Cell-Free Hydrogel System Based on a Tissue-Specific Extracellular Matrix for In Situ Adipose Tissue Regeneration

Well-designed scaffolds provide appropriate niches that can effectively recruit host cells and induce differentiation of recruited cells into the desired cell types, facilitating in situ tissue regeneration. Here we report a tissue-specific extracellular matrix (ECM) hydrogel composed of adipose-derived soluble ECM (sECM) and methylcellulose (MC) as a cell-free scaffold system for adipose tissue regeneration. The sECM–MC hydrogels showed a thermosensitive sol–gel phase transition and rapidly formed a soft hydrogel with a stiffness of 3.8 kPa at body temperature. An in vivo study showed that the sECM–MC hydrogel facilitated the infiltration of host cell populations, particularly adipose-derived stem cells (ASCs) and adipose tissue macrophages (ATMs) that directly contribute to the adipose tissue regeneration. Moreover, the hydrogel significantly enhanced host-derived adipogenesis and angiogenesis without exogenous cells or bioactive molecules. Our results indicate that the sECM–MC hydrogels provide mechanical and biochemical cues for host-derived adipose regeneration. Overall, the sECM–MC hydrogels are a highly promising cell-free therapeutic approach for in situ adipose tissue regeneration

A bilayer composite composed of TiO₂-incorporated electrospun chitosan membrane and human extracellular matrix sheet as a wound dressing

<div><p>We designed bilayer composites composed of an upper layer of titanium dioxide (TiO₂)-incorporated chitosan membrane and a sub-layer of human adipose-derived extracellular matrix (ECM) sheet as a wound dressing for full-thickness wound healing. The dense and fibrous top layer, which aims to protect the wound from bacterial infection, was prepared by electrospinning of chitosan solution followed by immersion in TiO₂ solution. The sponge-like sub-layer, which aims to promote new tissue regeneration, was prepared with acellular ECM derived from human adipose tissue. Using a modified drop plate method, there was a 33.9 and 69.6% reduction in viable <i>Escherichia coli</i> and <i>Staphylococcus aureus</i> on the bilayer composite, respectively. In an <i>in vivo</i> experiment using rats, the bilayer composites exhibited good biocompatibility and provided proper physicochemical and compositional cues at the wound site. Changes in wound size and histological examination of full-thickness wounds showed that the bilayer composites induced faster regeneration of granulation tissue and epidermis with less scar formation, than control wounds. Overall results suggest that the TiO₂-incorporated chitosan/ECM bilayer composite can be a suitable candidate as a wound dressing, with an excellent inhibition of bacterial penetration and wound healing acceleration effects.</p></div

Injectable and Thermosensitive Soluble Extracellular Matrix and Methylcellulose Hydrogels for Stem Cell Delivery in Skin Wounds

Extracellular matrix (ECM) provides structural support and biochemical cues for tissue development and regeneration. Here we report a thermosensitive hydrogel composed of soluble ECM (sECM) and methylcellulose (MC) for injectable stem cell delivery. The sECM was prepared by denaturing solid ECM extracted from human adipose tissue and then blended with a MC solution. At low temperatures, the sECM-MC solution displayed a viscous solution state in which the loss modulus (<i>G</i>″) was predominant over the storage modulus (<i>G</i>′). With increasing temperature, <i>G</i>′ increased dramatically and eventually exceeded <i>G</i>″ around 34 °C, characteristic of the transition from a liquid-like state to an elastic gel-like state. After a single injection of the stem cell-embedded hydrogel in full thickness cutaneous wound, the wound healed rapidly through re-epithelialization and neovascularization with minimum scar formation. The overall results suggest that in-situ-forming sECM-MC hydrogels are a promising injectable vehicle for stem cell delivery and tissue regeneration

Foamy histiocyte with intracellular fatty lobules and neo-vascularization in the injected sECM/MC hydrogels.

<p>(A) Focal capillary ingrowths (arrowhead) into the injection site. Cell aggregations with intracellular fatty lobules in the injection area (asterisk). (B) RAM 11 positive cells (dark brown) are foamy histiocytes, which increased as a function of time.</p

Laryngoscopic images after injection laryngoplasty of sECM/MC hydrogels into the left paralyzed vocal fold.

<p>The sECM/MC hydrogel injection group exhibited a straight and medialized vocal fold (white arrowhead) while the control group had a curved and lateralized vocal fold (black arrowhead), which is likely due to denervation of the recurrent laryngeal nerve.</p

Human Adipose Tissue Derived Extracellular Matrix and Methylcellulose Hydrogels Augments and Regenerates the Paralyzed Vocal Fold - Fig 3

Standard hematoxylin and eosin (H&E) staining of rabbit larynx after injection laryngoplasty into the left paralyzed vocal fold (A) and the quantitative analysis of remaining volume of sECM/MC hydrogels (B). (A) Histological examination of the injected biomaterials at 8 weeks post procedure. Area of the laryngeal intrinsic muscle was smaller on the denervated side in the control group (green dotted line) than on the contralateral normal side. In the sECM/MC group, the laryngeal muscle area was compensated for by the injected sECM/MC hydrogel (brown dotted line). The injected sECM/MC hydrogel (arrowhead) induced no significant inflammatory response including neutrophils or lymphocytes aggregation in the surrounding muscle (*), lamina propria (†), or epithelium (‡). (B) Quantitative analysis of remaining sECM/MC hydrogel volume (p = 0.501 using Kruskal-Wallis test).</p

Human Adipose Tissue Derived Extracellular Matrix and Methylcellulose Hydrogels Augments and Regenerates the Paralyzed Vocal Fold - Fig 2

<p><b>Representative serial images of high-speed camera recording at 8 weeks (A), the asymmetric index using videokymograms (B) and the results of asymmetry index for vocal functional analysis (C)</b>. (A) Normal and symmetrical vocal contacts showed no change in the vibration of vocal mucosa in sECM/MC groups relative to the control group. (B) The maximum distance in the left denervated vocal fold (<i>a</i>) was compared to the right vocal fold (<i>b</i>) using a videokymogram to generate an asymmetry index. The asymmetry index was calculated as follows: Asymmetry index = <i>a</i> / <i>b</i>. (C) The mean asymmetry index of the sECM/MC hydrogel group (1.020 ± 0.069) and the control group (0.787 ± 0.102) are shown (<i>p</i> = 0.047 using a Mann-Whitney U test). In diseased conditions, the index deviates from the value of 1.0</p

Stem Cell-Derived Extracellular Vesicle-Bearing Dermal Filler Ameliorates the Dermis Microenvironment by Supporting CD301b-Expressing Macrophages

Hyaluronic acid-based hydrogels (Hyal-Gels) have the potential to reduce wrinkles by physically volumizing the skin. However, they have limited ability to stimulate collagen generation, thus warranting repeated treatments to maintain their volumizing effect. In this study, stem cell-derived extracellular vesicle (EV)-bearing Hyal-Gels (EVHyal-Gels) were prepared as a potential dermal filler, ameliorating the dermis microenvironment. No significant differences were observed in rheological properties and injection force between Hyal-Gels and EVHyal-Gels. When locally administered to mouse skin, Hyal-Gels significantly extended the biological half-life of EVs from 1.37 d to 3.75 d. In the dermis region, EVHyal-Gels induced the overexpression of CD301b on macrophages, resulting in enhanced proliferation of fibroblasts. It was found that miRNAs, such as let-7b-5p and miR-24-3p, were significantly involved in the change of macrophages toward the CD301bhi phenotype. The area of the collagen layer in EVHyal-Gel-treated dermis was 2.4-fold higher than that in Hyal-Gel-treated dermis 4 weeks after a single treatment, and the collagen generated by EVHyal-Gels was maintained for 24 weeks in the dermis. Overall, EVHyal-Gels have the potential as an antiaging dermal filler for reprogramming the dermis microenvironment

Bioorthogonal Copper Free Click Chemistry for Labeling and Tracking of Chondrocytes <i>In Vivo</i>

Establishment of an appropriate cell labeling and tracking method is essential for the development of cell-based therapeutic strategies. Here, we are introducing a new method for cell labeling and tracking by combining metabolic gylcoengineering and bioorthogonal copper-free Click chemistry. First, chondrocytes were treated with tetraacetylated N-azidoacetyl-d-mannosamine (Ac₄ManNAz) to generate unnatural azide groups (-N₃) on the surface of the cells. Subsequently, the unnatural azide groups on the cell surface were specifically conjugated with near-infrared fluorescent (NIRF) dye-tagged dibenzyl cyclooctyne (DBCO-650) through bioorthogonal copper-free Click chemistry. Importantly, DBCO-650-labeled chondrocytes presented strong NIRF signals with relatively low cytotoxicity and the amounts of azide groups and DBCO-650 could be easily controlled by feeding different amounts of Ac₄ManNAz and DBCO-650 to the cell culture system. For the <i>in vivo</i> cell tracking, DBCO-650-labeled chondrocytes (1 × 10⁶ cells) seeded on the 3D scaffold were subcutaneously implanted into mice and the transplanted DBCO-650-labeled chondrocytes could be effectively tracked in the prolonged time period of 4 weeks using NIRF imaging technology. Furthermore, this new cell labeling and tracking technology had minimal effect on cartilage formation <i>in vivo</i>

Bioorthogonal Copper Free Click Chemistry for Labeling and Tracking of Chondrocytes <i>In Vivo</i>

Establishment of an appropriate cell labeling and tracking method is essential for the development of cell-based therapeutic strategies. Here, we are introducing a new method for cell labeling and tracking by combining metabolic gylcoengineering and bioorthogonal copper-free Click chemistry. First, chondrocytes were treated with tetraacetylated N-azidoacetyl-d-mannosamine (Ac₄ManNAz) to generate unnatural azide groups (-N₃) on the surface of the cells. Subsequently, the unnatural azide groups on the cell surface were specifically conjugated with near-infrared fluorescent (NIRF) dye-tagged dibenzyl cyclooctyne (DBCO-650) through bioorthogonal copper-free Click chemistry. Importantly, DBCO-650-labeled chondrocytes presented strong NIRF signals with relatively low cytotoxicity and the amounts of azide groups and DBCO-650 could be easily controlled by feeding different amounts of Ac₄ManNAz and DBCO-650 to the cell culture system. For the <i>in vivo</i> cell tracking, DBCO-650-labeled chondrocytes (1 × 10⁶ cells) seeded on the 3D scaffold were subcutaneously implanted into mice and the transplanted DBCO-650-labeled chondrocytes could be effectively tracked in the prolonged time period of 4 weeks using NIRF imaging technology. Furthermore, this new cell labeling and tracking technology had minimal effect on cartilage formation <i>in vivo</i>