Bioengineered Dual-Targeting Protein Nanocage for Stereoscopical Loading of Synergistic Hydrophilic/Hydrophobic Drugs to Enhance Anticancer Efficacy

A biocompatible and modifiable protein nanocarrier is a promising candidate for tumor targeted drug delivery. However, it is challenging to effectively load hydrophobic drugs, not to mention to upload both hydrophilic and hydrophobic drugs on one protein nanocarrier. Here, an amphiphilic multi-drug loading protein nanocage (Am-PNCage) is presented which is generated by replacing the fifth helix of human H-ferritin (HFn) subunit with a functional motif composed of hydrophobic–hydrophilic-RGD peptides. The Am-PNCage possesses a dual targeting property resulting from the intrinsic CD71 targeting ability of HFn and the integrin α vβ3 targeting ability of displayed RGD peptides. Through the hydrophilic drug entry channel in the protein nanocage and hydrophobic peptides displayed on the outer surface, amphiphilic epirubicin (132)/camptothecin (50) are stereoscopically loaded into the inner cavity/outer protein shell, respectively, for one Am-PNCage, exhibiting cascade drug release pattern. The dual-targeted Am-PNCage promotes the loaded drugs penetrating various 3D tumor models in vitro, as well as traversing the brain blood barrier and accumulating in brain tumors in vivo. Moreover, the drug loaded Am-PNCage shows reduced side effects and significantly enhances synergistic efficacy against brain tumor, metastatic liver cancers, and drug resistant breast tumor. Thus, the Am-PNCage represents a novel promising protein nanocarrier for targeted combination chemotherapy.     

Injection and Self‐Assembly of Bioinspired Stem Cell‐Laden Gelatin/Hyaluronic Acid Hybrid Microgels Promote Cartilage Repair In Vivo

In this paper, a novel bioinspired stem cell‐laden microgel and related in vivo cartilage repair strategy are proposed. In particular, herein the preparation of new stem cell‐laden microgels, which can be injected into the chondral defect site in a minimally invasive way, and more importantly, capable of in situ self‐assembly into 3D macroporous scaffold without external stimuli, is presented. Specifically, thiolated gelatin (Gel‐SH) and vinyl sulfonated hyaluronic acid (HA‐VS) are first synthesized, and then stem cell‐laden gelatin/hyaluronic acid hybrid microgels (Gel‐HA) are generated by mixing Gel‐SH, HA‐VS, and bone mesenchymal stem cells (BMSCs) together via droplet‐based microfluidic approach, followed by gelation through fast and efficient thiol‐Michael addition reaction. The encapsulated BMSCs show high viability, proliferation, and chondrogenic differentiation potential in the microgels. Moreover, the in vitro test proves that BMSC‐laden Gel‐HA microgels are injectable without sacrificing BMSC viability, and more importantly, can self‐assemble into cartilage‐like scaffolds via cell–cell interconnectivity. In vivo experiments further confirm that the self‐assembled microgels can inhibit vascularization and hypertrophy. The Gel‐HA microgels and relevant cartilage repair strategy, i.e., injecting BMSC‐laden microgels separately and reconstructing chondral defect structure by microgel self‐assembly, provides a simple and effective method for cartilage tissue engineering and regenerative medicine.     

Interaction of Human Mesenchymal Stem Cells with Soft Nanocomposite Hydrogels Based on Polyethylene Glycol and Dendritic Polyglycerol

Keeping the stemness of human mesenchymal stem cells (hMSCs) and their adipocyte differentiation potential is critical for clinical use. However, these features are lost on traditional substrates. hMSCs have often been studied on stiff materials whereas culturing hMSCs in their native niche increases their potential. Herein, a patterned hydrogel nanocomposite with the stiffness of liver tissues is obtained without any molding process. To investigate hMSCs' mechanoresponse to the material, the RGD spacing units and the stiffness of the hydrogels are dually tuned via the linker length. This work suggests that hMSCs' locomotion is influenced by the nature of the hydrogel layer (bulk or thin film). Contrary to on bulk surfaces, cell traction occurs during cell spreading on thin films. In addition, hMSCs' spreading behavior varies from shorter to longer linker‐based hydrogels, where on both surfaces hMSCs maintains their stemness as well as their adipogenic differentiation potential with a higher number of adipocytes for nanocomposites with a longer polymer linker. Overall, this work addresses the need for a new alternative for hMSCs culture allowing the cells to differentiate exclusively into adipocytes. This material represents a cell‐responsive platform with a tissue‐mimicking architecture given by the mechanical and morphological properties of the hydrogel.     

Tracking of Transplanted Human Mesenchymal Stem Cells in Living Mice using Near‐Infrared Ag2S Quantum Dots

Stem cell therapeutics has emerged as a novel regenerative therapy for tissue repair in the last decade. However, dynamically tracking the transplanted stem cells in vivo remains a grand challenge for stem cell‐based regeneration medicine in full understanding the function and the fate of the stem cells. Herein, Ag₂S quantum dots (QDs) in the second near‐infrared window (NIR‐II, 1.0–1.4 μm) are employed for dynamically tracking of human mesenchymal stem cells (hMSCs) in vivo with high sensitivity and high spatial and temporal resolution. As few as 1000 Ag₂S QDs‐labeled hMSCs are detectable in vivo and their fluorescence intensity can maintain up to 30 days. The in situ translocation and dynamic distribution of transplanted hMSCs in the lung and liver can be monitored up to 14 days with a temporal resolution of 100 ms. The in vivo high‐resolution imaging indicates the heparin‐facilitated translocation of hMSCs from lung to liver as well as the long‐term retention of hMSCs in the liver contribute to the treatment of liver failure. The novel NIR‐II imaging offers a possibility of tracking stem cells in living animals with both high spatial and temporal resolution, and encourages the future clinical applications in imaging‐guided cell therapies.     

Integrated and Bifunctional Bilayer 3D Printing Scaffold for Osteochondral Defect Repair

Bioinspired scaffolds with two distinct regions resembling stratified anatomical architecture provide potential strategies for osteochondral defect repair and are studied in preclinical animals. However, delamination of the two layers often causes tissue disjunction between the regenerated cartilage and subchondral bone, leading to few commercially available clinical applications. This study develops an integrated poly(ε-caprolactone) (PCL)-based scaffold for repairing osteochondral defects. An extracellular matrix (ECM)-incorporated 3D printing composite scaffold (ECM/PCL) coated with ECM hydrogel (E-co-E/PCL) is fabricated as the upper layer, and magnesium oxide nanoparticles coated with polydopamine (MgO@PDA)-incorporated composite scaffold (MD/PCL) is fabricated using 3D printing as the bottom layer. The physicochemical and mechanical properties of the bilayer scaffold meet the requirements in designing and fabricating the osteochondral scaffold, especially a strong interface possessed between the two layers. By in vitro study, the integrated scaffold stimulates proliferation, chondrogenic differentiation, and osteogenic differentiation of human bone mesenchymal stem cells. Moreover, the integrated bilayer scaffold exhibits well repair ability to facilitate simultaneous regeneration of cartilage and subchondral bone after implanting into the osteochondral defect in rats. In addition, cartilage “tidemarks” completely regenerated after 12 weeks of implantation of the bilayer scaffold, which indicates no tissue disjunctions formed between the regenerated cartilage and subchondral bone.     

Unfolding the Mystery Behind the Onset of Chondrocyte Hypertrophy during Chondrogenesis: Toward Designing Advanced Permanent Cartilage-mimetic Biomaterials

Successful recapitulation of the anatomical microarchitecture and biomechanics of the native articular cartilage under in vitro culture conditions is still an elusive topic of research. The major roadblock lies in maintaining the stable chondrogenic phenotype in vivo or under long-term in vitro conditions. Tissue engineers worldwide has coined this aberrant loss of permanent cartilage characteristics to transient cartilage form as “chondrocyte hypertrophy”. Although the following has been validated through the expression of a few known markers but very little is understood regarding the molecular mechanism that dwells underneath. This review summarizes the precise aetiology behind the development and progression of the hypertrophic phenotype in chondrocytes under in vitro chondrogenic conditions. Based on the current literature survey, it is deciphered that the type of cell utilized (chondrocytes or stem cells), the chondrogenic culture conditions (growth factors/biochemical mediators) and the culture microenvironment (oxygen tension, mechanical loading) during chondrogenesis have a direct correlation with the dysregulated activity of the chondrogenic signaling pathways corroborating the onset of hypertrophic maturation of chondrocytes. Furthermore, it is critically analyzed whether to completely inhibit these hypertrophy-inducing signaling pathways or apply a brake in terms of time-dependent dose due to their functional duality role in chondrogenesis.     

Three Dimensional Graphene Foam/Polymer Hybrid as a High Strength Biocompatible Scaffold

Graphene foam (GrF)/polylactic acid–poly‐ε‐caprolactone copolymer (PLC) hybrid (GrF‐PLC) scaffold is synthesized in order to utilize both the desirable properties of graphene and that of foams such as excellent structural characteristics and a networked 3‐D structure for cells to proliferate in. The hybrid scaffold is synthesized by a dip‐coating method that enables retention of the porous 3D structure. The excellent wettability of PLC with graphene foam along with the formation of PLC bridges leads to a ≈3700% enhancement in strength and a ≈3100% increase in ductility in the GrF‐PLC scaffold. Biocompatibility of both graphene foam and GrF‐PLC scaffold is demonstrated by culturing of human mesenchymal stem cells (hMSCs) for 28 days, a period over which cell proliferation is robust. The hMSCs are differentiated in chondrogenic media and supported chondrogenesis in both scaffolds. The demand for aggrecan extracellular matrix protein synthesis is reduced in hybrids due to improved bearing of cell‐induced loads, this may be critical for ensuring adequate cellular distribution and layering of extracellular matrix. Hence, the unique mechanical and biotolerant properties of the GrF‐PLC scaffold are suited for musculoskeletal tissue engineering applications, such as the growth of de novo cartilage to replace cartilage lost due to injury or osteoarthritis.     

人骨髓间充质干细胞的分离培养及其形态学特点

利用Percoll梯度分离、贴壁筛选法及单克隆培养法分离、培养人骨髓间充质干细胞（mesenehymal stem cells,MSCs）;采用流式细胞术检测MSCs免疫学表型;采用细胞组织化学和免疫荧光技术观察MSCs的一般形态学特点;利用透射电镜和扫描电镜观察MSCs的超微结构。结果表明,分离、培养出高度同源性的MSCs具有独特的细胞免疫学表型,即CD73、CD105、CD44、CD166、CD90阳性,CD34、CD31、CD45阴性;细胞组织化学MSCs PAS-过碘酸雪夫氏染色阳性,苏丹黑-B（SB）及碱性磷酸酶（AKP）染色阴性;免疫荧光见细胞CD73、CD105阳性;透射电镜下MSCs表面有微绒毛,相邻细胞间有缝隙连接,细胞器丰富;扫描电镜下MSCs呈长梭形、鱼群状排列,表面有许多微绒毛。实验结果为MSCs的诱导分化及应用提供了形态学基础。     

Dissociated and Reconstituted Cartilage Microparticles in Densified Collagen Induce Local hMSC Differentiation

Current use of decellularized articular cartilage as a regenerative platform suffers from limited implant diffusion characteristics and cellular infiltration. Attempts to address this concern using decellularized cartilage microparticles allow for customized implant shape, tailored porosity, and improved cell infiltration. However, these developments utilize severe crosslinking agents that adversely affect cell differentiation, and fail to attain clinically relevant mechanical properties required for the implant survival. These issues have been overcome through the formation of a composite approach, combining the advantages of mature, decellularized tissue with tunable features of a reconstituted collagen hydrogel system. Through the application of a plastic compression regime, cellularized composite structures are formed that exceeded the percolation threshold of the cartilage microparticles and exhibited clinically relevant mechanical properties. Chemical reduction and mechanical reconstitution methods to investigate the contributions of glycosaminoglycan and collagenous components to chondrogenic induction and matrix properties have been utilized. With the inclusion of human mesenchymal stem cells into the composite system, microenvironment‐dependent cell morphology and phenotype when in contact with cartilage microparticles are shown. This work demonstrates a cartilage microparticle composite matrix with clinically relevant mechanical properties, and chondrogenic differentiation of human mesenchymal stem that infiltrate both native and chemically reduced cartilage microparticles.     

Protamine Functionalized Single‐Walled Carbon Nanotubes for Stem Cell Labeling and In Vivo Raman/Magnetic Resonance/Photoacoustic Triple‐Modal Imaging

Stem cells have shown great potential in regenerative medicine and attracted tremendous interests in recent years. Sensitive and reliable methods for stem cell labeling and in vivo tracking are thus urgently needed. Here, a novel approach to label human mesenchymal stem cells (hMSCs) with single‐walled carbon nanotubes (SWNTs) for in vivo tracking by triple‐modal imaging is presented. It is shown that polyethylene glycol (PEG) functionalized SWNTs conjugated with protamine (SWNT‐PEG‐PRO) exhibit extremely efficient cell entry into hMSCs, without affecting their proliferation and differentiation. The strong inherent resonance Raman scattering of SWNTs is used for in vitro and in vivo Raman imaging of SWNT‐PEG‐PRO‐labeled hMSCs, enabling ultrasensitive in vivo detection of as few as 500 stem cells administrated into mice. On the other hand, the metallic catalyst nanoparticles attached on nanotubes can be utilized as the T2‐contrast agent in magnetic resonance (MR) imaging of SWNT‐labeled hMSCs. Moreover, in vivo photoacoustic imaging of hMSCs in mice is also demonstrated. The work reveals that SWNTs with appropriate surface functionalization have the potential to serve as multifunctional nanoprobes for stem cell labeling and multi‐modal in vivo tracking.     

One‐Step Covalent Immobilization of β‐Cyclodextrin on sp2 Carbon Surfaces for Selective Trace Amount Probing of Guests

The modification of solid surfaces with supramolecular hosts is a powerful method to tailor interfacial properties and confer chemical selectivity, but often involves multistep protocols that hinder facile upscaling. Here, the one‐step covalent modification of highly oriented pyrolytic graphite (HOPG) with a β‐cyclodextrin (β‐CD) derivative, which efficiently forms inclusion complexes with hydrophobic guests of suitable size, is demonstrated. The grafted β‐CD‐HOPG surface is investigated toward electrochemical detection of ferrocene and dopamine. The enrichment of the analytes at the electrode surface, through inclusion in β‐CD, leads to an enhanced electrochemical response and an improved detection limit. Furthermore, the modified β‐CD‐HOPG electrode discriminates analytes that form host–guest complexes with β‐CD against a 100‐fold higher background of electroactive substances that do not. Atomic force microscopy, scanning tunneling microscopy, and Raman spectroscopy confirm the covalent nature of the modification and reveal high stability toward solvent rinsing, ultrasonication, and temperatures up to 140 °C. The one‐step covalent modification therefore holds substantial promise for the routine production of inexpensive, yet robust and highly performant electrochemical sensors. Beyond electrochemical sensor development, our strategy is valuable to prepare materials where accurate spatial positioning of functional units and efficient current collection are crucial, e.g. in photoelectrodes or electrocatalysts.     

3D Superelastic Scaffolds Constructed from Flexible Inorganic Nanofibers with Self‐Fitting Capability and Tailorable Gradient for Bone Regeneration

Repair of bone defects with irregular shapes or at soft tissue insertion sites faces a huge challenge. Scaffolds capable of adapting to bone cavities, generating stiffness gradients, and inducing osteogenesis are necessary. Herein, a superelastic 3D ceramic fibrous scaffold is developed by assembly of intrinsically rigid, structurally flexible electrospun SiO₂ nanofibers with chitosan as bonding sites (SiO₂ NF‐CS) via a lyophilization technique. SiO₂ NF‐CS scaffolds exhibit excellent elasticity (full recovery from 80% compression), fast recovery rate (>500 mm min^?1), and good fatigue resistance (>10 000 cycles of compression) in an aqueous medium. SiO₂ NF‐CS scaffolds induce human mesenchymal stem cell (hMSC) elongation and differentiation into osteoblasts. In vivo self‐fitting capability is demonstrated by implanting compressed SiO₂ NF‐CS scaffolds into different shaped mandibular defects in rabbits, with a spontaneous recovery and full filling of defects. Rat calvarial defect repair validates enhanced bone formation and vascularization by cell (hMSC) histomorphology analysis. Further, subchondral bone scaffolds with gradations in SiO₂ nanofibers are developed, leading to a stiffness gradient and spatially chondrogenic and osteogenic differentiation of hMSCs. This work presents a type of 3D ceramic fibrous scaffold, which can closely match bone defects with irregular shapes or at different implant sites, and is promising for clinical translation.     

Multifunctional Shell–Core Nanoparticles for Treatment of Multidrug Resistance Hepatocellular Carcinoma

Multidrug resistance (MDR) is the main obstruction against the chemotherapy for hepatocellular carcinoma. Herein, a biodegradable multifunctional tumor‐targeted core–shell structural nanocarrier (RGD peptide functionalized nanoparticles, RGD‐NPs) is reported for treating MDR hepatocellular carcinoma, which consists of three components: pH‐triggered calcium phosphate shell, long circulation phosphatidylserine‐polyethylene glycol (PS‐PEG) core, and an active targeting ligand RGD peptide. Drug‐resistance inhibitor (verapamil, VER) and chemotherapeutic agent (mitoxantrone, MIT) are separately encapsulated into the outer shell layer and inner core layer to obtain VER and MIT loaded RGD‐NPs (VM‐RGD‐NPs). Due to the shell–core structure, the VER and MIT can release sequentially, thus synergistically weakening the efflux effect to MIT by MDR cells. Also, the calcium phosphate can trigger lysosomal escaping through the varied pH value. Together with the optimized internalization pathway in MDR tumor cells, the increased intracellular effective chemotherapeutic drug concentration can be realized, thus achieving the improved curative effect. In this system, the PEG extends the circulation time in vivo. Also, the peptide RGD distinctly increases the affinity to MDR tumors with respect to nontargeted nanoparticles. As a consequence, VM‐RGD‐NPs exhibit a significant synergistic effect toward the MDR hepatocellular carcinoma, providing a promising therapeutic approach for MDR tumor.     

3D Printing of Bilineage Constructive Biomaterials for Bone and Cartilage Regeneration

Owing to the different biological properties of articular cartilage and subchondral bone, it remains significant challenge to construct a bi‐lineage constructive scaffold. In this study, manganese (Mn)‐doped β‐TCP (Mn‐TCP) scaffolds with varied Mn contents are prepared by a 3D‐printing technology. The effects of Mn on the physicochemical properties, bioactivity, and corresponding mechanism for stimulating osteochondral regeneration are systematically investigated. The incorporation of Mn into β‐TCP lowers the lattices parameters and crystallization temperatures, but improves the scaffold density and compressive strength. The ionic products from Mn‐TCP significantly improve the proliferation of both rabbit chondrocytes and mesenchymal stem cells (rBMSCs), as well as promote the differentiation of chondrocytes and rBMSCs. The in vivo study shows that Mn‐TCP scaffolds distinctly improve the regeneration of subchondral bone and cartilage tissues as compared to TCP scaffolds, upon transplantation in rabbit osteochondral defects for 8 and 12 weeks. The mechanism is closely related to the Mn²⁺ ions significantly stimulated the proliferation and differentiation of chondrocytes through activating HIF pathway and protected chondrocytes from the inflammatory osteoarthritis environment by activating autophagy. These findings suggest that 3D‐printing of Mn‐containing scaffolds with improved physicochemical properties and bilineage bioactivities represents an intelligent strategy for regenerating osteochondral defects.     

Conjugated Polymer Nanodots as Ultrastable Long‐Term Trackers to Understand Mesenchymal Stem Cell Therapy in Skin Regeneration

Stem cell–based therapies hold great promise in providing desirable solutions for diseases that cannot be effectively cured by conventional therapies. To maximize the therapeutic potentials, advanced cell tracking probes are essential to understand the fate of transplanted stem cells without impairing their properties. Herein, conjugated polymer (CP) nanodots are introduced as noninvasive fluorescent trackers with high brightness and low cytotoxicity for tracking of mesenchymal stem cells (MSCs) to reveal their in vivo behaviors. As compared to the most widely used commercial quantum dot tracker, CP nanodots show significantly better long‐term tracking ability without compromising the features of MSCs in terms of proliferation, migration, differentiation, and secretome. Fluorescence imaging of tissue sections from full‐thickness skin wound–bearing mice transplanted with CP nanodot‐labeled MSCs suggests that paracrine signaling of the MSCs residing in the regenerated dermis is the predominant contribution to promote skin regeneration, accompanied with a small fraction of endothelial differentiation. The promising results indicate that CP nanodots could be used as next generation of fluorescent trackers to reveal the currently ambiguous mechanisms in stem cell therapies through a facile and effective approach.     

Direct 3D Printing of High Strength Biohybrid Gradient Hydrogel Scaffolds for Efficient Repair of Osteochondral Defect

The emerging 3D printing technique allows for tailoring hydrogel‐based soft structure tissue scaffolds for individualized therapy of osteochondral defects. However, the weak mechanical strength and uncontrollable swelling intrinsic to conventional hydrogels restrain their use as bioinks. Here, a high‐strength thermoresponsive supramolecular copolymer hydrogel is synthesized by one‐step copolymerization of dual hydrogen bonding monomers, N‐acryloyl glycinamide, and N‐[tris(hydroxymethyl)methyl] acrylamide. The obtained copolymer hydrogels demonstrate excellent mechanical properties—robust tensile strength (up to 0.41 MPa), large stretchability (up to 860%), and high compressive strength (up to 8.4 MPa). The rapid thermoreversible gel ? sol transition behavior makes this copolymer hydrogel suitable for direct 3D printing. Successful preparation of 3D‐printed biohybrid gradient hydrogel scaffolds is demonstrated with controllable 3D architecture, owing to shear thinning property which allows continuous extrusion through a needle and also immediate gelation of fluid upon deposition on the cooled substrate. Furthermore, this biohybrid gradient hydrogel scaffold printed with transforming growth factor beta 1 and β‐tricalciumphosphate on distinct layers facilitates the attachment, spreading, and chondrogenic and osteogenic differentiation of human bone marrow stem cells (hBMSCs) in vitro. The in vivo experiments reveal that the 3D‐printed biohybrid gradient hydrogel scaffolds significantly accelerate simultaneous regeneration of cartilage and subchondral bone in a rat model.     

Double-Lock Nanomedicines Enable Tumor-Microenvironment-Responsive Selective Antitumor Therapy

A hybrid nanocarrier for reducing the off-target adverse effects of chemotherapy via selective drug delivery to the tumor cells is reported. The model active agent, combretastatin A4 (CA4) phosphate is deposited onto the magnetite (Fe₃O₄) nanoparticles as the core, followed by lipid coating as the shell. Upon nanocarrier administration and biodistribution to the tumor site, the high level of adenosine triphosphate in the extracellular space of tumor induces the cargo release via phosphate displacement. Then, the CA4 phosphate is dephosphorylated by the alkaline phosphatase that is overexpressed at the plasma membrane of certain tumor cells, resulting in enhanced intracellular uptake of hydrophobic CA4. These sequential two-step unlocking processes enable the preferable accumulation of CA4 within tumor cells. Such approach is applicable to a wide range of chemotherapeutics and is promising for efficacy enhancement and side-effect reduction of antitumor chemotherapy.     

Dual Roles of Graphene Oxide in Chondrogenic Differentiation of Adult Stem Cells: Cell‐Adhesion Substrate and Growth Factor‐Delivery Carrier

Here, it is shown that graphene oxide (GO) can be utilized as both a cell‐adhesion substrate and a growth factor protein‐delivery carrier for the chondrogenic differentiation of adult stem cells. Conventionally, chondrogenic differentiation of stem cells is achieved by culturing cells in pellets and adding the protein transforming growth factor‐β3 (TGF‐β3), a chondrogenic factor, to the culture medium. However, pellets mainly provide cell‐cell interaction and diffusional limitation of TGF‐β3 may occur inside the pellet both of these factors may limit the chondrogenic differentiation of stem cells. In this study, GO sheets (size = 0.5–1 μm) were utilized to adsorb fibronectin (FN, a cell‐adhesion protein) and TGF‐β3 and were then incorporated in pellets of human adipose‐derived stem cells (hASCs). The hybrid pellets of hASC‐GO enhanced the chondrogenic differentiation of hASCs by adding the cell‐FN interaction and supplying TGF‐β3 effectively. This method may provide a new platform for stem cell culture for regenerative medicine.     

An NIR‐II Fluorescence/Dual Bioluminescence Multiplexed Imaging for In Vivo Visualizing the Location,Survival, and Differentiation of Transplanted Stem Cells

The in vivo distribution, viability, and differentiation capability of transplanted stem cells are vital for the therapeutic efficacy of stem cell–based therapy. Herein, an NIR‐II fluorescence/dual bioluminescence multiplexed imaging method covering the visible and the second near‐infrared window from 400 to 1700 nm is successfully developed for in vivo monitoring the location, survival, and osteogenic differentiation of transplanted human mesenchymal stem cells (hMSCs) in a calvarial defect mouse model. The exogenous Ag₂S quantum dot–based fluorescence imaging in the second near‐infrared window is applied for visualizing the long‐term biodistribution of transplanted hMSCs. Endogenous red firefly luciferase (RFLuc)‐based bioluminescence imaging (BLI) and the collagen type 1 promoter–driven Gaussia luciferase (GLuc)‐based BLI are employed to report the survival and osteogenic differentiation statuses of the transplanted hMSCs. Meanwhile, by integrating the three imaging channels, multiple dynamic biological behaviors of transplanted hMSCs and the promotion effects of immunosuppression and the bone morphogenetic protein 2 on the survival and osteogenic differentiation of transplanted hMSCs are directly observed. The novel multiplexed imaging method can greatly expand the capability for multifunctional analysis of the fates and therapeutic capabilities of the transplanted stem cells, and aid in the improvement of stem cell–based regeneration therapies and their clinical translation.     

Magnesium Gradient-Based Hierarchical Scaffold for Dual-Lineage Regeneration of Osteochondral Defect

Osteochondral regeneration remains a great challenge due to the limited self-healing ability and the complexity of its hierarchical structure and composition. Mg²⁺ and hypoxia are two effective modulators in boosting chondrogenesis. To this end, a double-layered scaffold (D) consisting of a hydrogel layer on a porous cryogel is fabricated to mimic the hierarchical structure of osteochondral tissue. An Mg²⁺ gradient is incorporated into the double-layered scaffold with hypoxia-mimicking deferoxamine (DFO) embedded in the hydrogel (D-Mg-DFO), which remarkably augments the dual-lineage regeneration of both cartilage and subchondral bone. The higher Mg²⁺ supplementation from the upper hydrogel, associated with its hypoxia-mimicking situation and small pore size, exhibits promotive effects on chondrogenic differentiation. The lower Mg²⁺ supplementation from the bottom cryogel, associated with its interconnected macroporous structure, achieves multiple contributions in stem cell migration from bone marrow cavity, matrix mineralization, and osteogenesis. Furthermore, rabbits’ trochlea osteochondral defects are established to evaluate the regenerative outcome. Compared to control scaffolds containing only Mg²⁺ or DFO, the D-Mg-DFO scaffold presents the best regenerative effect under the synergistic contribution of multiple factors. Overall, this work provides a new design of scaffold toward an effective repair of cartilage defect.