In Vitro Study of a Novel Vibrio alginolyticus-Based Collagenase for Future Medical Application.

Mesenchymal stem cells extracted from adipose tissue are particularly promising given the ease of harvest by standard liposuction and reduced donor site morbidity. This study proposes a novel enzymatic method for isolating stem cells using Vibrio alginolyticus collagenase, obtaining a high-quality product in a reduced time. Initially, the enzyme concentration and incubation time were studied by comparing cellular yield, proliferation, and clonogenic capacities. The optimized protocol was phenotypically characterized, and its ability to differentiate in the mesodermal lineages was evaluated. Subsequently, that protocol was compared with two Clostridium histolyticum-based collagenases, and other tests for cellular integrity were performed to evaluate the enzyme's effect on expanded cells. The best results showed that using a concentration of 3.6 mg/mL Vibrio alginolyticus collagenase allows extracting stem cells from adipose tissue after 20 min of enzymatic reaction like those obtained with Clostridium histolyticum-based collagenases after 45 min. Moreover, the extracted cells with Vibrio alginolyticus collagenase presented the phenotypic characteristics of stem cells that remain after culture conditions. Finally, it was seen that Vibrio alginolyticus collagenase does not reduce the vitality of expanded cells as Clostridium histolyticum-based collagenase does. These findings suggest that Vibrio alginolyticus collagenase has great potential in regenerative medicine, given its degradation selectivity by protecting vital structures for tissue restructuration.

Highly Pluripotent Adipose-Derived Stem Cell-Enriched Nanofat: A Novel Translational System in Stem Cell Therapy.

Fat graft is widely used in plastic and reconstructive surgery. The size of the injectable product, the unpredictable fat resorption rates, and subsequent adverse effects make it tricky to inject untreated fat into the dermal layer. Mechanical emulsification of fat tissue, which Tonnard introduced, solves these problems, and the product obtained was called nanofat. Nanofat is widely used in clinical and aesthetic settings to treat facial compartments, hypertrophic and atrophic scars, wrinkle attenuation, skin rejuvenation, and alopecia. Several studies demonstrate that the tissue regeneration effects of nanofat are attributable to its rich content of adipose-derived stem cells. This study aimed to characterize Hy-Tissue Nanofat product by investigating morphology, cellular yield, adipose-derived stem cell (ASC) proliferation rate and clonogenic capability, immunophenotyping, and differential potential. The percentage of SEEA3 and CD105 expression was also analyzed to establish the presence of multilineage-differentiating stress-enduring (MUSE) cell. Our results showed that the Hy-Tissue Nanofat kit could isolate 3.74 × 104 ± 1.31 × 104 proliferative nucleated cells for milliliter of the treated fat. Nanofat-derived ASC can grow in colonies and show high differentiation capacity into adipocytes, osteocytes, and chondrocytes. Moreover, immunophenotyping analysis revealed the expression of MUSE cell antigen, making this nanofat enriched of pluripotent stem cell, increasing its potential in regenerative medicine. The unique characteristics of MUSE cells give a simple, feasible strategy for treating a variety of diseases.

Correction: De Francesco et al. In Vitro Characterization of Canine Microfragmented Adipose Tissue Non-Enzymatically Extracted from the Thigh and Lumbar Regions. Animals 2021, 11, 3231.

Reetuparna Biswas was not included as an author in the original publication [...].

Tissue-Material Integration and Biostimulation Study of Collagen Acellular Matrices.

BACKGROUND: Breast reconstruction after mastectomy using silicone implants is a surgical procedure that occasionally leads to capsular contracture formation. This phenomenon constitutes an important and persistent cause of morbidity, and no successful therapies are available to date. Recently, the use of acellular membranes as a protective material for silicone prostheses has been gaining attention due to their ability to prevent this adverse outcome. For this reason, the evaluation of the tissue-material integration and the induced biostimulation by acellular membranes results crucial. Evaluation of in vivo tissue integration and biostimulation induced by three different natural acellular collagen membranes. METHODS: Scanning electron microscopy was performed to analyse the membrane porosity and cells-biomaterial interaction in vitro, both in dry and wet conditions. Adipose-derived stem cells were cultured in the presence of membranes, and the colonisation capacity and differentiation potential of cells were assessed. In vivo tests and ex vivo analyses have been performed to evaluate dermal integration, absorption degree and biostimulation induced by the evaluated membrane. RESULTS: Analysis performed in vitro on the three different acellular dermal matrices evidenced that porosity and the morphological structure of membranes influence the liquid swelling ratio, affecting the cell mobility and the colonisation capacity. Moreover, the evaluated membranes influenced in different manner the adipose derived stem cells differentiation and their survival. In vivo investigation indicated that the absorption degree and the fluid accumulation surrounding the implant were membrane-dependent. Finally, ex vivo analysis confirmed the membrane-dependent behavior revealing different degree of tissue integration and biostimulation, such as adipogenic stimulation. CONCLUSION: The physico-chemical characteristics of the membranes play a key role in the biostimulation of the cellular environment inducing the development of well-organized adipose tissue.

In Vitro Characterization of Canine Microfragmented Adipose Tissue Non-Enzymatically Extracted from the Thigh and Lumbar Regions.

Within the adult canine population, disabilities and symptoms including joint pain and functional impairment are commonly observed in articular cartilage lesions and present a challenging feat in the operating room. Clinical settings require less invasive and more minimally manipulated measures facilitated by innovative and advanced technology. Mesenchymal stem cells have recently been proposed and, furthermore, autologous adipose tissue administration via injection has emerged as a new albeit somewhat controversial therapeutic tool. The purpose of this study is to characterize canine autologous micro-fragmented adipose tissue (micrografts) by mechanical approach without substantial manipulations. Adipose tissue samples collected from six dogs were processed by a Rigenera device and by enzymatic digestion from two different body regions (lumbar and thigh region). Interestingly, the immunophenotypic analysis attested that cells from Rigenera® were highly positive for the mesenchymal stem cells markers CD73 and CD90, less positive for hematopoietic CD45 and CD34, and negative for MHC class II antibodies (which play a role in immune responses). Finally, the Rigenera® technology obtained micrografts with a 35% higher expression of the IL10 gene with relevant anti-inflammatory activities compared to the enzymatic digestion protocol. This evidence suggests a potential improved clinical outcome capable of modulating inflammation and immune responses.

In Vitro Characterization of Adipose Stem Cells Non-Enzymatically Extracted from the Thigh and Abdomen.

Autologous fat grafting is a surgical technique in which adipose tissue is transferred from one area of the body to another, in order to reconstruct or regenerate damaged or injured tissues. Before reinjection, adipose tissue needs to be purified from blood and cellular debris to avoid inflammation and preserve the graft viability. To perform this purification, different enzymatic and mechanical methods can be used. In this study, we characterized in vitro the product of a closed automatic device based on mechanical disaggregation, named Rigenera®, focusing on two sites of adipose tissue harvesting. At first, we optimized the Rigenera® operating timing, demonstrating that 60 s of treatment allows a higher cellular yield, in terms of the cell number and growth rate. This result optimizes the mechanical disaggregation and it can increase the clinical efficiency of the final product. When comparing the extracted adipose samples from the thigh and abdomen, our results showed that the thigh provides a higher number of mesenchymal-like cells, with a faster replication rate and a higher ability to form colonies. We can conclude that by collecting adipose tissue from the thigh and treating it with the Rigenera® device for 60 s, it is possible to obtain the most efficient product.

Simple and Rapid Non-Enzymatic Procedure Allows the Isolation of Structurally Preserved Connective Tissue Micro-Fragments Enriched with SVF.

The stromal vascular fraction (SVF) consists of a heterogeneous population of stem and stromal cells, generally obtained from adipose tissue by enzymatic digestion. For human cell-based therapies, mechanical process methods to obtain SVF represent an advantageous approach because they have fewer regulatory restrictions for their clinical use. The aim of this study was to characterize a novel commercial system for obtaining SVF from adipose tissue by a mechanical approach without substantial manipulations. Lipoaspirate samples collected from 27 informed patients were processed by a simple and fast mechanical system (by means of Hy-Tissue SVF). The Hy-Tissue SVF product contained a free cell fraction and micro-fragments of stromal connective tissue. The enzymatic digestion of the micro-fragments increased the yield of free cells (3.2 times) and CFU-F (2.4 times). Additionally, 10% of free cells from SVF were positive for CD34+, suggesting the presence of endothelial cells, pericytes, and potential adipose-derived stem cells (ADSC). Moreover, the SVF cells were able to proliferate and differentiate in vitro toward adipocytes, osteocytes, and chondrocytes. The immunophenotypic analysis of expanded cells showed positivity for typical mesenchymal stem cell markers. The Hy-Tissue SVF system allows the isolation of stromal vascular fraction, making this product of potential interest in regenerative medicine.