Extrusion bioprinting of cellular aggregates improves mesenchymal stem cell proliferation and differentiation.

3D extrusion bioprinting brings the prospect of stem cell-based therapies in regenerative medicine. These bioprinted stem cells are expected to proliferate and differentiate to form the desired organoids into 3D structures, which is critical for complex tissue construction. However, this strategy is hampered by low reproducible cell number and viability, and organoid immaturity due to incomplete differentiation of stem cells. Hence, we apply a novel extrusion-based bioprinting process with cellular aggregates (CA) bioink, in which the encapsulated cells are precultured in hydrogels to undergo aggregation. In this study, alginate-gelatin-collagen (Alg-Gel-Col) hydrogel containing mesenchymal stem cells (MSCs) were precultured for 48 h to form CA bioink and resulted in high cell viability and printing fidelity. Meanwhile, MSCs in CA bioink showed high proliferation, stemness and lipogenic differentiative potential in contrast to that in single cell (SC) bioink and hanging drop cell spheroid (HDCS) bioink, which indicated the considerable potential for complex tissue construction. In addition, the printability and efficacy of human umbilical cord MSCs (hUC-MSCs) were further confirmed the translational potential of this novel bioprinting method.

Effects of in situ cross-linked graphene oxide-containing gelatin methacrylate anhydride hydrogel on wound vascularization of full-thickness skin defect in mice / 中华烧伤杂志

Objective: To prepare graphene oxide (GO)-containing gelatin methacrylate anhydride (GelMA) hydrogel and to investigate the effects of in situ photopolymerized GO-GelMA composite hydrogel in wound vascularization of full-thickness skin defect in mice. Methods: The experimental study method was used. The 50 μL of 0.2 mg/mL GO solution was evenly applied onto the conductive gel, and the structure and size of GO were observed under field emission scanning electron microscope after drying. Human skin fibroblasts (HSFs) were divided into 0 μg/mL GO (without GO solution, the same as below) group, 0.1 μg/mL GO group, 1.0 μg/mL GO group, 5.0 μg/mL GO group, and 10.0 μg/mL GO group treated with GO of the corresponding final mass concentration, and the absorbance value was detected using a microplate analyzer after 48 h of culture to reflect the proliferation activity of cells (n=6). HSFs and human umbilical vein vascular endothelial cells (HUVECs) were divided into 0 μg/mL GO group, 0.1 μg/mL GO group, 1.0 μg/mL GO group, and 5.0 μg/mL GO group treated with GO of the corresponding final mass concentration, and the migration rates of HSFs at 24 and 36 h after scratching (n=5) and HUVECs at 12 h after scratching (n=3) were detected by scratch test, and the level of vascular endothelial growth factor (VEGF) secreted by HSFs after 4, 6, and 8 h of culture was detected by enzyme-linked immunosorbent assay method (n=3). The prepared GO-GelMA composite hydrogels containing GO of the corresponding final mass concentration were set as 0 μg/mL GO composite hydrogel group, 0.1 μg/mL GO composite hydrogel group, 1.0 μg/mL GO composite hydrogel group, and 5.0 μg/mL GO composite hydrogel group to observe their properties before and after cross-linking, and to detect the release of GO after soaking with phosphate buffer solution for 3 and 7 d (n=3). The full-thickness skin defect wounds were made on the back of 16 6-week-old female C57BL/6 mice. The mice treated with in situ cross-linked GO-GelMA composite hydrogel containing GO of the corresponding final mass concentration were divided into 0 μg/mL GO composite hydrogel group, 0.1 μg/mL GO composite hydrogel group, 1.0 μg/mL GO composite hydrogel group, and 5.0 μg/mL GO composite hydrogel group according to the random number table, with 4 mice in each group. The general condition of wound was observed and the wound healing rate was calculated on 3, 7, and 14 d of treatment, the wound blood perfusion was detected by laser Doppler flowmetry on 3, 7, and 14 d of treatment and the mean perfusion unit (MPU) ratio was calculated, and the wound vascularization on 7 d of treatment was observed after hematoxylin-eosin staining and the vascular density was calculated (n=3). The wound tissue of mice in 0 μg/mL GO composite hydrogel group and 0.1 μg/mL GO composite hydrogel group on 7 d of treatment was collected to observe the relationship between the distribution of GO and neovascularization by hematoxylin-eosin staining (n=3) and the expression of VEGF by immunohistochemical staining. Data were statistically analyzed with analysis of variance for repeated measurement, one-way analysis of variance, and Tukey's method. Results: GO had a multilayered lamellar structure with the width of about 20 μm and the length of about 50 μm. The absorbance value of HSFs in 10.0 μg/mL GO group was significantly lower than that in 0 μg/mL GO group after 48 h of culture (q=7.64, P<0.01). At 24 h after scratching, the migration rates of HSFs were similar in the four groups (P>0.05); at 36 h after scratching, the migration rate of HSFs in 0.1 μg/mL GO group was significantly higher than that in 0 μg/mL GO group, 1.0 μg/mL GO group, and 5.0 μg/mL GO group (with q values of 7.48, 10.81, and 10.20, respectively, P<0.01). At 12 h after scratching, the migration rate of HUVECs in 0.1 μg/mL GO group was significantly higher than that in 0 μg/mL GO group, 1.0 μg/mL GO group, and 5.0 μg/mL GO group (with q values of 7.11, 8.99, and 14.92, respectively, P<0.01), and the migration rate of HUVECs in 5.0 μg/mL GO group was significantly lower than that in 0 μg/mL GO group and 1.0 μg/mL GO group (with q values of 7.81 and 5.33, respectively, P<0.05 or P<0.01 ). At 4 and 6 h of culture, the VEGF expressions of HSFs in the four groups were similar (P>0.05); at 8 h of culture, the VEGF expression of HSFs in 0.1 μg/mL GO group was significantly higher than that in 0 μg/mL GO group and 5.0 μg/mL GO group (with q values of 4.75 and 4.48, respectively, P<0.05). The GO-GelMA composite hydrogels in the four groups were all red liquid before cross-linking, which turned to light yellow gel after cross-linking, with no significant difference in fluidity. The GO in the GO-GelMA composite hydrogel of 0 μg/mL GO composite hydrogel group had no release of GO at all time points; the GO in the GO-GelMA composite hydrogels of the other 3 groups was partially released on 3 d of soaking, and all the GO was released on 7 d of soaking. From 3 to 14 d of treatment, the wounds of mice in the 4 groups were covered with hydrogel dressings, kept moist, and gradually healed. On 3, 7, and 14 d of treatment, the wound healing rates of mice in the four groups were similar (P>0.05). On 3 d of treatment, the MPU ratio of wound of mice in 0.1 μg/mL GO composite hydrogel group was significantly higher than that in 0 μg/mL GO composite hydrogel group, 1.0 μg/mL GO composite hydrogel group, and 5.0 μg/mL GO composite hydrogel group (with q values of 10.70, 11.83, and 10.65, respectively, P<0.05 or P<0.01). On 7 and 14 d of treatment, the MPU ratios of wound of mice in the four groups were similar (P>0.05). The MPU ratio of wound of mice in 0.1 μg/mL GO composite hydrogel group on 7 d of treatment was significantly lower than that on 3 d of treatment (q=14.38, P<0.05), and that on 14 d of treatment was significantly lower than that on 7 d of treatment (q=27.78, P<0.01). On 7 d of treatment, the neovascular density of wound of mice on 7 d of treatment was 120.7±4.1 per 200 times of visual field, which was significantly higher than 61.7±1.3, 77.7±10.2, and 99.0±7.9 per 200 times of visual field in 0 μg/mL GO composite hydrogel group, 1.0 μg/mL GO composite hydrogel group, and 5.0 μg/mL GO composite hydrogel group (with q values of 12.88, 7.79, and 6.70, respectively, P<0.01), and the neovascular density of wound of mice in 1.0 μg/mL GO composite hydrogel group and 5.0 μg/mL GO composite hydrogel group was significantly higher than that in 0 μg/mL GO composite hydrogel group (with q values of 5.10 and 6.19, respectively, P<0.05). On 7 d of treatment, cluster of new blood vessels in wound of mice in 0.1 μg/mL GO composite hydrogel group was significantly more than that in 0 μg/mL GO composite hydrogel group, and the new blood vessels were clustered near the GO; a large amount of VEGF was expressed in wound of mice in 0.1 μg/mL GO composite hydrogel group in the distribution area of GO and new blood vessels. Conclusions: GO with mass concentration lower than 10.0 μg/mL had no adverse effect on proliferation activity of HSFs, and GO of 0.1 μg/mL can promote the migration of HSFs and HUVECs, and can promote the secretion of VEGF in HSFs. In situ photopolymerized of GO-GelMA composite hydrogel dressing can promote the wound neovascularization of full-thickness skin defect in mice and increase wound blood perfusion in the early stage, with GO showing an enrichment effect on angiogenesis, and the mechanism may be related to the role of GO in promoting the secretion of VEGF by wound cells.

Bioactive nanoparticle reinforced alginate/gelatin bioink for the maintenance of stem cell stemness.

Mesenchymal cells (MSCs) are an attractive option as seed cells for bioprinting. However, loss of stemness and undesired differentiation reduces their effectiveness. In this study, 12 nm bioactive nanoparticles (BNPs) which could release silicon (Si) ions were used to enhance the properties of alginate/gelatin hydrogel bioink to maintain MSC stemness. By specifically leveraging biochemical signals of BNPs, bioink with defined stiffness towards osteogenic and adipogenic potential, independent of pore structure, were designed by incorporating with different concentration of BNPs. These bioink were characterized by printability, mechanical and rheological properties as well as osteogenic and adipogenic potentials. Notably, the effect of 2% BNPs addition in alginate/gelatin hydrogel on MSC stemness maintenance was confirmed by the expression of stemness markers. At higher concentrations of BNPs (5%), printability was impacted by the gelling process. We further confirmed the enhanced stemness maintenance by sweat gland lineage commitment of bioprinted MSCs in vitro. Overall, our study proved that alginate/gelatin hydrogel bioink reinforced by BNPs in the optimal concentrations could retain MSC stemness as well as support MSC growth and prolong the desired differentiation. These findings may provide a new approach to achieve the ideal therapeutic potential of MSCs in 3D bioprinting application.

Bone marrow-derived mesenchymal stem cell attenuates skin fibrosis development in mice.

Recent studies showed that mesenchymal stem cell (MSC) transplantation significantly alleviated tissue fibrosis; however, little is known about the efficacy on attenuating cutaneous scar formation. In this study, we established a dermal fibrosis model induced by bleomycin and evaluated the benefit of bone marrow-derived mesenchymal stem cells (BM-MSCs) on skin fibrosis development. Tracing assay of green fluorescent protein (GFP(+) )BM-MSCs showed that the cells disappeared gradually within 24 hours upon administration, which hinted the action of BM-MSCs in vivo was exerted in the initial phase of repair in this model. Therefore, we repeatedly transplanted syngeneic BM-MSCs in the process of skin fibrosis formation. After 3 weeks, it was found that BM-MSC-treated lesional skin demonstrated a unanimous basket-weave organisation of collagen arrangement similar to normal skin, with few inflammatory cells. In addition, lesional skin with BM-MSC treatment exhibited a significant down-regulation of transforming growth factor-ß1 (TGF-ß1), type I collagen and heat-shock protein 47 (HSP47), with higher expression of matrix metalloproteinases (MMPs)-2, -9 and -13. Further experiments showed that α-smooth muscle actin (α-SMA) positive cells, the most reliable marker of myofibroblasts, apparently decreased after BM-MSC transplantation, which revealed that BM-MSCs could attenuate myofibroblast proliferation and differentiation as well as matrix production. Taken together, these findings suggested that BM-MSCs can inhibit the formation process of bleomycin-induced skin fibrosis, alleviate inflammation and favour the remodelling of extracellular matrix.

Mesenchymal stem cells: a revolution in therapeutic strategies of age-related diseases.

The great evolutionary biologist Theodosius Dobzhansky once said: "Nothing in biology makes sense except in the light of evolution". Aging is a complex biological phenomenon and the factors governing the process of aging and age-related diseases are only beginning to be understood, oxidative stress, telomere shortening in DNA components and genetic changes were shown to be the mainly regulating mechanisms during the recent decades. Although a considerable amount of both animal and clinical data that demonstrate the extensive and safe use of mesenchymal stromal cells (MSCs) is available, the precise summarization and identification of MSCs in age-related diseases remains a challenge. Along this line, this review discussed several typical age-related diseases for which MSCs have been proved to confer protection and put forward a hypothesis for the association among MSCs and age-related diseases from an evolutionary perspective. Above all, we hope further and more research efforts could be aroused to elucidate the role and mechanisms that MSCs involved in the age-related diseases.