Tissue adhesive, ROS scavenging and injectable PRP-based 'plasticine' for promoting cartilage repair.

Platelet-rich plasma (PRP) that has various growth factors has been used clinically in cartilage repair. However, the short residence time and release time at the injury site limit its therapeutic effect. The present study fabricated a granular hydrogel that was assembled from gelatin microspheres and tannic acid through their abundant hydrogen bonding. Gelatin microspheres with the gelatin concentration of 10 wt% and the diameter distribution of 1-10 µm were used to assemble by tannic acid to form the granular hydrogel, which exhibited elasticity under low shear strain, but flowability under higher shear strain. The viscosity decreased with the increase in shear rate. Meanwhile, the granular hydrogel exhibited self-healing feature during rheology test. Thus, granular hydrogel carrying PRP not only exhibited well-performed injectability but also performed like a 'plasticine' that possessed good plasticity. The granular hydrogel showed tissue adhesion ability and reactive oxygen species scavenging ability. Granular hydrogel carrying PRP transplanted to full-thickness articular cartilage defects could integrate well with native cartilage, resulting in newly formed cartilage articular fully filled in defects and well-integrated with the native cartilage and subchondral bone. The unique features of the present granular hydrogel, including injectability, plasticity, porous structure, tissue adhesion and reactive oxygen species scavenging provided an ideal PRP carrier toward cartilage tissue engineering.

Pluronic F127-Lipoic Acid Adhesive Nanohydrogel Combining with Ce3+/Tannic Acid/Ulinastatin Nanoparticles for Promoting Wound Healing.

Developing strong anti-inflammatory wound dressings is of great significance for protecting inflammatory cutaneous wounds and promoting wound healing. The present study develops a nanocomposite Pluronic F127 (F127)-based hydrogel dressing with injectable, tissue adhesive, and anti-inflammatory performance. Briefly, Ce3+/tannic acid/ulinastatin nanoparticles (Ce3+/TA/UTI NPs) are fabricated. Meanwhile, α-lipoic acid is bonded to the ends of F127 to prepare F127-lipoic acid (F127LA) and its nanomicelles. Due to the gradual viscosity change instead of mutation during phase transition, the mixed Ce3+/TA/UTI NPs and F127LA nanomicelles show well-performed injectability at 37 °C and can form a semisolid composite nanohydrogel that can tightly attach to the skin at 37 °C. Furthermore, ultraviolet (UV) irradiation without a photoinitiator transforms the semisolid hydrogel into a solid hydrogel with well-performed elasticity and toughness. The UV-cured composite nanohydrogel acts as a bioadhesive that can firmly adhere to tissues. Due to the limited swelling property, the hydrogel can firmly adhere to tissues in a wet environment, which can seal wounds and provide a reliable physical barrier for the wounds. Ce3+/TA/UTI NPs in the hydrogel exhibit lipopolysaccharide (LPS)-scavenging ability and reactive oxygen species (ROS)-scavenging ability and significantly reduce the expression of inflammatory factors in wounds at the early stage, accelerating LPS-induced wound healing.

Tissue adhesive indocyanine green-locking granular gel-mediated photothermal therapy combined with checkpoint inhibitor for preventing postsurgical recurrence and metastasis of colorectal cancer.

Developing effective therapy to inhibit postoperative recurrence and metastasis of colorectal cancer (CRC) is challenging and significant to reduce mortality and morbidity. Here, a granular hydrogel, assembled from gelatin microgels by dialdehyde starch and interpenetrated with in situ polymerized poly(sulfobetaine methacrylate-co-N-isopropylacrylamide) (P(SBMA-co-NIPAM)), is prepared to load and lock Food and Drug Administration (FDA)-approved indocyanine green (ICG) with definite photothermal function and biosafety for photothermal therapy (PTT) combining with checkpoint inhibitor. The presence of P(SBMA-co-NIPAM) endows granular hydrogel with high retention to water-soluble ICG, preventing easy diffusion and rapid scavenging of ICG. The ICG-locking granular hydrogel can be spread and adhered onto the surgery site at wet state in vivo, exerting a persistent and stable PTT effect. Combined with αPD-L1 treatment, ICG-locking granular hydrogel-mediated PTT can eradicate postsurgery residual and metastatic tumors, and prevent long-term tumor recurrence. Further mechanistic studies indicate that combination treatment effectively promotes dendritic cells maturation in lymph nodes, enhances the number and infiltration of CD8+ T and CD4+ T cells in tumor tissue, and improves memory T cell number in spleen, thus activating the antitumor immune response. Overall, ICG-locking gel-mediated PTT is expected to exhibit broad clinical applications in postoperative treatment of cancers, like CRC.

Injectable, self-healing poly(amino acid)-hydrogel based on phenylboronate ester bond for osteochondral tissue engineering.

A new generation of osteochondral integrated scaffolds is needed for articular osteochondral regeneration, which can not only facilitate the accurate construction of osteochondral scaffolds in a minimally invasive manner but also firmly combine the subchondral bone layer and cartilage layer. Herein, an osteochondral integrated hydrogel scaffold was constructed by the poly(L-glutamic acid) (PLGA) based self-healing hydrogels with phenylboronate ester (PBE) as the dynamic cross-linking. The bone layer self-healing hydrogel (hydrogel O-S) was prepared by physically blending nanohydroxyapatite into the self-healing hydrogel PLGA-PBE-S, which was fabricated by 3-aminophenylboronic acid/glycidyl methacrylate-modified PLGA (PLGA-GMA-PBA) and 3-amino-1,2-propanediol/N-(2-aminoethyl) acrylamide-modified PLGA (PLGA-ADE-AP). The cartilage layer self-healing hydrogel (hydrogel C-S) was prepared by PLGA-GMA-APBA and glucosamine- modified PLGA-ADE-AP (PLGA-ADE-AP-G). Excellent injectability and self-healing profiles of hydrogel O-S and C-S were observed, the self-healing efficiencies were 97.02% ± 1.06% and 99.06% ± 0.57%, respectively. Based on the injectability and spontaneous healing on the interfaces of hydrogel O-S and C-S, the osteochondral hydrogel (hydrogel OC) was conveniently constructed in a minimally invasive manner. In addition,in situphotocrosslinking was used to enhance the mechanical strength and stability of the osteochondral hydrogel. The osteochondral hydrogels exhibited good biodegradability and biocompatibility. The osteogenic differentiation genes BMP-2, ALPL, BGLAP and COL I of adipose-derived stem cells (ASCs) in the bone layer of the osteochondral hydrogel were significantly expressed, and the chondrogenic differentiation genes SOX9, aggrecan and COL II of ASCs in the cartilage layer of the osteochondral hydrogel were obviously upregulated after 14 d of induction. The osteochondral hydrogels could effectively promote repair of osteochondral defects after 3 months post-surgery.

Building a Poly(amino acid)/Chitosan-Based Self-Healing Hydrogel via Host-Guest Interaction for Cartilage Regeneration.

Cartilage injury is a very common joint disease, and cartilage repair is a great challenge in clinical treatment due to the specific structure of cartilage tissue and its microenvironment in vivo. The injectable self-healing hydrogel is a very promising candidate as a cartilage repair material because of its special network structure, high water retention and self-healing properties. In this work, a self-healing hydrogel cross-linked by host-guest interaction between cyclodextrin and cholic acid was developed. The host material was composed of ß-cyclodextrin and 2-hydroxyethyl methacrylate-modified poly(l-glutamic acid) (P(LGA-co-GM-co-GC)), while the guest material was chitosan modified by cholic acid, glycidyl methacrylate, and (2,3-epoxypropyl)trimethylammonium chloride (EPTAC) (QCSG-CA). The host-guest interaction self-healing hydrogels, named as HG hydrogels (HG gel), exhibited excellent injectability and self-healable property, and the self-healing efficiency was greater than 90%. Furthermore, in order to enhance the mechanical properties and slow down the degradation of the HG gel in vivo, the second network was constructed by photo-cross-linking in situ. Biocompatibility tests showed that the enhanced multi-interaction hydrogel (MI gel) was extremely suitable for cartilage tissue engineering both in vitro and in vivo. In addition, the adipose derived stem cells (ASCs) in MI gel were able to differentiate cartilage effectively in vitro in the presence of inducing agents. Subsequently, the MI gel without ASCs was transplanted into rat cartilage defects in vivo for the regeneration of cartilage. After 3 months postimplantation, new cartilage tissue was successfully regenerated in a rat cartilage defect. All results indicated that the injectable self-healing host-guest hydrogels have important potential applications in cartilage injury repair.

Comparative Physiological and Transcriptome Analysis Reveals Potential Pathways and Specific Genes Involved in Waterlogging Tolerance in Apple Rootstocks.

Apple (Malus × domestica Borkh.) is one of the most cultivated fruit crops in China. Apple trees frequently encounter waterlogging stress, mainly due to excess rainfall, soil compaction, or poor soil drainage, results in yellowing leaves and declined fruit quality and yield in some regions. However, the mechanism underlying the response to waterlogging has not been well elucidated. Therefore, we performed a physiological and transcriptomic analysis to examine the differential responses of two apple rootstocks (waterlogging-tolerant M. hupehensis and waterlogging-sensitive M. toringoides) to waterlogging stress. The results showed that M. toringoides displayed more severe leaf chlorosis during the waterlogging treatment than M. hupehensis. Compared with M. hupehensis, the more severe leaf chlorosis induced by waterlogging stress in M. toringoides was highly correlated with increased electrolyte leakage and superoxide radicals, hydrogen peroxide accumulation, and increased stomata closure. Interestingly, M. toringoides also conveyed a higher ethylene production under waterlogging stress. Furthermore, RNA-seq revealed that a total of 13,913 common differentially expressed genes (DEGs) were differentially regulated between M. hupehensis and M. toringoides under waterlogging stress, especially those DEGs involved in the biosynthesis of flavonoids and hormone signaling. This suggests a possible link of flavonoids and hormone signaling to waterlogging tolerance. Taken together, our data provide the targeted genes for further investigation of the functions, as well as for future molecular breeding of waterlogging-tolerant apple rootstocks.

Highly tough and elastic microspheric gel for transarterial catheter embolization in treatment of liver metastasis tumor.

Transarterial embolization is a widely recognized clinical treatment method for liver tumors. Given that the soft and easily damaged features of embolic particles may limit tumor embolization efficiency, the present study carries out an attempt of fabricating tough and elastic microspheric gel for promoting embolization efficiency. To promote the toughness of hydrogel, poly(ethylene glycol)-co-poly(ε-caprolactone)-co-poly(ethylene glycol) (PPP) and PPP with two terminal double bonds (PPPDA) are co-assembled into nano-micelles, which are connected with methacrylated chitosan (CSMA) to fabricate microspheric gels via microfluidic technology. Lowering double bond density of micelles promotes the freedom degree of micelles, significantly enhancing hydrogel toughness. To compensate for the strength loss caused by the decrease of double bond density of micelles, phytic acid (PA) are employed to interact with CS to form a physical network, further improving hydrogel strength and toughness. The CS-PPPDA&PPP-PA microspheric gels exhibit higher blocking effect in vitro. A rabbit VX2 liver metastasis tumor model is prepared to verify the embolization efficacy of CS-PPPDA&PPP-PA microspheric gels. Compared with clinical used microspheres, fewer CS-PPPDA&PPP-PA microspheric gels can achieve enough embolization efficiency. After embolization for 14 days, CS-PPPDA&PPP-PA microspheric gels exhibit improved tumor necrosis rate and promoted tumor cells apoptosis with reduced inflammation in surrounding tissues, confirming advanced embolic efficiency of tough microgels.

The engineered CRISPR-Mb2Cas12a variant enables sensitive and fast nucleic acid-based pathogens diagnostics in the field.

Existing CRISPR/Cas12a-based diagnostic platforms offer accurate and vigorous monitoring of nucleic acid targets, but have the potential to be further optimized for more efficient detection. Here, we profiled 16 Cas12a orthologs, focusing on their trans-cleavage activity and their potential as diagnostic enzymes. We observed the Mb2Cas12a has more robust trans-cleavage activity than other orthologs, especially at lower temperatures. An engineered Mb2Cas12a-RRVRR variant presented robust trans-cleavage activity and looser PAM constraints. Moreover, we found the existing one-pot assay, which simultaneously performed Recombinase Polymerase Amplification (RPA) and Cas12a reaction in one system, resulted in the loss of single-base discrimination during diagnosis. Therefore, we designed a reaction vessel that physically separated the RPA and Cas12a steps while maintaining a closed system. This isolated but closed system made diagnostics more sensitive and specific and effectively prevented contamination. This shelved Mb2Cas12a-RRVRR variant-mediated assay detected various targets in less than 15 min and exhibited equal or greater sensitivity than qPCR when detecting bacterial pathogens, plant RNA viruses and genetically modified crops. Overall, our findings further improved the efficiency of the current CRISPR-based diagnostic system and undoubtedly have great potential for highly sensitive and specific detection of multiple sample types.

Hyaluronic acid-g-lipoic acid granular gel for promoting diabetic wound healing.

Diabetic patients are prone to developing chronic inflammation after trauma and have persistent nonhealing wounds. Reactive oxygen species (ROS) and recurrent bacterial infections at the site of long-term wounds also further delay skin wound healing and tissue regeneration. In this study, a granular gel (which exhibits ROS scavenging and antibacterial properties) is fabricated based on hyaluronic acid-g-lipoic acid (HA-LA). Briefly, HA-LA is synthesized to fabricate HA-LA microgels, which are further assembled by Ag+ via its coordination effect with disulfide in dithiolane to form a granular gel. The extrudable bulk granular gel possesses a shear-thinning feature and is immediately restored to a solid state after extrusion, and this can be easily applied to the whole wound area. Therefore, the grafted LA not only allows for the construction of the granular gel but also removes excess ROS from the microenvironment. Additionally, the presence of Ag+ realizes the assembly of microgels and has antibacterial effects. In vivo experiments show that the HA-LA granular gel eliminates excessive ROS at the wound site and up-regulates the secretion of reparative growth factors, thus, accelerating common and diabetic wound healing significantly. Therefore, the ROS-scavenging granular gel that can be applied to the wound surface with chronic inflammation demonstrates strong clinical utility.

A "T.E.S.T." hydrogel bioadhesive assisted by corneal cross-linking for in situ sutureless corneal repair.

Corneal transplantation is an effective clinical treatment for corneal diseases, which, however, is limited by donor corneas. It is of great clinical value to develop bioadhesive corneal patches with functions of "Transparency" and "Epithelium & Stroma generation", as well as "Suturelessness" and "Toughness". To simultaneously meet the "T.E.S.T." requirements, a light-curable hydrogel is designed based on methacryloylated gelatin (GelMA), Pluronic F127 diacrylate (F127DA) & Aldehyded Pluronic F127 (AF127) co-assembled bi-functional micelles and collagen type I (COL I), combined with clinically applied corneal cross-linking (CXL) technology for repairing damaged cornea. The patch formed after 5 min of ultraviolet irradiation possesses transparent, highly tough, and strongly bio-adhesive performance. Multiple cross-linking makes the patch withstand deformation near 600% and exhibit a burst pressure larger than 400 mmHg, significantly higher than normal intraocular pressure (10-21 mmHg). Besides, the slower degradation than GelMA-F127DA&AF127 hydrogel without COL I makes hydrogel patch stable on stromal beds in vivo, supporting the regrowth of corneal epithelium and stroma. The hydrogel patch can replace deep corneal stromal defects and well bio-integrate into the corneal tissue in rabbit models within 4 weeks, showing great potential in surgeries for keratoconus and other corneal diseases by combining with CXL.

An anti-bacterial porous shape memory self-adaptive stiffened polymer for alveolar bone regeneration after tooth extraction.

The regeneration of alveolar bone after tooth extraction is critical for the placement of dental implants. Developing a rigid porous scaffold with defect shape adaptability is of great importance but challenging for alveolar bone regeneration. Herein, we design and synthesize a biocompatible poly(l-glutamic acid)-g-poly(ε-caprolactone) (PLGA-g-PCL) porous shape memory (SM) polymer. The PLGA-g-PCL is then copolymerized with acryloyl chloride grafted poly(ω-pentadecalactone) (PPDLDA) having a higher phase transition temperature than shape recovery temperature to maintain stiffness after shape recovery to resist chewing force. The hybrid polydopamine/silver/hydroxyapatite (PDA/Ag/HA) is coated to the surface of (PLGA-g-PCL)-PPDL scaffold to afford the anti-bacterial activity. The porous SM scaffold can be deformed into a compact size and administered into the socket cavity in a minimally invasive mode, and recover its original shape with a high stiffness at body temperature, fitting well in the socket defect. The SM scaffold exhibits robust antibacterial activity against Staphylococcus aureus (S. aureus). The porous microstructure and cytocompatibility of PLGA allow for the ingrowth and proliferation of stem cells, thus facilitating osteogenic differentiation. The micro-CT and histological analyses demonstrate that the scaffold boosts efficient new bone regeneration in the socket of rabbit mandibular first premolar. This porous shape memory self-adaptive stiffened polymer opens up a new avenue for alveolar bone regeneration.

A Simple and Efficient Strategy for Preparing a Cell-Spheroid-Based Bioink.

Cell spheroids are a promising bioprinting building block that can mimic several physiological conditions in embryonic development. However, it remains challenging to efficiently prepare cell-spheroid-based bioink (Sph-bioink) with favorable printability and spheroid fusion ability. In this work, a poly(N-isopropylacrylamide) (PNIPAAm)-based porous hydrogel is developed as an "all-in-one" platform for Sph-bioink preparation. On the one hand, the nonadhesive porous structure in hydrogels is an effective tool for fabricating adipose-derived stem cell (ASC) spheroids in high yield, and the hydrogel itself also serves as a "carrier" for conveniently transferring cell spheroids to the bioprinter. On the other hand, the integration of redox/thermo-responsiveness allows the hydrogel to shift from a solid spheroid-making tool to an extrudable bioprinting medium that is sensitive to temperature. These features enabled a simple procedure for preparing Sph-bioink, in which the cell spheroids were densely packed to retain fusion capability. The present study also demonstrates that ASC spheroids formed in hydrogels have good biological preservation and superior chondrogenic differentiation, and verified the feasibility of using Sph-bioink to build custom-shaped mature cartilage. In conclusion, this strategy provides a simple, efficient, and standardized approach for Sph-bioink preparation, making it possible to produce tissue-engineered constructs with accelerated maturation and functionalization.

Solid multifunctional granular bioink for constructing chondroid basing on stem cell spheroids and chondrocytes.

Stem cell spheroids are advanced building blocks to produce chondroid. However, the multi-step operations including spheroids preparation, collection and transfer, the following 3D printing and shaping limit their application in 3D printing. The present study fabricates an 'ALL-IN-ONE' bioink based on granular hydrogel to not only produce adipose derived stem cell (ASC) spheroids, but also realize the further combination of chondrocytes and the subsequent 3D printing. Microgels (6-10µm) grafted with ß-cyclodextrin (ß-CD) (MGß-CD) were assembled and crosslinked byin-situpolymerized poly (N-isopropylacrylamide) (PNIPAm) to form bulk granular hydrogel. The host-guest action between ß-CD of microgels and PNIPAm endows the hydrogel with stable, shear-thinning and self-healing properties. After creating caves, ASCs aggregate spontaneously to form numerous spheroids with diameter of 100-200µm inside the hydrogel. The thermosensitive porous granular hydrogel exhibits volume change under different temperature, realizing further adsorbing chondrocytes. Then, the granular hydrogel carrying ASC spheroids and chondrocytes is extruded by 3D printer at room temperature to form a tube, which can shrink at cell culture temperature to enhance the resolution. The subsequent ASC spheroids/chondrocytes co-culture forms cartilage-like tissue at 21 din vitro, which further matures subcutaneouslyin vivo, indicating the application potential of the fully synthetic granular hydrogel ink toward organoid culture.

A rice tubulin tyrosine ligase like 12 regulates phospholipase D activity and tubulin synthesis.

All plant α-tubulins encode a C-terminal tyrosine. An elusive tubulin tyrosine carboxypeptidase can cleave off, and a tubulin tyrosine ligase (TTL) re-ligate this tyrosine. The biological function of this cycle remains unclear but may correlate with microtubule stability. To get insight into the functional context of this phenomenon, we used cold-induced elimination of microtubules as experimental model. In previous work, we had analysed a rice TTL-like 12 (OsTTLL12), the only potential candidate of plant TTL. To follow the effect of OsTTLL12 upon microtubule responses in vivo, we expressed OsTTLL12-RFP into tobacco BY-2 cells stably overexpressing NtTUA3-GFP. We found that overexpression of OsTTLL12-RFP made microtubules disappear faster in response to cold stress, accompanied with more rapid Ca2+ influx, culminating in reduced cold tolerance. Treatment with different butanols indicated that α-tubulin detyrosination/tyrosination differently interacts with phospholipase D (PLD) dependent signalling. In fact, rice PLDα1 decorated microtubules and increased detyrosinated α-tubulin. Unexpectedly, overexpression of the two proteins (OsTTLL12-RFP, NtTUA3-GFP) mutually regulated the accumulation of their transcripts, leading us to a model, where tubulin detyrosination feeds back upon tubulin transcripts and defines a subset of microtubules for interaction with PLD dependent stress signalling.

Anti-microtubule activity of the traditional Chinese medicine herb Northern Ban Lan (Isatis tinctoria) leads to glucobrassicin.

Traditional Chinese medicine (TCM) belongs to the most elaborate and extensive systems of plant-based healing. The herb Northern Ban Lan (Isatis tinctoria) is famous for its antiviral and anti-inflammatory activity. Although numerous components isolated from I. tinctoria have been characterized so far, their modes of action have remained unclear. Here, we show that extracts from I. tinctoria exert anti-microtubular activity. Using time-lapse microscopy in living tobacco BY-2 (Nicotiana tabacum L. cv Bright Yellow 2) cells expressing green fluorescent protein-tubulin, we use activity-guided fractionation to screen out the biologically active compounds of I. tinctoria. Among 54 fractions obtained from either leaves or roots of I. tinctoria by methanol (MeOH/H2 O 8:2), or ethyl acetate extraction, one specific methanolic root fraction was selected, because it efficiently and rapidly eliminated microtubules. By combination of further purification with ultra-high-performance liquid chromatography and high-resolution tandem mass spectrometry most of the bioactivity could be assigned to the glucosinolate compound glucobrassicin. Glucobrassicin can also affect microtubules and induce apoptosis in HeLa cells. In the light of these findings, the antiviral activity of Northern Ban Lan is discussed in the context of microtubules being hijacked by many viral pathogens for cell-to-cell spread.

Mussel-Inspired Bisphosphonated Injectable Nanocomposite Hydrogels with Adhesive, Self-Healing, and Osteogenic Properties for Bone Regeneration.

Injectable hydrogels have received much attention because of the advantages of simulation of the natural extracellular matrix, microinvasive implantation, and filling and repairing of complex shape defects. Yet, for bone repair, the current injectable hydrogels have shown significant limitations such as the lack of tissue adhesion, deficiency of self-healing ability, and absence of osteogenic activity. Herein, a strategy to construct mussel-inspired bisphosphonated injectable nanocomposite hydrogels with adhesive, self-healing, and osteogenic properties is developed. The nano-hydroxyapatite/poly(l-glutamic acid)-dextran (nHA/PLGA-Dex) dually cross-linked (DC) injectable hydrogels are fabricated via Schiff base cross-linking and noncovalent nHA-BP chelation. The chelation between bisphosphonate ligands (alendronate sodium, BP) and nHA favors the uniform dispersion of the latter. Moreover, multiple adhesion ligands based on catechol motifs, BP, and aldehyde groups endow the hydrogels with good tissue adhesion. The hydrogels possess excellent biocompatibility and the introduction of BP and nHA both can effectively promote viability, proliferation, migration, and osteogenesis differentiation of MC3T3-E1 cells. The incorporation of BP groups and HA nanoparticles could also facilitate the angiogenic property of endothelial cells. The nHA/PLGA-Dex DC hydrogels exhibited considerable biocompatibility despite the presence of a certain degree of inflammatory response in the early stage. The successful healing of a rat cranial defect further proves the bone regeneration ability of nHA/PLGA-Dex DC injectable hydrogels. The developed tissue adhesive osteogenic injectable nHA/PLGA-Dex hydrogels show significant potential for bone regeneration application.

A rice tubulin tyrosine ligase-like 12 protein affects the dynamic and orientation of microtubules.

The detyrosination/retyrosination cycle is the most common post-translational modification of α-tubulin. Removal of the conserved C-terminal tyrosine of α-tubulin by a still elusive tubulin tyrosine carboxypeptidase, and religation of this tyrosine by a tubulin tyrosine ligase (TTL), are probably common to all eukaryotes. Interestingly, for plants, the only candidates qualifying as potential TTL homologs are the tubulin tyrosine ligase-like 12 proteins. To get insight into the biological functions of these potential TTL homologs, we cloned the rice TTL-like 12 protein (OsTTLL12) and generated overexpression OsTTLL12-RFP lines in both rice and tobacco BY-2 cells. We found, unexpectedly, that overexpression of this OsTTLL12-RFP increased the relative abundance of detyrosinated α-tubulin in both coleoptile and seminal root, correlated with more stable microtubules. This was independent of the respective orientation of cortical microtubule, and followed by correspondingly changing growth of coleoptiles and seminal roots. A perturbed organization of phragmoplast microtubules and disoriented cell walls were further characteristics of this phenotype. Thus, the elevated tubulin detyrosination in consequence of OsTTLL12 overexpression affects structural and dynamic features of microtubules, followed by changes in the axiality of cell plate deposition and, consequently, plant growth.

A bilayered scaffold with segregated hydrophilicity-hydrophobicity enables reconstruction of goat hierarchical temporomandibular joint condyle cartilage.

Temporomandibular joint (TMJ) supports chewing, talking or other daily oral activities. So far, it still remains a great challenge to treat the defected TMJ condyle cartilage through tissue engineering technology. Herein, a bilayered scaffold is designed to fully reconstruct the different cartilage matrices of TMJ condyle under same induction condition. The bilayered scaffold with segregated hydrophobicity-hydrophilicity in top and bottom layer is prepared from a low and high content of polyethylene glycol (PEG) crosslinked poly (L-glutamic acid)-g-polycaprolactone (PLGA-g-PCL). The hydrophobic aggregates in top layer support the adhesion and spread of bone mesenchymal stem cells (BMSCs), thus inducing the differentation towards fibrocartilage; while aggregates (spheroids) are formed on the hydrophlic bottom layer, showing a preferable hyaline differentiation pathway under same chondrogenic induction in vitro. After 14 d in vitro induction, the scaffold/BMSCs construct is implanted in goat TMJ condyle defects. The post-operative outcome after 2 months demonstrates that the defects are fully covered by neo-cartilage. And the regenerated hierarchical TMJ condyle cartilage perfectly consist of ordered fibrocartilage and hyaline cartilage, which is same as natural condyle cartilage. These results corroborate that this bilayered scaffold with segregated hydrophilicity-hydrophobicity carrying induced BMSCs is a promising for treatment of TMJ condyle cartilage defects.

Designer Hydrogel with Intelligently Switchable Stem-Cell Contact for Incubating Cartilaginous Microtissues.

Stem-cell-derived organoid can resemble in vivo tissue counterpart and mimic at least one function of tissue or organ, possessing great potential for biomedical application. The present study develops a hydrogel with cell-responsive switch to guide spontaneous and sequential proliferation and aggregation of adipose-derived stem cells (ASCs) without inputting artificial stimulus for in vitro constructing cartilaginous microtissues with enhanced retention of cell-matrix and cell-cell interactions. Polylactic acid (PLA) rods are surface-aminolyzed by cystamine, followed by being involved in the amidation of poly(( l-glutamic acid) and adipic acid dihydrazide (ADH) to form a hydrogel. Along with tubular pore formation in hydrogel after dissolution of PLA rods, aminolyzed PLA molecules with disulfide bonds on rod surfaces are covalently transferred to the tubular pore surfaces of poly(l-glutamic acid)/ADH hydrogel. Because PLA attaches cells, while poly(l-glutamic acid)/ADH hydrogel repels cells, ASCs are found to adhere and proliferate on the tubular pore surfaces of hydrogel first and then cleave disulfide bonds by secreting molecules containing thiol, thus inducing desorption of PLA molecules and leading to their spontaneous detachment and aggregation. Associated with chondrogenic induction by TGF-ß1 and IGF-1 in vitro for 28 days, the hydrogel as an all-in-one incubator produces well-engineered columnar cartilage microtissues from ASCs, with the glycosaminoglycans (GAGs) and collagen type II (COL II) deposition achieving 64 and 69% of those in chondrocytes pellet, respectively. The cartilage microtissues further matured in vivo for 8 weeks to exhibit extremely similar histological features and biomechanical performance to native hyaline cartilage. The GAGs and COL II content, as well as compressive modulus of the matured tissue show no significant difference with native cartilage. The designer hydrogel may hold a promise for long-term culture of other types of stem cells and organoids.

In Situ Precipitation of Cluster and Acicular Hydroxyapatite onto Porous Poly(γ-benzyl-l-glutamate) Microcarriers for Bone Tissue Engineering.

Bone tissue engineering scaffold based on microcarriers provides an effective approach for the repair of irregular bone defects. The implantation of microcarriers by injection can reduce surgical trauma and fill various irregular shaped bone defects. Microcarriers with porous structure and osteogenic properties have shown great potential in promoting the repair of bone defects. In this study, two kinds of hydroxyapatite/poly-(γ-benzyl-l-glutamate) (HA/PBLG) microcarriers were constructed by emulsion/in situ precipitation method and their structures and properties were studied. First, PBLG porous microcarriers were prepared by an emulsion method. Surface carboxylation of PBLG microcarriers was performed to promote the deposition of HA on PBLG microcarriers. Next, the modified porous PBLG microcarriers were used as the matrix, combined with the in situ precipitation method; the cluster HA and acicular HA were precipitated onto the surface of porous microcarriers in the presence of ammonia water and tri(hydroxymethyl)aminomethane (Tris) solution, respectively. The micromorphology, composition, and element distribution of the two kinds of microcarriers were characterized by TEM, SEM, and AFM. Adipose stem cells (ADSCs) were cultured on the cluster HA/PBLG and acicular HA/PBLG microcarriers, respectively. ADSCs could grow and proliferate normally on both kinds of microcarriers wherein the acicular HA/PBLG microcarriers were more favorable for early cell adhesion and showed a beneficial effect on mineralization and osteogenic differentiation of ADSCs. Successful healing of a rabbit femur defect verified the bone regeneration ability of acicular HA/PBLG microcarriers.