ABSTRACT
Objective:To prepare human decellular adipose tissue matrix (DAT) as injectable homogenate by a specially developed high-speed soft tissue homogenizer with controllable temperature, and to investigate its cellular compatibility and filling effect.Methods:From March 2019 to March 2021, DAT was obtained in the 940th Hospital from normal adipose tissue after decellular treatment. The DAT was mixed with normal saline at the rate of 1: 12. The specially developed high-speed soft tissue homogenizer with controllable temperature was used to conduct homogenization at 803×g for 10 min. The produced DAT homogenizer could pass through the 27G needle smoothly. DAT homogenate was observed under scanning electron microscope and its cytocompatibility was detected. Finally, granular fat, DAT homogenate and DAT homogenate + ADSCs were injected into the back of rats respectively, and the filling effect, angiogenesis ability and inflammatory infiltration were compared.Results:After decellular treatment, adipose tissue changed into DAT without cellular residue. The particle size of DAT homogenate was about (749.91±136.79) nm, which was prepared by the specially developed temperature controlled high-speed soft tissue homogenater. The adhesion rate was (92.16±1.00) %, and the A value increased with time. The cell growth was good, and the homogenate had no toxicity to the cells. In vivo experiment, the filling effect of DAT homogenate and DAT homogenate + ADSCs was significantly better than that of granulated fat group ( P<0.01). In the granulated fat group, a large number of adipocytes were necrotic and fused to form oil sacs, while DAT homogenate, DAT homogenate + ADSCs showed no obvious degradation, and some adipocytes were generated. The results of CD31 staining indicated that the number of microvessels in DAT homogenate group and DAT homogenate + ADSCs group was higher than that in granulated fat group ( P<0.01). The results of CD68 staining indicated that the inflammatory infiltration in DAT homogenate group and DAT homogenate + ADSCs group was lower than that in granule fat group ( P<0.01). Conclusions:Using the self-developed temperature controlled high-speed soft tissue homogenate device, DAT could be prepared into DAT homogenate that can pass through 27G needle. It has good biocompatibility and filling advantages, and injective process is simple, and the trauma is small, and so it could be used as filling material.
ABSTRACT
Objective: To explore the possibility of constructing tissue engineered adipose by adipose tissue derived extracellular vesicles (hAT-EV) combined with decellularized adipose tissue (DAT) scaffolds, and to provide a new therapy for soft tissue defects. Methods: The adipose tissue voluntarily donated by the liposuction patient was divided into two parts, one of them was decellularized and observed by HE and Masson staining and scanning electron microscope (SEM). Immunohistochemical staining and Western blot detection for collagen type Ⅰ and Ⅳ and laminin were also employed. Another one was incubated with exosome-removed complete medium for 48 hours, then centrifuged to collect the medium and to obtain hAT-EV via ultracentrifugation. The morphology of hAT-EV was observed by transmission electron microscopy; the nanoparticle tracking analyzer (NanoSight) was used to analyze the size distribution; Western blot was used to analyse membrane surface protein of hAT-EV. Adipose derived stem cells (ADSCs) were co-cultured with PKH26 fluorescently labeled hAT-EV, confocal fluorescence microscopy was used to observe the uptake of hAT-EV by ADSCs. Oil red O staining was used to evaluate adipogenic differentiation after hAT-EV and ADSCs co-cultured for 15 days. The DAT was scissored and then injected into the bilateral backs of 8 C57 mice (6-week-old). In experimental group, 0.2 mL hAT-EV was injected weekly, and 0.2 mL PBS was injected weekly in control group. After 12 weeks, the mice were sacrificed, and the new fat organisms on both sides were weighed. The amount of new fat was evaluated by HE and peri-lipoprotein immunofluorescence staining to evaluate the ability of hAT-EV to induce adipogenesis in vivo. Results: After acellularization of adipose tissue, HE and Masson staining showed that DAT was mainly composed of loosely arranged collagen with no nucleus; SEM showed that no cells and cell fragments were found in DAT, and thick fibrous collagen bundles could be seen; immunohistochemical staining and Western blot detection showed that collagen type Ⅰ and Ⅳ and laminin were retained in DAT. It was found that hAT-EV exhibited a spherical shape of double-layer envelope, with high expressions of CD63, apoptosis-inducible factor 6 interacting protein antibody, tumor susceptibility gene 101, and the particle size of 97.9% hAT-EV ranged from 32.67 nmto 220.20 nm with a peak at 91.28 nm. Confocal fluorescence microscopy and oil red O staining showed that hAT-EV was absorbed by ADSCs and induced adipogenic differentiation. In vivo experiments showed that the wet weight of fat new organisms in the experimental group was significantly higher than that in the control group ( t=2.278, P=0.048). HE staining showed that the structure of lipid droplets in the experimental group was more than that in the control group, and the collagen content in the control group was higher than that in the experimental group. The proportion of new fat in the experimental group was significantly higher than that in the control group ( t=4.648, P=0.017). Conclusion: DAT carrying hAT-EV can be used as a new method to induce adipose tissue regeneration and has a potential application prospect in the repair of soft tissue defects.
ABSTRACT
Objective: To preliminary explore the effect of decellularized adipose tissue (DAT) combined with vacuum sealing drainage (VSD) on wound inflammation in pigs. Methods: The DAT was prepared through the process of freeze-thaw, enzymatic digestion, organic solvent extraction, and vacuum freeze-drying. The appearance of DAT was observed before and after freeze-drying. HE staining was used to observe its structure and acellular effect. Eighteen male Bama minipigs were recruited, and four dorsal skin soft tissue wounds in diameter of 4 cm were made on each pig and randomly divided into 4 groups for different treatments. The wounds were treated with DAT combined with VSD in DAT/VSD group, DAT in DAT group, VSD in VSD group, and sterile gauze dressing in control group. HE staining was performed at 3, 7, 10, and 14 days after treatment. Moreover, the expressions of inflammatory factors [interleukin 1β (IL-1β), IL-6, and tumor necrosis factor α (TNF-α)], as well as the phenotypes of M1 and M2 macrophage phenotypic markers [inducible nitric oxide synthase (iNOS) and arginase 1 (ARG-1)] were detected by real-time fluorescence quantitative PCR (qRT-PCR). ELISA was used to determine the content of iNOS and ARG-1. Results: General observation and HE staining showed that DAT obtained in this study had a loose porous structure without cells. The neutrophils of wounds were significantly less in DAT/VSD group than in control group and DAT group ( P0.05) between DAT/VSD group and VSD group. And the neutrophils were significantly less in DAT/VSD group than in other three groups ( P<0.05) at 7, 10, and 14 days. The mRNA expressions of IL-1β, IL-6, TNF-α, and iNOS were significantly lower in DAT/VSD group than in other three groups at 3, 7, 10, and 14 days ( P<0.05), while the mRNA expression of ARG-1 was significantly higher in DAT/VSD group than in other three groups ( P<0.05). ELISA showed that the content of iNOS was significantly lower in DAT/VSD group than in other three groups at 3, 7, 10, and 14 days ( P<0.05), while the content of ARG-1 was significantly higher in DAT/VSD group than in other three groups ( P<0.05). Conclusion: DAT combined with VSD can significantly reduce inflammatory cell infiltration during wound healing, regulate the expressions of inflammatory factors and macrophage phenotype, and the effect is better than single use of each and conventional dressing change.
ABSTRACT
Extracellular matrix plays an important role in cell proliferation, differentiation and gene expression. It is similar to the three-dimensional structure of native tissue, which has been widely used in tissue engineering as a scaffold. Decellularized adipose tissue matrix is derived from adipose tissue, which is abundant and easily obtainable.It alsohas the function of promoting adipogenesis and angiogenesis. It provides a good microenvironment for adipose derived stem cells, and has great potential for the repair and reconstruction of soft tissue defects.In this paper, the preparation methods, the effects of the different forms and applications of decellularized adipose extracellular matrix scaffolds are reviewed.