Application of functionalized hyaluronic acid hydrogel with the activity of regulating the behaviors of stem cells in repairing rabbit knee articular cartilage.

Using injectable hydrogels loaded with mesenchymal stem cells (MSCs) to repair chondral defects is a new trend of cartilage tissue engineering in recent years. In this study, hyaluronic acid (HA) hydrogels containing the system of sustained-release Kartogenin (KGN) and modified by RGD and HAV peptides were used to facilitate repair of cartilage defect in the knee joint of rabbits. Different groups of implants were injected into osteochondral defects, and samples were taken 4 weeks after operation. Through the qualitative and quantitative analysis of Micro-CT, it can be seen that both FH (unloaded cell group) and R + FH groups (allogeneic cell group) can repair osteochondral defects well, and the amount of bone formation is high, which is close to the intact cartilage groups. Macroscopic observation and histological staining analysis showed that except for the intact cartilage group, FH group obtained the highest score. The morphology of the cartilage tissue in the FH groups was more regular and continuous than that in R + FH and H + FH (xenogeneic cell group) groups, approaching that of native cartilage. Immunohistochemical staining of Collagen II (Col II) showed that the expression and morphology of Col II in FH groups were similar to those in intact cartilage tissue. Interestingly, through in vivo experiments, this functionalized hyaluronic acid hydrogel can effectively promote the rapid repair of rabbit knee cartilage defects within one month.

A chondrogenesis induction system based on a functionalized hyaluronic acid hydrogel sequentially promoting hMSC proliferation, condensation, differentiation, and matrix deposition.

Hydrogel scaffolds are widely used in cartilage tissue engineering as a natural stem cell niche. In particular, hydrogels based on multiple biological signals can guide behaviors of mesenchymal stem cells (MSCs) during neo-chondrogenesis. In the first phase of this study, we showed that functionalized hydrogels with grafted arginine-glycine-aspartate (RGD) peptides and lower degree of crosslinking can promote the proliferation of human mesenchymal stem cells (hMSCs) and upregulate the expression of cell receptor proteins. Moreover, grafted RGD and histidine-alanine-valine (HAV) peptides in hydrogel scaffolds can regulate the adhesion of the intercellular at an early stage. In the second phase, we confirmed that simultaneous use of HAV and RGD peptides led to greater chondrogenic differentiation compared to the blank control and single-peptide groups. Furthermore, the controlled release of kartogenin (KGN) can better facilitate cell chondrogenesis compared to other groups. Interestingly, with longer culture time, cell condensation was clearly observed in the groups with RGD and HAV peptide. In all groups with RGD peptide, significant matrix deposition was observed, accompanied by glycosaminoglycan (GAG) and collagen (Coll) production. Through in vitro and in vivo experiments, this study confirmed that our hydrogel system can sequentially promote the proliferation, adhesion, condensation, chondrogenic differentiation of hMSCs, by mimicking the cell microenvironment during neo-chondrogenesis.

Tripeptide-based macroporous hydrogel improves the osteogenic microenvironment of stem cells.

Due to the ability to combine multiple osteogenic induction "cues" at the same time, hydrogels are widely used in the three-dimensional (3D) culture of human mesenchymal stem cells (hMSCs) and osteoinduction. However, the survival and proliferation of stem cells in a 3D culture system are limited, which reduces their osteogenic differentiation efficiency. In addition, the cells inside the hydrogel are prone to apoptosis due to hypoxia, which is a serious challenge for tissue engineering based on stem cells. In this study, a tripeptide-based macroporous alginate hydrogel was prepared to improve the osteogenic microenvironment of stem cells. The arginine-glycine-aspartate (RGD) peptide promoted the adhesion and proliferation of stem cells, and the degradation of gelatin microspheres (GMs) produced a macroporous structure to enhance further the migration and aggregation of stem cells. Mesoporous silica nanoparticles (MSNs) sustained-release bone-forming peptide-1 (BFP-1) induced osteogenic differentiation, and the sustained release of the QK peptide from the GMs promoted angiogenesis. In vitro experiments have shown that this functionalized hydrogel stimulates the proliferation of hMSCs, encourages larger cell cluster formation, and enhances the osteogenic differentiation efficiency. The released QK facilitates the proliferation and migration of endothelial cells. In vivo experiments have also verified that this system has a better osteogenic effect, and more blood vessels were observed inside the hydrogel, than in other systems. In general, this research has led to the development of a tripeptide macroporous hydrogel that can simultaneously promote osteogenesis and angiogenesis, showing great promise for applications of 3D cultures and stem cell-based tissue engineering.

Multifunctional Immunoliposomes Combining Catalase and PD-L1 Antibodies Overcome Tumor Hypoxia and Enhance Immunotherapeutic Effects Against Melanoma.

BACKGROUND: Immune checkpoint blockades (ICBs) are a promising treatment for cancers such as melanoma by blocking important inhibitory pathways that enable tumor cells to evade immune attack. Programmed death ligand 1 monoclonal antibodies (aPDL1s) can be used as an ICB to significantly enhance the effectiveness of tumor immunotherapy by blocking the PD-1/PD-L1 inhibitory pathway. However, the effectiveness of aPDL1s may be limited by low selectivity in vivo and immunosuppressed tumor microenvironment including hypoxia. PURPOSE: To overcome the limitations, we develop a multifunctional immunoliposome, called CAT@aPDL1-SSL, with catalase (CAT) encapsulated inside to overcome tumor hypoxia and aPDL1s modified on the surface to enhance immunotherapeutic effects against melanoma. METHODS: The multifunctional immunoliposomes (CAT@aPDL1-SSLs) are prepared using the film dispersion/post-insertion method. The efficacy of CAT@aPDL1-SSLs is verified by multiple experiments in vivo and in vitro. RESULTS: The results of this study suggest that the multifunctional immunoliposomes preserve and protect the enzyme activity of CAT and ameliorate tumor hypoxia. Moreover, the enhanced cellular uptake of CAT@aPDL1-SSLs in vitro and their in vivo biodistribution suggest that CAT@aPDL1-SSLs have great targeting ability,resulting in improved delivery and accumulation of immunoliposomes in tumor tissue.Finally, by activating and increasing the infiltration of CD8+ T cells at the tumor site, CAT@aPDL1-SSLs inhibit the growth of tumor and prolong survival time of mice,with low systemic toxicity. CONCLUSION: In conclusion, the multifunctional immunoliposomes developed and proposed in this study are a promising candidate for melanoma immunotherapy, and could potentially be combined with other cancer therapies like radiotherapy and chemotherapy to produce positive outcomes.

Synergistic Effects of Kartogenin and Transforming Growth Factor-ß3 on Chondrogenesis of Human Umbilical Cord Mesenchymal Stem Cells In Vitro.

OBJECTIVE: To explore the effect of kartogenin (KGN) on proliferation and chondrogenic differentiation of human umbilical cord mesenchymal stem cells (hUCMSC) in vitro, and the synergistic effects of KGN and transforming growth factor (TGF)-ß3 on hUCMSC. METHODS: Human umbilical cord mesenchymal stem cells were isolated and cultured. Then the differentiation properties were identified by flow cytometry analysis. HUCMSC were divided into four groups: control group, KGN group, TGF-ß3 group, and TK group (with TGF-ß3 and KGN added into the medium simultaneously). Cells in all groups were induced for 21 days using the suspension ball culture method. Hematoxylin and eosin, immunofluorescence, and Alcian blue staining were used to analyze chondrogenic differentiation. Real-time reverse transcriptase polymerase chain reaction was performed to investigate genes associated with chondrogenic differentiation. RESULT: Hematoxylin and eosin staining showed that cells in the TGF-ß3 group and the TK group had formed cartilage-like tissue after 21 days of culture. The results of immunofluorescence and Alcian blue staining showed that compared with the control group, cells in the KGN and TGF-ß3 groups demonstrated increased secretion of aggrecan after 21 days of culture. In addition, cells in the group combining KGN with TGF-ß3 (5.587 ± 0.27, P < 0.01) had more collagen II secretion than cells in the TGF-ß3 alone group (2.86 ± 0.141, P < 0.01) or the KGN group (1.203 ± 0.215, P < 0.01). The expression of aggrecan (2.468 ± 0.097, P < 0.05) and SRY-Box 9 (4.08 ± 0.13, P < 0.05) in cells in the group combining KGN with TGF-ß3 was significantly higher than those in the TGF-ß3 group (2.216 ± 0.09, 3.02 ± 0.132, P < 0.05).' CONCLUSION: The combination of KGN and TGF-ß3 had synergistic effects and induced hUCMSC chondrogenesis. This could represent a new approach for clinical application and studies on cartilage repair and regeneration.

[Preparation and evaluation of carboxymethyl chitosan/sodium alginate hydrogel for cartilage tissue engineering].

OBJECTIVE: This study aimed to optimize the preparation of carboxymethyl chitosan/sodium alginate (CMCS/OSA) compound hydrogels. This study also aimed to investigate the applicability of the hydrogels in cartilage tissue engi-neering. METHODS: Three groups of CMCS/OSA composite hydrogels with amino-to-aldehyde ratios of 2∶1, 1∶1 and 1∶2 were prepared. The microstructure, physical properties, and cell biocompatibility of the three groups of CMCS/OSA com-posite hydrogels were evaluated. Samples were subjected to scanning electron microscopy, rheological test, adhesion tension test, swelling rate test, and cell experiments to identify the CMCS/OSA composite hydrogel with the cross-linking degree that can meet the requirements for scaffolds in cartilage tissue engineering. RESULTS: The experimental results showed that the CMCS/OSA hydrogel with a amine-to-aldhyde ratio of 1∶1 had good porosity, suitable gelling time, strong adhesive force, stable swelling rate, and good cellular biocompatibility. CONCLUSIONS: The CMCS/OSA compound hydrogel prepared with a 1∶1 ratio of amino and aldehyde groups has potential applications in cartilage tissue engineering.

Silk fibroin/cartilage extracellular matrix scaffolds with sequential delivery of TGF-ß3 for chondrogenic differentiation of adipose-derived stem cells.

A 3-D scaffold that simulates the microenvironment in vivo for regenerating cartilage is ideal. In this study, we combined silk fibroin and decellularized cartilage extracellular matrix by temperature gradient-guided thermal-induced phase separation to produce composite scaffolds (S/D). Resulting scaffolds had remarkable mechanical properties and biomimeticstructure, for a suitable substrate for attachment and proliferation of adipose-derived stem cells (ADSCs). Moreover, transforming growth factor ß3 (TGF-ß3) loaded on scaffolds showed a controlled release profile and enhanced the chondrogenic differentiation of ADSCs during the 28-day culture. The S/D scaffold itself can provide a sustained release system without the introduction of other controlled release media, which has potential for commercial and clinical applications. The results of toluidine blue, Safranin O, and immunohistochemical staining and analysis of collagen II expression showed maintenance of a chondrogenic phenotype in all scaffolds after 28-day culture. The most obvious phenomenon was with the addition of TGF-ß3. S/D composite scaffolds with sequential delivery of TGF-ß3 may mimic the regenerative microenvironment to enhance the chondrogenic differentiation of ADSCs in vitro.