Project description:OBJECTIVE: To assess the safety and efficacy as well as determination of the molecular mechanism of action of topical allogeneic ADSC therapy in the treatment of ulcers in patients with neuropathic diabetic foot. RESEARCH DESIGN AND METHODS: We examined a total of 57 patients with neuropathic diabetic foot in grades IA and IIA according to the University of Texas classification. Finally 23 patients received treatment with standard therapy and fibrin glue (fibrin gel group) and 23 received treatment with standard therapy and ADSC suspended in tissue glue (fibrin gel + ADSC group). The primary outcome was determining the time needed to reduce the wound size by 50% from the initial size in both groups by assessing the wound surface, the evaluation of treatment safety, expressed as the occurrence of any adverse events; the evaluation of symptoms improvement (e.g. pain, itching, etc.) assessed by Visual Analogue Scale (VAS) and analysis of the expression of selected pro-angiogenic and immunomodulatory factors in the wounds treated in both study groups. RESULTS:The ADSC group benefited from the use of stem cells in terms of accelerated wound healing and greater likelihood of complete healing of the wound. In a second week after administration of therapy, the difference between the groups was already statistically significant (p = 0,04). The time to the 50% reduction of the size of the wound in the fibrin gel + ADSC group was significantly shorter (18.9 +/- 2.2 days ) than in the group receiving fibrin gel (39.8 +/- 6.9 days ), p = 0.0035. CONCLUSIONS: The results of the preliminary analysis show that the use of allogeneic ADSCs in the treatment of neuropathic diabetic foot ulcers is a safe method of therapy, which allows to improve the effects of wound healing. Our study indicates also the association of higher expression of selected proteins expression intensifying the healing process through a faster transition to the proliferation phase with a faster reduction in wound area, and long-term efficacy of allogeneic ADSC cells.

Project description:Diabetes Mellitus (DM) is a chronic, severe disease rapidly increasing in incidence and prevalence and is associated with numerous complications. Patients with DM are at high risk of developing diabetic foot ulcers (DFU) that often lead to lower limb amputations, long term disability, and a shortened lifespan. Despite this, the effects of DM on human foot skin biology are largely unknown. Thus, the focus of this study was to determine whether DM changes foot skin biology predisposing it for healing impairment and development of DFU. Foot skin samples were collected from 20 patients receiving corrective foot surgery and, using a combination of multiple molecular and cellular approaches we performed comparative analyses of non-ulcerated non-neuropathic diabetic foot skin (DFS) and healthy non-diabetic foot skin (NFS). MicroRNA (miR) profiling of laser captured epidermis and primary dermal fibroblasts from both DFS and NFS samples identified 5 miRs de-regulated in the epidermis of DFS though none reached statistical significance. MiR-31-5p and miR-31-3p were most profoundly induced. Although none were significantly regulated in diabetic fibroblasts, miR-29c-3p showed a trend of up-regulation, which was confirmed by qPCR in a prospective set of 20 skin samples. Gene expression profiling of full thickness biopsies identified 36 de-regulated genes in DFS (>2 fold-change, unadjusted p-value ≤ 0.05). Of this group, three out of seven tested genes were confirmed by qPCR: SERPINB3 was up-regulated whereas OR2A4 and LGR5 were down-regulated in DFS. However no morphological differences in histology, collagen deposition, and number of blood vessels or lymphocytes were found. No difference in proliferative capacity was observed by quantification of Ki67 positive cells in epidermis. These findings suggest DM causes only subtle changes to foot skin. Since morphology, mRNA and miR levels were not affected in a major way, additional factors, such as neuropathy, vascular complications, or duration of DM, may further compromise tissue’s healing ability leading to development of DFUs.

Project description:Diabetes Mellitus (DM) is a chronic, severe disease rapidly increasing in incidence and prevalence and is associated with numerous complications. Patients with DM are at high risk of developing diabetic foot ulcers (DFU) that often lead to lower limb amputations, long term disability, and a shortened lifespan. Despite this, the effects of DM on human foot skin biology are largely unknown. Thus, the focus of this study was to determine whether DM changes foot skin biology predisposing it for healing impairment and development of DFU. Foot skin samples were collected from 20 patients receiving corrective foot surgery and, using a combination of multiple molecular and cellular approaches we performed comparative analyses of non-ulcerated non-neuropathic diabetic foot skin (DFS) and healthy non-diabetic foot skin (NFS). MicroRNA (miR) profiling of laser captured epidermis and primary dermal fibroblasts from both DFS and NFS samples identified 5 miRs de-regulated in the epidermis of DFS though none reached statistical significance. MiR-31-5p and miR-31-3p were most profoundly induced. Although none were significantly regulated in diabetic fibroblasts, miR-29c-3p showed a trend of up-regulation, which was confirmed by qPCR in a prospective set of 20 skin samples. Gene expression profiling of full thickness biopsies identified 36 de-regulated genes in DFS (>2 fold-change, unadjusted p-value ≤ 0.05). Of this group, three out of seven tested genes were confirmed by qPCR: SERPINB3 was up-regulated whereas OR2A4 and LGR5 were down-regulated in DFS. However no morphological differences in histology, collagen deposition, and number of blood vessels or lymphocytes were found. No difference in proliferative capacity was observed by quantification of Ki67 positive cells in epidermis. These findings suggest DM causes only subtle changes to foot skin. Since morphology, mRNA and miR levels were not affected in a major way, additional factors, such as neuropathy, vascular complications, or duration of DM, may further compromise tissue’s healing ability leading to development of DFUs. foot skin biopsies obtained from diabetic and non-diabetic people were thoroughly washed in DMEM supplemented with 2× Penicillin/Streptomycin/Fungizone, and gentamicin (50mg/L). After, the epidermis was removed from specimens, the dermis was finely minced and placed in 12-well plates. For the establishment of fibroblast cultures, DMEM (Life Technologies) supplemented with 10% FBS, HEPES (1.9mg/ml), streptomycin (100 μg/ml), penicillin (100 U/ml), and Fungizone Antimycotic (0.25 µg/mL) was used. RNA was isolated from cells (passage 1 or 2) using miRNeasy Mini Kit (QIAGEN). miR expression profiles of DFF and NFF fibroblasts were generated by nanoString nCounter miR Expression Assays (NanoString Technologies, Seattle, WA, USA) and analyzed using nSolver2.0 software following manufacturer's instructions. Data were normalized to gene controls included in the assays.

			Action	DRS
	SRR15974200_1.fastq.gz	Fastqsanger.gz
	SRR15974200_2.fastq.gz	Fastqsanger.gz
	SRR15974201_1.fastq.gz	Fastqsanger.gz
	SRR15974201_2.fastq.gz	Fastqsanger.gz
	SRR15974202_1.fastq.gz	Fastqsanger.gz

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