mice. Methods: Biocuratives containing MSCs were constructed by 3D bioprinting, and applied to skin wounds on the back of streptozotocin (STZ)-induced type 1 diabetic (T1D) mice. The healing process, after the application of biocuratives with or without MSCs was histologically analyzed. In parallel, genes related to growth factors, mast cells (MC), M1 and M2 macrophage profiles were evaluated by RT-PCR. Macrophages were characterized by flow cytometry, and MC by toluidine blue staining and flow cytometry. Results: Mice with T1D exhibited fewer skin MC and delayed wound healing when compared to the non-diabetic group. Treatment with the biocuratives containing MSCs accelerated wound healing and improved skin collagen deposition in diabetic mice. Increased TGF-ß gene expression and M2 macrophage-related markers were also detected in skin of diabetic mice that received MSCs-containing biocuratives. Finally, MSCs upregulated IL-33 gene expression and augmented the number of MC in the skin of diabetic mice. Conclusion: These results reveal the therapeutic potential of biocuratives containing MSCs in the healing of skin wounds in diabetic mice, providing a scientific base for future treatments in diabetic patients.

Bone marrow-derived products: A classification proposal - bone marrow aspirate, bone marrow aspirate concentrate or hybrid?

Degenerative musculoskeletal disorders are one of the top causes of pain and disability in the adult population. Current available alternatives to mitigate symptoms include conservative treatments such as the administration of pharmacological agents and an educative approach towards lifestyle modification. The use of certain analgesics, such as opiates and corticosteroids, delivers short term results but do not address the etiological source of pain and disability. Also, prolonged use of such medications may cause additional complications. Therefore, the demand for musculoskeletal tissue regeneration has led to an alternative approach referred to as "orthobiologics". This alternative is based on cellular and molecular components capable of inducing and promoting tissue repair. Bone marrow (BM) aspirate (BMA) and concentrate are well-known orthobiologics used to treat musculoskeletal conditions. Orthobiologics derived from the BM have been discussed in the literature; however, the lack of standardization regarding collection and processing protocols presents a challenge for generalization of study outcomes and determination of efficacy. Since BM-derived orthobiologics have not yet been classified, to our knowledge, this manuscript proposes the ACH classification system, which speaks to BMA (A), BMA and concentrate (C) and hybrid (H), which combines A and C. This classification proposes and describes 8 parameters that are relevant for the quality of biological products. The more parameters used would imply greater characterization and complexity of the evaluation of the biological product used. The ACH classification envisages a necessary contribution to the comprehension of both clinical procedures and research outcomes, ultimately ushering in a standardization of best practice.

Hyaluronic acid and fibrin from L-PRP form semi-IPNs with tunable properties suitable for use in regenerative medicine.

Autologous leukocyte- and platelet-rich plasma (L-PRP) combined with hyaluronic acid (HA) has been widely used in local applications for cartilage and bone regeneration. The association between L-PRP and HA confers structural and rheological changes that differ among individual biomaterials but has not been investigated. Therefore, the standardization and characterization of L-PRP-HA are important to consider when comparing performance results to improve future clinical applications. To this end, we prepared semi-interpenetrating polymer networks (semi-IPNs) of L-PRP and HA and characterized their polymerization kinetics, morphology, swelling ratio, stability and rheological behavior, which we found to be tunable according to the HA molar mass (MM). Mesenchymal stem cells derived from human adipose tissue (h-AdMSCs) seeded in the semi-IPNs had superior viability and chondrogenesis and osteogenesis capabilities compared to the viability and capabilities of fibrin. We have demonstrated that the preparation of the semi-IPNs under controlled mixing ensured the formation of cell-friendly hydrogels rich in soluble factors and with tunable properties according to the HA MM, rendering them suitable for clinical applications in regenerative medicine.

Correction to: Priming approaches to improve the efficacy of mesenchymal stromal cell-based therapies.

The original article [1] contained an error in the presentation of the first author's name, Nádia de Cássia Noronha. This has now been corrected.

Priming approaches to improve the efficacy of mesenchymal stromal cell-based therapies.

Multipotent mesenchymal stromal cells (MSC) have been widely explored for cell-based therapy of immune-mediated, inflammatory, and degenerative diseases, due to their immunosuppressive, immunomodulatory, and regenerative potentials. Preclinical studies and clinical trials have demonstrated promising therapeutic results although these have been somewhat limited. Aspects such as low in vivo MSC survival in inhospitable disease microenvironments, requirements for ex vivo cell overexpansion prior to infusions, intrinsic differences between MSC and different sources and donors, variability of culturing protocols, and potency assays to evaluate MSC products have been described as limitations in the field. In recent years, priming approaches to empower MSC have been investigated, thereby generating cellular products with improved potential for different clinical applications. Herein, we review the current priming approaches that aim to increase MSC therapeutic efficacy. Priming with cytokines and growth factors, hypoxia, pharmacological drugs, biomaterials, and different culture conditions, as well as other diverse molecules, are revised from current and future perspectives.

DPP-4 Inhibition Leads to Decreased Pancreatic Inflammatory Profile and Increased Frequency of Regulatory T Cells in Experimental Type 1 Diabetes.

Sitagliptin is a dipeptidyl peptidase-4 inhibitor (iDPP-4), which has been used for type 2 diabetes treatment. Recently, iDPP-4 has been described as a promising treatment of type 1 diabetes (T1D) but is still necessary to evaluate immune effects of sitagliptin. C57BL/6 mice were induced by multiple low doses of streptozotocin. Diabetes incidence, insulin, glucagon, glucagon-like peptide-1 (GLP-1) serum levels, and inflammatory cytokine levels were quantified in pancreas homogenate after 30 and 90 days of treatment. In addition, frequencies of inflammatory and regulatory T cell subsets were determined in the spleen and in the pancreatic lymph nodes. iDPP-4 decreased blood glucose level while increased GLP-1 and insulin levels. After long-term treatment, treated diabetic mice presented decreased frequency of CD4+CD26+ T cells and increased percentage of CD4+CD25hiFoxp3+ T cells in the spleen. Besides, pancreatic lymph nodes from diabetic mice treated with iDPP-4 presented lower percentage of CD11b+ cells and decreased levels of inflammatory cytokines in the pancreas. Treatment of type 1 diabetic mice with iDPP-4 improved metabolic control, decreased inflammatory profile in the pancreatic microenvironment, and increased systemic regulatory T cell frequency. Therefore, we suggest the long-term use of sitagliptin as a feasible and effective therapy for T1D.

Multipotent mesenchymal stromal cells from patients with newly diagnosed type 1 diabetes mellitus exhibit preserved in vitro and in vivo immunomodulatory properties.

BACKGROUND: Type 1 diabetes mellitus (T1D) is characterized by autoimmune responses resulting in destruction of insulin-producing pancreatic beta cells. Multipotent mesenchymal stromal cells (MSCs) exhibit immunomodulatory potential, migratory capacity to injured areas and may contribute to tissue regeneration by the secretion of bioactive factors. Therefore, MSCs are considered as a promising approach to treat patients with different autoimmune diseases (AID), including T1D patients. Phenotypical and functional alterations have been reported in MSCs derived from patients with different AID. However, little is known about the properties of MSCs derived from patients with T1D. Since autoimmunity and the diabetic microenvironment may affect the biology of MSCs, it becomes important to investigate whether these cells are suitable for autologous transplantation. Thus, the aim of the present study was to evaluate the in vitro properties and the in vivo therapeutic efficacy of MSCs isolated from bone marrow of newly diagnosed T1D patients (T1D-MSCs) and to compare them with MSCs from healthy individuals (C-MSCs). METHODS: T1D-MSCs and C-MSCs were isolated and cultured until third passage. Then, morphology, cell diameter, expression of surface markers, differentiation potential, global microarray analyses and immunosuppressive capacity were in vitro analyzed. T1D-MSCs and C-MSCs therapeutic potential were evaluated using a murine experimental model of streptozotocin (STZ)-induced diabetes. RESULTS: T1D-MSCs and C-MSCs presented similar morphology, immunophenotype, differentiation potential, gene expression of immunomodulatory molecules and in vitro immunosuppressive capacity. When administered into diabetic mice, both T1D-MSCs and C-MSCs were able to reverse hyperglycemia, improve beta cell function and modulate pancreatic cytokine levels. CONCLUSIONS: Thus, bone marrow MSCs isolated from T1D patients recently after diagnosis are not phenotypically or functionally impaired by harmful inflammatory and metabolic diabetic conditions. Our results provide support for the use of autologous MSCs for treatment of newly diagnosed T1D patients.

Xenogeneic Mesenchymal Stromal Cells Improve Wound Healing and Modulate the Immune Response in an Extensive Burn Model.

Major skin burns are difficult to treat. Patients often require special care and long-term hospitalization. Besides specific complications associated with the wounds themselves, there may be impairment of the immune system and of other organs. Mesenchymal stromal cells (MSCs) are a recent therapeutic alternative to treat burns, mainly aiming to accelerate the healing process. Several MSC properties favor their use as therapeutic approach, as they promote angiogenesis, stimulate regeneration, and enhance the immunoregulatory function. Moreover, since patients with extensive burns require urgent treatment and because the expansion of autologous MSCs is a time-consuming process, in this present study we chose to evaluate the therapeutic potential of xenogeneic MSCs in the treatment of severe burns in rats. MSCs were isolated from mouse bone marrow, expanded in vitro, and intradermally injected in the periphery of burn wounds. MSC-treated rats presented higher survival rates (76.19%) than control animals treated with PBS (60.86%, p < 0.05). In addition, 60 days after the thermal injury, the MSC-treated group showed larger proportion of healed areas within the burn wounds (90.81 ± 5.05%) than the PBS-treated group (76.11 ± 3.46%, p = 0.03). We also observed that CD4(+) and CD8(+) T cells in spleens and in damaged skin, as well as the percentage of neutrophils in the burned area, were modulated by MSC treatment. Plasma cytokine (TGF-ß, IL-10, IL-6, and CINC-1) levels were also altered in the MSC-treated rats, when compared to controls. Number of injected GFP(+) MSCs progressively decreased over time, and 60 days after injection, few MSCs were still detected in the skin of treated animals. This study demonstrates the therapeutic effectiveness of intradermal application of MSCs in a rat model of deep burns, providing basis for future regenerative therapies in patients suffering from deep burn injuries.

Therapeutic efficacy and biodistribution of allogeneic mesenchymal stem cells delivered by intrasplenic and intrapancreatic routes in streptozotocin-induced diabetic mice.

INTRODUCTION: Mesenchymal stromal/stem cells (MSCs) are multipotent cells that have the ability to express and secrete a wide range of immunomodulatory molecules, cytokines, growth factors and antiapoptotic proteins. MSCs modulate both innate and adaptive immune responses making them potential candidates for the treatment of patients with type 1 diabetes mellitus (T1D). However, one problem frequently associated with the systemic MSCs administration is the entrapment of the cells mainly in the lungs. In this sense, trying to avoid the lung barrier, the purpose of this study was to evaluate the long-term therapeutic efficacy and biodistribution of allogeneic adipose tissue-derived MSCs (ADMSCs) injected via two different delivery routes (intrasplenic/I.Sp and intrapancreatic/I.Pc) in a murine model of diabetes induced by streptozotocin (STZ). METHODS: Experimental diabetes was induced in C57BL/6 male mice by multiple low-doses of STZ. MSCs were isolated from adipose tissue (ADMSCs) of Balb/c mice. A single dose of 1x10(6) ADMSCs was microinjected into the spleen or into the pancreas of diabetic mice. Control group received injection of PBS by I.Sp or I.Pc delivery routes. Glycemia, peripheral glucose response, insulin-producing ß cell mass, regulatory T cell population, cytokine profile and cell biodistribution were evaluated after ADMSCs/PBS administration. RESULTS: ADMSCs injected by both delivery routes were able to decrease blood glucose levels and improve glucose tolerance in diabetic mice. ADMSCs injected by I.Sp route reverted hyperglycemia in 70% of diabetic treated mice, stimulating insulin production by pancreatic ß cells. Using the I.Pc delivery route, 42% of ADMSCs-treated mice responded to the therapy. Regulatory T cell population remained unchanged after ADMSCs administration but pancreatic TGF-ß levels were increased in ADMSCs/I.Sp-treated mice. ADMSCs administrated by I.Sp route were retained in the spleen and in the liver and ADMSCs injected by I.Pc route remained in the pancreas. However, ADMSCs injected by these delivery routes remained only few days in the recipients. CONCLUSION: Considering the potential role of MSCs in the treatment of several disorders, this study reports alternative delivery routes that circumvent cell entrapment into the lungs promoting beneficial therapeutic responses in ADMSCs-treated diabetic mice.

Combining xanthan and chitosan membranes to multipotent mesenchymal stromal cells as bioactive dressings for dermo-epidermal wounds.

The association between tridimensional scaffolds to cells of interest has provided excellent perspectives for obtaining viable complex tissues in vitro, such as skin, resulting in impressive advances in the field of tissue engineering applied to regenerative therapies. The use of multipotent mesenchymal stromal cells in the treatment of dermo-epidermal wounds is particularly promising due to several relevant properties of these cells, such as high capacity of proliferation in culture, potential of differentiation in multiple skin cell types, important paracrine and immunomodulatory effects, among others. Membranes of chitosan complexed with xanthan may be potentially useful as scaffolds for multipotent mesenchymal stromal cells, given that they present suitable physico-chemical characteristics and have adequate tridimensional structure for the adhesion, growth, and maintenance of cell function. Therefore, the purpose of this work was to assess the applicability of bioactive dressings associating dense and porous chitosan-xanthan membranes to multipotent mesenchymal stromal cells for the treatment of skin wounds. The membranes showed to be non-mutagenic and allowed efficient adhesion and proliferation of the mesenchymal stromal cells in vitro. In vivo assays performed with mesenchymal stromal cells grown on the surface of the dense membranes showed acceleration of wound healing in Wistar rats, thus indicating that the use of this cell-scaffold association for tissue engineering purposes is feasible and attractive.

Dynamic changes of the Th17/Tc17 and regulatory T cell populations interfere in the experimental autoimmune diabetes pathogenesis.

A balance between proinflammatory (Th17 and Tc17) and anti-inflammatory (regulatory T cells) subsets of T cells is essential to maintain immunological tolerance and prevent the onset of several autoimmune diseases, including type 1 diabetes. However, the kinetics of these subsets and disease severity during the streptozotocin (STZ)-induced diabetes course has not been determined. Thus, susceptible C57BL/6 mice were administrated with multiple low doses of STZ and we evaluated the frequency/absolute number of these T cell subsets in the pancreatic lymph nodes (PLNs) and spleen and Th1, Th17, Treg cytokine production in the pancreatic tissue. At different time points of the disease progression (6, 11, 18 and 25 days after the last STZ administration), the histopathological alterations were also evaluated by H&E and immunohistochemistry staining. During the initial phase of diabetes development (day 6), we noted increased numbers of CD4(+) and CD8(+) T cells in spleen and PLNs. At the same time, the frequencies of Th17 and Tc17 cells in PLNs were also enhanced. In addition, the early augment of interferon gamma (IFN-γ), tumoral necrosis factor (TNF-α), IL-6 and IL-17 levels in pancreatic tissue correlated with pancreatic islet inflammation and mild ß-cell damage. Notably, the absolute number of Treg cells increased in PLNs during over time when compared to control group. Interestingly, increased IL-10 levels were associated with control of the inflammatory process during the late phase of the type 1 diabetes (day 25). In agreement, mice lacking the expression of IL-17 receptor (Il17r) showed impairment in STZ-induced diabetes progression, reduced peri-insulitis and beta cells preservation when compared with wild-type mice. Our findings suggest that dynamic changes of pathogenic Th17/Tc17 and regulatory T cell subsets numbers is associated with early strong inflammation in the pancreatic islets followed by late regulatory profile during the experimental STZ-induced diabetes course.