Aptamer-Modified Tetrahedral Framework Nucleic Acid Synergized with TGF-ß3 to Promote Cartilage Protection in Osteoarthritis by Enhancing Chondrogenic Differentiation of MSCs.

Characterized by progressive and irreversible degeneration of the articular cartilage (AC), osteoarthritis (OA) is the most common chronic joint disease, and there is no cure for OA at present. Recent studies suggest that enhancing the recruitment of endogenous mesenchymal stem cells (MSCs) to damaged cartilage is a promising therapeutic strategy for cartilage repair. Tetrahedral framework nucleic acid (tFNA) is a novel DNA nanomaterial and has shown great potential in the field of biomedical science. Transforming growth factor-beta 3 (TGF-ß3), a vital member of the highly conserved TGF-ß superfamily, is considered to induce chondrogenesis. A 66-base DNA aptamer named HM69 is reported to identify and recruit MSCs. In this study, aptamer HM69-modified tFNAs were successfully self-assembled and used to load TGF-ß3 when the disulfide bonds combined. We confirmed the successful synthesis of the final composition, HM69-tFNA@TGF-ß3 (HTT), by PAGE, dynamic light scattering, and atomic force microscopy. The results of in vitro experiments showed that HTT effectively induced MSC proliferation, migration, and chondrogenic differentiation. In addition, HTT-treated MSCs were shown to protect the OA chondrocytes. In DMM mice, the injection of HTT improved the therapeutic outcome of mouse pain symptoms and AC degeneration. In conclusion, this study innovatively used the disulfide bonds combined with TGF-ß3 and tFNA, and an additional sequence HM69 was loaded on tFNA for the better-targeted recruitment of MSCs. HTT demonstrated its role in promoting the chondrogenesis of MSCs and cartilage protection, indicating that it might be promising for OA therapy.

Cartilage regeneration using transforming growth factor-beta 3-loaded injectable crosslinked hyaluronic acid hydrogel.

Cartilage defects can be difficult to heal, potentially leading to complications such as osteoarthritis. Recently, a tissue engineering approach that uses scaffolds and growth factors has been proposed to regenerate new cartilage tissues. Herein, we investigated the application of hyaluronic acid (HA) gel loaded with transforming growth factor-beta 3 (TGF-ß3) for enhanced cartilage regeneration. We assessed the clinical conditions required to efficiently enhance the ability of the modified HA gel to repair defective cartilage. Based on our findings, the prepared HA gel exhibited good physicochemical and mechanical properties and was non-toxic and non-inflammatory. Moreover, HA gel-loaded TGF-ß3 (HAT) had improved biocompatibility and promoted the synthesis of cartilage-specific matrix and collagen, further improving its ability to repair defects. The application of HAT resulted in an initial burst release of HA, which degraded slowly in vivo. Finally, HAT combined with microfracture-inducing bone marrow stem cells could significantly improve the cartilage microenvironment and regeneration of cartilage defects. Our results indicate that HA is a suitable material for developing growth factor carriers, whereas HAT is a promising candidate for cartilage regeneration. Furthermore, this differentiated strategy provides a rapid and effective clinical approach for next-generation cartilage regeneration.

Dramatic activation of an antibody by a single amino acid change in framework.

Antibody function is typically entirely dictated by the Complementarity Determining Regions (CDRs) that directly bind to the antigen, while the framework region acts as a scaffold for the CDRs and maintains overall structure of the variable domain. We recently reported that the rabbit monoclonal antibody 4A11 (rbt4A11) disrupts signaling through both TGFß2 and TGFß3 (Sun et al. in Sci Transl Med, 2021. https://doi.org/10.1126/scitranslmed.abe0407 ). Here, we report a dramatic, unexpected discovery during the humanization of rbt4A11 where, two variants of humanized 4A11 (h4A11), v2 and v7 had identical CDRs, maintained high affinity binding to TGFß2/3, yet exhibited distinct differences in activity. While h4A11.v7 completely inhibited TGFß2/3 signaling like rbt4A11, h4A11.v2 did not. We solved crystal structures of TGFß2 complexed with Fab fragments of h4A11.v2 or h4A11.v7 and identified a novel interaction between the two heavy chain molecules in the 2:2 TGFb2:h4A11.v2-Fab complex. Further characterization revealed that framework residue variations at either position 19, 79 or 81 (Kabat numbering) of the heavy chain strikingly converts h4A11.v2 into an inhibitory antibody. Our work suggests that in addition to CDRs, framework residues and interactions between Fabs in an antibody could be engineered to further modulate activity of antibodies.

An alginate-poly(acrylamide) hydrogel with TGF-ß3 loaded nanoparticles for cartilage repair: Biodegradability, biocompatibility and protein adsorption.

Current implantable materials are limited in terms of function as native tissue, and there is still no effective clinical treatment to restore articular impairments. Hereby, a functionalized polyacrylamide (PAAm)-alginate (Alg) Double Network (DN) hydrogel acting as an articular-like tissue is developed. These hydrogels sustain their mechanical stability under different temperature (+4 °C, 25 °C, 40 °C) and humidity conditions (60% and 75%) over 3 months. As for the functionalization, transforming growth factor beta-3 (TGF-ß3) encapsulated (NPTGF-ß3) and empty poly(lactide-co-glycolide) (PLGA) nanoparticles (PLGA NPs) are synthesized by using microfluidic platform, wherein the mean particle sizes are determined as 81.44 ± 9.2 nm and 126 ± 4.52 nm with very low polydispersity indexes (PDI) of 0.194 and 0.137, respectively. Functionalization process of PAAm-Alg hydrogels with ester-end PLGA NPs is confirmed by FTIR analysis, and higher viscoelasticity is obtained for functionalized hydrogels. Moreover, cartilage regeneration capability of these hydrogels is evaluated with in vitro and in vivo experiments. Compared with the PAAm-Alg hydrogels, functionalized formulations exhibit a better cell viability. Histological staining, and score distribution confirmed that proposed hydrogels significantly enhance regeneration of cartilage in rats due to stable hydrogel matrix and controlled release of TGF-ß3. These findings demonstrated that PAAm-Alg hydrogels showed potential for cartilage repair and clinical application.

An improved strategy of TGFß3 expression in Escherichia coli: Exploiting folding modulators for a switch from misfolded to folded form.

Transforming growth factor beta 3 (TGFß3) exhibits a complex native structure featuring the presence of multiple disulfide bonds forming the active dimer. Consequently, its heterologous expression in microbial system invariably leads to inclusion body (IB) formation. In this study, we observed an interesting phenomenon of switching a significant fraction of misfolded TGFß3 to folded form by modulating the cellular protein folding machinery. We carried out co-expression experiments with chaperones and demonstrated the requirement of a coordinated action of DnaK-DnaJ-GrpE and GroESL, to achieve the native soluble conformation of TGFß3, during over-expression in E. coli. The novelty of this study lies in the fact that orchestration of a group of chaperones to work in concert for efficient folding and assembly of TGFß3-like cytokines has not been widely explored. Additionally, we have also demonstrated that presence of osmolytes (sorbitol or trehalose) in the growth media have an appreciable impact on the solubility of TGFß3. We have further shown a synergism between the effects of molecular chaperone and osmolytes on the solubility of TGFß3. We have confirmed the functionality of soluble TGFß3 by performing binding interactions with its cognate receptor TßRII. Our study delineates the fact that an effective combination of chaperones or optimum concentration of compatible osmolyte, can efficiently abrogate competing aggregation pathways and help attain the native conformation of a cysteine rich cytokine in a facile manner.

Restoration of cartilage defects using a superparamagnetic iron oxide-labeled adipose-derived mesenchymal stem cell and TGF-ß3-loaded bilayer PLGA construct.

Aim: We aimed to evaluate the capacity of the bilayer polylactic-co-glycolic acid (PLGA)/TGF-ß3/adipose-derived mesenchymal stem cell (ADSC) construct used to repair cartilage defects and the role of ADSCs in the repair process in vivo. Materials & methods: Defects were created surgically on the femoropatellar groove of knee joints in 64 rabbits. All the rabbits were randomly divided into four groups: defect group, PLGA group, PLGA/TGF-ß3 group and PLGA/TGF-ß3/ADSC group. In vivo MRI and Prussian blue staining were applied. Quantitative real-time PCR and western blot methods were used to analyze the gene and protein expression. Results & conclusion: The result showed that TGF-ß3 could effectively stimulate the expressions of aggrecan, collagen type II and SRY-related HMG box 9 (SOX9). The bilayer PLGA/TGF-ß3/ADSC construct showed a promising repair effect.

Sustained release of TGF-ß3 from polysaccharide nanoparticles induces chondrogenic differentiation of human mesenchymal stromal cells.

Medical treatment of certain diseases and biomedical implants are tending to use delivery systems on the nanoscale basis for biologically active factors including drugs (e. g. antibiotics) or growth factors. Nanoparticles are a useful tool to deliver bioactive substances of different chemical nature directly to the site where it is required in the patient. Here we developed three innovative delivery systems based on different polysaccharides in order to induce a sustained release of TGF-ß3 to mediate chondrogenesis of human mesenchymal stromal cells. We were able to encapsulate the protein into nanoparticles and subsequently release TGF-ß3 from these particles. The protein was still active and was able to induce chondrogenic differentiation of human mesenchymal stromal cells.

Promoted chondrogenesis of hMCSs with controlled release of TGF-ß3 via microfluidics synthesized alginate nanogels.

The field of cartilage tissue engineering has been evolved in the last decade and a myriad of scaffolding biomaterials and bioactive agents have been proposed. Controlled release of growth factors encapsulated in the polymeric nanomaterials has been of interest notably for the repair of damaged articular cartilage. Here, we proposed an on-chip hydrodynamic flow focusing microfluidic approach for synthesis of alginate nanogels loaded with the transforming growth factor beta 3 (TGF-ß3) through an ionic gelation method in order to achieve precise release profile of these bioactive agents during chondrogenic differentiation of mesenchymal stem cells (MSCs). Alginate nanogels with adjustable sizes were synthesized by fine-tuning the flow rate ratio (FRR) in the microfluidic device consisting of cross-junction microchannels. The result of present study showed that the proposed approach can be a promising tool to synthesize bioactive -loaded polymeric nanogels for applications in drug delivery and tissue engineering.

Transforming Growth Factor-ß3/Chitosan Sponge (TGF-ß3/CS) Facilitates Osteogenic Differentiation of Human Periodontal Ligament Stem Cells.

Periodontal disease is the main reason for tooth loss in adults. Tissue engineering and regenerative medicine are advanced technologies used to manage soft and hard tissue defects caused by periodontal disease. We developed a transforming growth factor-ß3/chitosan sponge (TGF-ß3/CS) to repair periodontal soft and hard tissue defects. We investigated the proliferation and osteogenic differentiation behaviors of primary human periodontal ligament stem cells (hPDLSCs) to determine the bioactivity and potential application of TGF-ß3 in periodontal disease. We employed calcein-AM/propidium iodide (PI) double labeling or cell membranes (CM)-Dil labeling coupled with fluorescence microscopy to trace the survival and function of cells after implantation in vitro and in vivo. The mineralization of osteogenically differentiated hPDLSCs was confirmed by measuring alkaline phosphatase (ALP) activity and calcium content. The levels of COL I, ALP, TGF-ßRI, TGF-ßRII, and Pp38/t-p38 were assessed by western blotting to explore the mechanism of bone repair prompted by TGF-ß3. When hPDLSCs were implanted with various concentrations of TGF-ß3/CS (62.5-500 ng/mL), ALP activity was the highest in the TGF-ß3 (250 ng/mL) group after 7 d (p < 0.05 vs. control). The calcium content in each group was increased significantly after 21 and 28 d (p < 0.001 vs. control). The optimal result was achieved by the TGF-ß3 (500 ng/mL) group. These results showed that TGF-ß3/CS promotes osteogenic differentiation of hPDLSCs, which may involve the p38 mitogen-activated protein kinase (MAPK) signaling pathway. TGF-ß3/CS has the potential for application in the repair of incomplete alveolar bone defects.

Coacervate-mediated exogenous growth factor delivery for scarless skin regeneration.

Although there are numerous medical applications to recover damaged skin tissue, scarless wound healing is being extensively investigated to provide a better therapeutic outcome. The exogenous delivery of therapeutic growth factors (GFs) is one of the engineering strategies for skin regeneration. This study presents an exogenous GF delivery platform developed using coacervates (Coa), a tertiary complex of poly(ethylene argininyl aspartate diglyceride) (PEAD) polycation, heparin, and cargo GFs (i.e., transforming growth factor beta 3 (TGF-ß3) and interleukin 10 (IL-10)). Coa encompasses the advantage of high biocompatibility, facile preparation, protection of cargo GFs, and sustained GF release. We therefore speculated that coacervate-mediated dual delivery of TGF-ß3/IL-10 would exhibit synergistic effects for the reduction of scar formation during physiological wound healing. Our results indicate that the exogenous administration of dual GF via Coa enhances the proliferation and migration of skin-related cells. Gene expression profiles using RT-PCR revealed up-regulation of ECM formation at early stage of wound healing and down-regulation of scar-related genes at later stages. Furthermore, direct injection of the dual GF Coa into the edges of damaged skin in a rat skin wound defect model demonstrated accelerated wound closure and skin regeneration after 3 weeks. Histological evaluation and immunohistochemical staining also revealed enhanced formation of the epidermal layer along with facilitated angiogenesis following dual GF Coa delivery. Based on these results, we conclude that polycation-mediated Coa fabrication and exogenous dual GF delivery via the Coa platform effectively augments both the quantity and quality of regenerated skin tissues without scar formation. STATEMENT OF SIGNIFICANCE: This study was conducted to develop a simple administration platform for scarless skin regeneration using polycation-based coacervates with dual GFs. Both in vitro and in vivo studies were performed to confirm the therapeutic efficacy of this platform toward scarless wound healing. Our results demonstrate that the platform developed by us enhances the proliferation and migration of skin-related cells. Sequential modulation in various gene expression profiles suggests a balanced collagen-remodeling process by dual GFs. Furthermore, in vivo histological evaluation demonstrates that our technique enhances clear epidermis formation with less scab and thicker woven structure of collagen bundle, similar to that of a normal tissue. We propose that simple administration of dual GFs with Coa has the potential to be applied as a clinical approach for fundamental scarless skin regeneration.