Identification of an Ultrathin Osteochondral Interface Tissue with Specific Nanostructure at the Human Knee Joint.

Cartilage adheres to subchondral bone via a specific osteochondral interface tissue where forces are transferred from soft cartilage to hard bone without conferring fatigue damage over a lifetime of load cycles. However, the fine structure and mechanical properties of the osteochondral interface tissue remain unclear. Here, we identified an ultrathin ∼20-30 µm graded calcified region with two-layered micronano structures of osteochondral interface tissue in the human knee joint, which exhibited characteristic biomolecular compositions and complex nanocrystals assembly. Results from finite element simulations revealed that within this region, an exponential increase of modulus (3 orders of magnitude) was conducive to force transmission. Nanoscale heterogeneity in the hydroxyapatite, coupled with enrichment of elastic-responsive protein-titin, which is usually present in muscle, endowed the osteochondral tissue with excellent mechanical properties. Collectively, these results provide novel insights into the potential design for high-performance interface materials for osteochondral interface regeneration.

"All-in-One" Gel System for Whole Procedure of Stem-Cell Amplification and Tissue Engineering.

Microsphere (MS)-based systems provides great advantages for cell expansion and transplantation due to their high surface-to-volume ratio and biomimetic environment. However, a MS-based system that includes cell attachment, proliferation, passage, harvest, cryopreservation, and tissue engineering together has not been realized yet. An "all-in-one" gel MS-based system is established for human adipose-derived mesenchymal stem cells (hADSCs), realizing real 3D culture with enhanced expansion efficiency and simplified serial cell culture operations, and construction of macrotissues with uniform cell distribution and specific function. A 3D digital light-processing technology is developed to fabricate gel MSs in an effective way. The printed MSs present a suitable environment with rough surface architecture and the mechanical properties of soft tissues, leading to high cell viability, attachment, proliferation, activity, and differentiation potential. Further, convenient standard operation procedures, including cell passage, detachment, and cryopreservation, are established for cell culture on the gel MSs. Finally, hADSCs-loaded gel MSs form macrotissues through a "bottom-up" approach, which demonstrates the potential applications for tissue engineering. These findings exhibit the feasibility and beauty of "all-in-one" stem cell culture and tissue engineering system.

Gel microspheres enhance the stemness of ADSCs by regulating cell-ECM interaction.

The gel microsphere culture system (GMCS) showed various advantages for mesenchymal stem cell (MSC) expansion and delivery, such as high specific surface area, small and regular shape, extensive adjustability, and biomimetic properties. Although various technologies and materials have been developed to promote the development of gel microspheres, the differences in the biological status of MSCs between the GMCS and the traditional Petri dish culture system (PDCS) are still unknown, hindering gel microspheres from becoming a culture system as widely used as petri dishes. In the previous study, an excellent "all-in-one" GMCS has been established for the expansion of human adipose-derived MSCs (hADSCs), which showed convenient cell culture operation. Here, we performed transcriptome and proteome sequencing on hADSCs cultured on the "all-in-one" GMCS and the PDCS. We found that hADSCs cultured in the GMCS kept in an undifferentiation status with a high stemness index, whose transcriptome profile is closer to the adipose progenitor cells (APCs) in vivo than those cultured in the PDCS. Further, the high stemness status of hADSCs in the GMCS was maintained through regulating cell-ECM interaction. For application, bilayer scaffolds were constructed by osteo- and chondro-differentiation of hADSCs cultured in the GMCS and the PDCS. The effect of osteochondral regeneration of the bilayer scaffolds in the GMCS group was better than that in the PDCS group. This study revealed the high stemness and excellent functionality of MSCs cultured in the GMCS, which promoted the application of gel microspheres in cell culture and tissue regeneration.

Seamless and early gap healing of osteochondral defects by autologous mosaicplasty combined with bioactive supramolecular nanofiber-enabled gelatin methacryloyl (BSN-GelMA) hydrogel.

Autologous mosaicplasty is a common approach used to treat osteochondral defects in clinical practice. Gap integration between host and transplanted plugs requires bone tissue reservation and hyaline cartilage regeneration without uneven surface, graft necrosis and sclerosis. However, poor gap integration is a serious concern, which eventually leads to deterioration of joint function. To deal with such complications, this study has developed a strategy to effectively enhance integration of the gap region following mosaicplasty by applying injectable bioactive supramolecular nanofiber-enabled gelatin methacryloyl (GelMA) hydrogel (BSN-GelMA). A rabbit osteochondral defect model demonstrated that BSN-GelMA achieved seamless osteochondral healing in the gap region between plugs of osteochondral defects following mosaicplasty, as early as six weeks. Moreover, the International Cartilage Repair Society score, histology score, glycosaminoglycan content, subchondral bone volume, and collagen II expression were observed to be the highest in the gap region of BSN-GelMA treated group. This improved outcome was due to bio-interactive materials, which acted as tissue fillers to bridge the gap, prevent cartilage degeneration, and promote graft survival and migration of bone marrow mesenchymal stem cells by releasing bioactive supramolecular nanofibers from the GelMA hydrogel. This study provides a powerful and applicable approach to improve gap integration after autologous mosaicplasty. It is also a promising off-the-shelf bioactive material for cell-free in situ tissue regeneration.

Mesenchymal Stem Cells-Hydrogel Microspheres System for Bone Regeneration in Calvarial Defects.

The repair of large bone defects in clinic is a challenge and urgently needs to be solved. Tissue engineering is a promising therapeutic strategy for bone defect repair. In this study, hydrogel microspheres (HMs) were fabricated to act as carriers for bone marrow mesenchymal stem cells (BMSCs) to adhere and proliferate. The HMs were produced by a microfluidic system based on light-induced gelatin of gelatin methacrylate (GelMA). The HMs were demonstrated to be biocompatible and non-cytotoxic to stem cells. More importantly, the HMs promoted the osteogenic differentiation of stem cells. In vivo, the ability of bone regeneration was studied by way of implanting a BMSC/HM system in the cranial defect of rats for 8 weeks. The results confirmed that the BMSC/HM system can induce superior bone regeneration compared with both the HMs alone group and the untreated control group. This study provides a simple and effective research idea for bone defect repair, and the subsequent optimization study of HMs will provide a carrier material with application prospects for tissue engineering in the future.

Grey Correlation Analysis of the Durability of Steel Fiber-Reinforced Concrete under Environmental Action.

The interface performance of steel fiber-reinforced concrete (SFRC) is a critical factor in determining mechanical properties and durability. The degradation of the concrete matrix and micro-structure interface is caused by environmental erosion, which shortens the service life of the structure design. Considering different volume contents of steel fiber (0%, 1%, 2%), the failure mechanism of SFRC under different environmental erosion conditions was studied through a laboratory test scheme. A total of six environmental factors are selected, including water, sodium chloride solution, sodium sulfate solution, dilute sulfuric acid solution, sodium hydroxide solution, and a freeze-thaw cycle. When subjected to different erosion concentrations and periods, micro-structure and axial bearing capacity deterioration laws are compared and analyzed. A durability equation related to fiber mixture ratio and strength is presented based on the experimental data and the numerical simulation method. The influence of different environments on steel fiber-reinforced concrete is analyzed, and the grey correlation degree of axial compressive strength is analyzed. The experimental results show that steel fiber can effectively improve the concrete axial bearing capacity, but different responses are observed under the various erosion conditions. A freeze-thaw cycle environment has the most significant impact on the axial compressive strength of concrete, followed by the sulfuric acid environment, and other environments have a weaker impact. The research results will provide a theoretical basis for predicting the performance deterioration of SFRC concerning other erosion conditions and periods.

"Musical dish" efficiently induces osteogenic differentiation of mesenchymal stem cells through music derived microstretch with variable frequency.

Nonuniform microstretching (NUMS) naturally occurs in real bone tissues in vivo, but its profound effects have not been identified yet. In order to explore the biological effects of NUMS and static stretch (uniform stretch [US]) on cells, a new "musical dish" device was developed. Musical signal was used to provide NUMS to cells. More stress fibers, arranging along the long axis of cells, were formed throughout the cells under NUMS, compared with US and untreated control group, although cell morphology did not show any alteration. Whole transcriptome sequencing revealed enhanced osteogenic differentiation of cells after NUMS treatment. Cells in the NUMS group showed a higher expression of bone-related genes, while genes related to stemness and other lineages were down-regulated. Our results give insights into the biological effects of NUMS and US on stem cell osteogenic differentiation, suggesting beneficial effects of micromechanical stimulus for osteogenesis. The newly developed device provides a basis for the development of NUMS derived rehabilitation technology to promote bone healing.

In-cytoplasm mitochondrial transplantation for mesenchymal stem cells engineering and tissue regeneration.

Stem cell therapies are unsatisfactory due to poor cell survival and engraftment. Stem cell used for therapy must be properly "tuned" for a harsh in vivo environment. Herein, we report that transfer of exogenous mitochondria (mito) to adipose-derived mesenchymal stem cells (ADSCs) can effectively boost their energy levels, enabling efficient cell engraftment. Importantly, the entire process of exogeneous mitochondrial endocytosis is captured by high-content live-cell imaging. Mitochondrial transfer leads to acutely enhanced bioenergetics, with nearly 17% of higher adenosine 5'-triphosphate (ATP) levels in ADSCs treated with high mitochondrial dosage and further results in altered secretome profiles of ADSCs. Mitochondrial transfer also induced the expression of 334 mRNAs in ADSCs, which are mainly linked to signaling pathways associated with DNA replication and cell division. We hypothesize that increase in ATP and cyclin-dependent kinase 1 and 2 expression might be responsible for promoting enhanced proliferation, migration, and differentiation of ADSCs in vitro. More importantly, mito-transferred ADSCs display prolonged cell survival, engraftment and horizontal transfer of exogenous mitochondria to surrounding cells in a full-thickness skin defect rat model with improved skin repair compared with nontreated ADSCs. These results demonstrate that intracellular mitochondrial transplantation is a promising strategy to engineer stem cells for tissue regeneration.

Advanced hydrogels for the repair of cartilage defects and regeneration.

Cartilage defects are one of the most common symptoms of osteoarthritis (OA), a degenerative disease that affects millions of people world-wide and places a significant socio-economic burden on society. Hydrogels, which are a class of biomaterials that are elastic, and display smooth surfaces while exhibiting high water content, are promising candidates for cartilage regeneration. In recent years, various kinds of hydrogels have been developed and applied for the repair of cartilage defects in vitro or in vivo, some of which are hopeful to enter clinical trials. In this review, recent research findings and developments of hydrogels for cartilage defects repair are summarized. We discuss the principle of cartilage regeneration, and outline the requirements that have to be fulfilled for the deployment of hydrogels for medical applications. We also highlight the development of advanced hydrogels with tailored properties for different kinds of cartilage defects to meet the requirements of cartilage tissue engineering and precision medicine.

Tissue-Adhesive Paint of Silk Microparticles for Articular Surface Cartilage Regeneration.

Current biomaterials and tissue engineering techniques have shown a promising efficacy on full-thickness articular cartilage defect repair in clinical practice. However, due to the difficulty of implanting biomaterials or tissue engineering constructs into a partial-thickness cartilage defect, it remains a challenge to provide a satisfactory cure in joint surface regeneration in the early and middle stages of osteoarthritis. In this study, we focused on a ready-to-use tissue-adhesive joint surface paint (JS-Paint) capable of promoting and enhancing articular surface cartilage regeneration. The JS-Paint is mainly composed of N-(2-aminoethyl)-4-(4-(hydroxymethyl)-2-methoxy-5-nitrosophenoxy) butanamide (NB)-coated silk fibroin microparticles and possess optimal cell adhesion, migration, and proliferation properties. NB-modified silk fibroin microparticles can directly adhere to the cartilage and form a smooth layer on the surface via the photogenerated aldehyde group of NB reacting with the -NH2 groups of the cartilage tissue. JS-Paint treatment showed a significant promotion of cartilage regeneration and restored the smooth joint surface at 6 weeks postsurgery in a rabbit model of a partial-thickness cartilage defect. These findings revealed that silk fibroin can be utilized to bring about a tissue-adhesive paint. Thus, the JS-Paint strategy has some great potential to enhance joint surface regeneration and revolutionize future therapeutics of early and middle stages of osteoarthritis joint ailments.

Single-Cell Profiles and Clinically Useful Properties of Human Mesenchymal Stem Cells of Adipose and Bone Marrow Origin.

BACKGROUND: Mesenchymal stem cells (MSCs) can be isolated from various tissues and can present themselves as a promising cell source for cell-based therapies. Although adipose- and bone marrow-derived mesenchymal stem cells have already been used in a considerable number of clinical trials for osteoarthritis treatment, systematic analyses from single- to bulk-cell resolution as well as clinical outcomes of these 2 MSCs are still insufficient. PURPOSE: To explore the characteristics and differences of adipose-derived stem cells (ADSCs) and bone marrow MSCs (BMSCs) at single- and bulk-cell levels, to study the clinical outcomes of these 2 cells on the treatment of osteoarthritis, and to provide potential guidance on the more precise clinical application of these MSCs. STUDY DESIGN: Controlled laboratory study and meta-analysis. METHODS: Same donor-derived ADSCs and BMSCs were isolated and cultured. Single- and bulk-cell assays were used to identify the characteristics of these 2 cells. Meta-analysis of clinical trials was done to compare the clinical therapeutic effects in osteoarthritis treatment with ADSCs and BMSCs. RESULTS: Single-cell RNA sequencing analysis showed that the population of ADSCs showed lower transcriptomic heterogeneity when compared with BMSCs. Additionally, as compared with BMSCs, ADSCs were less dependent on mitochondrial respiration for energy production. Furthermore, ADSCs had a lower expression level of human leukocyte antigen class I antigen and higher immunosuppression capacity when compared with the BMSC population. Meta-analysis of current clinical trials of osteoarthritis treatment with MSCs consistently showed that ADSCs are more stable than BMSCs in their therapeutic effect. CONCLUSION: These results provide basic biological insights into human ADSCs and BMSCs at the single-cell resolution. Findings indicated that ADSCs may be a more controllable stem cell source, may be more adaptable to surviving in the hypoxic articular cavity niche, and may exhibit superiority in regulating inflammation. Based on the meta-analysis results of the different characteristics of ADSCs and BMSCs, ADSCs were implicated as being a better cell source for osteoarthritis treatment. CLINICAL RELEVANCE: These results guide a more precise clinical application of adipose and bone marrow mesenchymal stem cells.

Genome-Wide Determination of Gene Essentiality by Transposon Insertion Sequencing in Yeast Pichia pastoris.

In many prokaryotes but limited eukaryotic species, the combination of transposon mutagenesis and high-throughput sequencing has greatly accelerated the identification of essential genes. Here we successfully applied this technique to the methylotrophic yeast Pichia pastoris and classified its conditionally essential/non-essential gene sets. Firstly, we showed that two DNA transposons, TcBuster and Sleeping beauty, had high transposition activities in P. pastoris. By merging their insertion libraries and performing Tn-seq, we identified a total of 202,858 unique insertions under glucose supported growth condition. We then developed a machine learning method to classify the 5,040 annotated genes into putatively essential, putatively non-essential, ambig1 and ambig2 groups, and validated the accuracy of this classification model. Besides, Tn-seq was also performed under methanol supported growth condition and methanol specific essential genes were identified. The comparison of conditionally essential genes between glucose and methanol supported growth conditions helped to reveal potential novel targets involved in methanol metabolism and signaling. Our findings suggest that transposon mutagenesis and Tn-seq could be applied in the methylotrophic yeast Pichia pastoris to classify conditionally essential/non-essential gene sets. Our work also shows that determining gene essentiality under different culture conditions could help to screen for novel functional components specifically involved in methanol metabolism.