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.     

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.     

Photoacoustic Imaging of Embryonic Stem Cell‐Derived Cardiomyocytes in Living Hearts with Ultrasensitive Semiconducting Polymer Nanoparticles

Human embryonic stem cell‐derived cardiomyocytes (hESC‐CMs) have become promising tools to repair injured hearts. To achieve optimal outcomes, advanced molecular imaging methods are essential to accurately track these transplanted cells in the heart. In this study, it is demonstrated for the first time that a class of photoacoustic nanoparticles (PANPs) incorporating semiconducting polymers (SPs) as contrast agents can be used in the photoacoustic imaging (PAI) of transplanted hESC‐CMs in living mouse hearts. This is achieved by virtue of two benefits of PANPs. First, strong photoacoustic (PA) signals and specific spectral features of SPs allow PAI to sensitively detect and distinguish a small number of PANP‐labeled cells (2000) from background tissues. Second, the PANPs show a high efficiency for hESC‐CM labeling without adverse effects on cell structure, function, and gene expression. Assisted by ultrasound imaging, the delivery and engraftment of hESC‐CMs in living mouse hearts can be assessed by PANP‐based PAI with high spatial resolution (≈100 µm). In summary, this study explores and validates a novel application of SPs as a PA contrast agent to track labeled cells with high sensitivity and accuracy in vivo, highlighting the advantages of integrating PAI and PANPs to advance cardiac regenerative therapies.     

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.     

Labeling of Adipose‐Derived Stem Cells by Oleic‐Acid‐Modified Magnetic Nanoparticles

The in vivo tracking of adipose derived stem cells (ASCs) is of essential concern when they are used as seed cells in tissue engineering. This study explores the feasibility of using magnetic nanoparticles (MNs), a type of contrast agents in magnetic resonance imaging (MRI), to label ASCs such that the labeled ASCs could be tracked in vivo by MRI non‐invasively and repeatedly. To do this, MNs of <10 nm surface‐coated with oleic acid are synthesized via a high‐temperature solution‐phase reaction. Cytotoxicity of the as‐synthesized MNs at concentrations up to 0.1 mg mL^?1 on 10⁴ cells mL^?1 ASCs is evaluated by LDH release. Since only minor cytotoxicity is detected, the effects of the labeling technique on cellular behaviors and uptake by labeled cells are investigated. Cell proliferation and differentiation with and without MNs are compared. The results show that proliferation of ASCs (10⁴ cells mL^?1) labeled by MNs (0.05 mg mL^?1) is significantly enhanced and dependent on the labeling time. The MNs are located in the vesicles within cytoplasm of ASCs. The cellular uptake reaches as high as ～180 pg/cell. Nevertheless, the labeled ASCs still maintained adipogenic and osteogenic differentiation. Hence, the feasibility of labeling ASCs by oleic acid coated MNs is ascertained and it was better to label the cells during their quiescent stage. The labeled ASCs can also be in vivo detected by MRI in a subcutaneous model in vivo. Further MRI tracking of the labeled ASCs in long‐term follow‐up would thus follow this current study.     

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.     

New Opportunities: Second Harmonic Generation of Boron‐Doped Graphene Quantum Dots for Stem Cells Imaging and Ultraprecise Tracking in Wound Healing

Second harmonic generation (SHG) has recently emerged, having the advantages of no bleaching, no blinking, and no signal saturation, as well as a high signal to‐noise ratio compared to fluorescence. Existing SHG probes are based on heavy metal or organic dye molecules, which have the shortcomings of toxicity, a large size, or photoinstability. To address the urgent need for long‐term tracking and imaging in organisms, boron‐doped graphene quantum dots (B‐GQDs), a highly biocompatible graphene based with a strong and photostable SHG signal is first synthesized and is further applied for stem cell imaging and tracking in wounds. The results demonstrate the possibility of stem cell internalization of B‐GQDs as a SHG probe and show no hindering of the stem cell's central physiological activities such as differentiation. Most importantly, B‐GQDs are successfully tracked in skin tissue in vivo after the labeled mouse mesenchymal stem cells being implanted over 35 days. This work will inspire the development of doped graphene quantum dot materials and promote the broad use of B‐GQDs in future molecular imaging, drug delivery, and stem cell therapy.     

Mesoporous Silica Coated Single‐Walled Carbon Nanotubes as a Multifunctional Light‐Responsive Platform for Cancer Combination Therapy

The development of cancer combination therapies, many of which rely on nanoscale theranostic agents, has received increasing attention in recent years. In this work, polyethylene glycol (PEG) modified mesoporous silica (MS) coated single‐walled carbon nanotubes (SWNTs) are fabricated and utilized as a multifunctional platform for imaging guided combination therapy of cancer. A model chemotherapy drug, doxorubicin (DOX), could be loaded into the mesoporous structure of the obtained SWNT@MS‐PEG nano‐carriers with high efficiency. Upon stimulation under near‐infrared (NIR) light, photothermally triggered drug release from DOX loaded SWNT@MS‐PEG is observed inside cells, resulting in a synergistic cancer cell killing effect. As revealed by both photoacoustic (PA) and magnetic resonance (MR) imaging, we further uncover efficient tumor accumulation of SWNT@MS‐PEG/DOX after intravenous injection into mice. In vivo combination therapy using this agent is further demonstrated in a mouse tumor model, achieving a remarkable synergistic anti‐tumor effect superior to that obtained by mono‐therapy. Our work presents a new type of theranostic nano‐platform, which could load therapeutic molecules with high efficiency, be responsive to external NIR stimulation, and at the same time serve as a diagnostic imaging agent.     

An Integrated Multifunctional Nanoplatform for Deep‐Tissue Dual‐Mode Imaging

The combination of biocompatible superparamagnetic and photoluminescent nanoparticles (NPs) is intensively studied as highly promising multifunctional (magnetic confinement and targeting, imaging, etc.) tools in biomedical applications. However, most of these hybrid NPs exhibit low signal contrast and shallow tissue penetration for optical imaging due to tissue‐induced optical extinction and autofluorescence, since in many cases, their photoluminescent components emit in the visible spectral range. Yet, the search for multifunctional NPs suitable for high photoluminescence signal‐to‐noise ratio, deep‐tissue imaging is still ongoing. Herein, a biocompatible core/shell/shell sandwich structured Fe₃O₄@SiO₂@NaYF₄:Nd³⁺ nanoplatform possessing excellent superparamagnetic and near‐infrared (excitation) to near‐infrared (emission), i.e., NIR‐to‐NIR photoluminescence properties is developed. They can be rapidly magnetically confined, allowing the NIR photoluminescence signal to be detected through a tissue as thick as 13 mm, accompanied by high T₂ relaxivity in magnetic resonance imaging. The fact that both the excitation and emission wavelengths of these NPs are in the optically transparent biological windows, along with excellent photostability, fast magnetic response, significant T₂‐contrast enhancement, and negligible cytotoxicity, makes them extremely promising for use in high‐resolution, deep‐tissue dual‐mode (optical and magnetic resonance) in vivo imaging and magnetic‐driven applications.     

“All‐in‐One” Nanoparticles for Trimodality Imaging‐Guided Intracellular Photo‐magnetic Hyperthermia Therapy under Intravenous Administration

Great efforts have been devoted so far to combine nano‐magnetic hyperthermia and nano‐photothermal therapy to achieve encouraging additive therapeutic performance in vitro and in vivo with limitation to direct intratumoral injection and no guidance of multimodality molecular imaging. In this study, a novel multifunctional theranostic nanoplatform (MNP@PES‐Cy7/2‐DG) consisting of magnetic nanoparticles (MNPs), poly(3,4‐ethylenedioxythiophene):poly(4‐styrenesulfonate) (PES), Cyanine7 (Cy7), and 2‐deoxyglucose (2‐DG)‐polyethylene glycol is developed. They are then applied to combined photo‐magnetic hyperthermia therapy under intravenous administration that is simultaneously guided by trimodality molecular imaging. Remarkably, nanoparticles are found aggregated mainly in the cytoplasm of tumor cells in vitro and in vivo, and exhibit stealth‐like behavior with a long second‐phase blood circulation half‐life of 20.38 ± 4.18 h. Under the guidance of photoacoustic/near‐infrared fluorescence/magnetic resonance trimodality imaging, tumors can be completely eliminated under intracellular photo‐magnetic hyperthermia therapy with additive therapeutic effect due to precise hyperthermia. This study may promote a further exploration of such a platform for clinical applications.     

Cancer Stem Cell‐Platelet Hybrid Membrane‐Coated Magnetic Nanoparticles for Enhanced Photothermal Therapy of Head and Neck Squamous Cell Carcinoma

Cell membrane–based nanosystems with desirable characteristics have been studied extensively for many therapeutic applications. However, current research has focused on single cell membrane, and multifunctional fused membrane materials from different membrane types are still rare. Herein, a platelet–cancer stem cell (CSC) hybrid membrane‐coated iron oxide magnetic nanoparticle (MN) {[CSC‐P]MN} is presented for the first time for the enhanced photothermal therapy of head and neck squamous cell carcinoma (HNSCC). Inherited from the original source cells, the platelet membrane shows immune evading ability due to the surface marker comprising a number of “don't eat me” signals, and the CSC membrane has homotypic targeting capabilities due to the specific surface adhesion molecules. The [CSC‐P]MNs possess superior characteristics for immune evasion, active cancer targeting, magnetic resonance imaging, and photothermal therapy. Compared with single cell membrane–coated MNs, [CSC‐P]MNs exhibit prolonged circulation times and enhanced targeting abilities. Moreover, the [CSC‐P]MNs exhibit a superior photothermal ability that provides excellent HNSCC tumor growth inhibition, particularly in an immunocompetent Tgfbr1/Pten conditional double knockout HNSCC mouse model that contains a more complex tumor microenvironment that is similar to the human HNSCC microenvironment. Collectively, this biomimetic multimembrane‐coated nanoplatform may provide enhanced antitumor efficacy in the complex tumor microenvironment.     

Hydroxyl‐Rich Polycation Brushed Multifunctional Rare‐Earth‐Gold Core–Shell Nanorods for Versatile Therapy Platforms

It is of great significance to develop a multifunctional imaging‐guided therapeutic platform with ideal resolution and sensitivity. Notably, rare‐earth (RE) nanoparticles are attractive candidates for multimodal imaging due to their unique optical and magnetic properties. In this work, a rational design of hierarchical nanohybrids employing RE‐Au hetero‐nanostructures is proposed. 1D RE nanorods are adopted as the core to facilitate cellular internalization with the coating of gold nanoshells for photothermal performances. Hydroxyl‐rich polycations with low cytotoxicity are grafted onto the surface of RE‐Au to produce RE‐Au‐PGEA (ethanolamine‐functionalized poly(glycidyl methacrylate)) nanohybrids with impressive gene transfection capability. Given the virtues of all the components, the feasibility of RE‐Au‐PGEA for multifunctional photoacoustic, computed tomography, magnetic resonance, upconversion luminescence imaging, and complementary photothermal therapy/gene therapy therapy is investigated in detail in vitro and in vivo. The visualization of the therapeutic processes with comprehensive information renders RE‐Au‐PGEA nanohybrid an intriguing platform to realize enhanced antitumor efficiency.     

Cypate‐Conjugated Porous Upconversion Nanocomposites for Programmed Delivery of Heat Shock Protein 70 Small Interfering RNA for Gene Silencing and Photothermal Ablation

Synergistic therapy is an accepted method of enhancing the efficacy of cancer therapies. In this study, cypate‐conjugated porous NaLuF₄ doped with Yb³⁺, Er³⁺, and Gd³⁺ is synthesized and its potential for upconversion luminescence/magnetic resonance dual‐modality molecular imaging for guiding oncotherapy is tested. Loading cypate‐conjugated upconversion nanoparticles (UCNP‐cy) with small interfering RNA gene against heat shock protein 70 (UCNP‐cy‐siRNA) enhances the cell damage. UCNP‐cy‐siRNA exhibits remarkable antitumor efficacy in vivo as a result of the synergistic effects of gene silencing and photothermal therapy, with low drug dose and minimal side effects. This result thus provides an explicit strategy for developing next‐generation multifunctional nanoplatforms for multimodal imaging‐guided synergistic oncotherapy.     

3D‐Printed Microrobotic Transporters with Recapitulated Stem Cell Niche for Programmable and Active Cell Delivery

Poor retention rate, low targeting accuracy, and spontaneous transformation of stem cells present major clinical barriers to the success of therapies based on stem cell transplantation. To improve the clinical outcome, efforts should focus on the active delivery of stem cells to the target tissue site within a controlled environment, increasing survival, and fate for effective tissue regeneration. Here, a remotely steerable microrobotic cell transporter is presented with a biophysically and biochemically recapitulated stem cell niche for directing stem cells towards a pre‐destined cell lineage. The magnetically actuated double‐helical cell microtransporters of 76 µm length and 20 µm inner cavity diameter are 3D printed where biological and mechanical information regarding the stem cell niche are encoded at the single‐cell level. Cell‐loaded microtransporters are mobilized inside confined microchannels along computer‐controlled trajectories under rotating magnetic fields. The mesenchymal stem cells are shown retaining their differentiation capacities to commit to the osteogenic lineage when stimulated inside the microswimmers in vitro. Such a microrobotic approach has the potential to enable the development of active microcarriers with embedded functionalities for controlled and precisely localized therapeutic cell delivery.     

Mussel Byssus‐Like Reversible Metal‐Chelated Supramolecular Complex Used for Dynamic Cellular Surface Engineering and Imaging

Inspired by the load‐bearing biostructures in nature, a multifunctional shell for encapsulating cell using the polyphenol–metal complexes is fabricated. The artificial shell is formed by cross‐linking of tannic acid and iron ion on cell surface. It can protect cells from unfriendly environments, including UV light irradiation and reactive oxygen damage. With the hybrid property of polyphenol and metal liands, the shell provides a versatile platform for cell surface engineering. The magnetic nanoparticles, DNA molecules, as well as the magnetic resonance imaging agents are easily incorporated into the shell. More interestingly, unlike the traditional passive coatings, here the shell can be controllably disassembled under external stimuli. The dynamic coating is used as a reversible element to regulate cell division and surface modification. The cell viability and protein expression experiments further confirm that the shell formation and degradation processes are biocompatible. This multifunctional coating strategy is applicable to multiple living cell types, including yeast cells, Escherichia coli bacteria, and mammalian cells. Therefore, this platform would be useful for living cell based fundamental research and biological applications.     

Wireless Manipulation of Magnetic/Piezoelectric Micromotors for Precise Neural Stem‐Like Cell Stimulation

Precise neural electrical stimulation, which is a means of promoting neuronal regeneration, is a promising solution for patients with neurotrauma and neurodegenerative diseases. In this study, wirelessly controllable targeted motion and precise stimulation at the single‐cell level using S.platensis@Fe₃O₄@tBaTiO₃ micromotors are successfully demonstrated for the first time. A highly versatile and multifunctional biohybrid soft micromotor is fabricated via the integration of S.platensis with magnetic Fe₃O₄ nanoparticles and piezoelectric BaTiO₃ nanoparticles. The results show that this micromotor system can achieve navigation in a highly controllable manner under a low‐strength rotating magnetic field. The as‐developed system can achieve single‐cell targeted motion and then precisely induce the differentiation of the targeted neural stem‐like cell by converting ultrasonic energy to an electrical signal in situ owing to the piezoelectric effect. This new approach toward the high‐precision stimulation of neural stem‐like cells opens up new applications for micromotors and has excellent potential for precise neuronal regenerative therapies.     

MRI‐Visible siRNA Nanomedicine Directing Neuronal Differentiation of Neural Stem Cells in Stroke

A major challenge in stroke treatment is the restoration of neural circuit in which neuron function plays a central role. Although transplantation of exogenous neural stem cells (NSCs) is admittedly a promising therapeutical means, the treatment outcome is greatly affected due to the poor NSCs differentiation into neurons caused by myelin associated inhibitory factors binding to Nogo‐66 receptor (NgR). Herein, a nanoscale polymersome is developed to codeliver superparamagnetic iron oxide nanoparticles and siRNA targeting NgR gene (siNgR) into NSCs. This multifunctional nanomedicine directs neuronal differentiation of NSCs through silencing the NgR gene and meanwhile allows a noninvasive monitoring of NSC migration with magnetic resonance imaging. An improved recovery of neural function is achieved in rat ischemic stroke model. The results demonstrate the great potential of the multifunctional siRNA nanomedicine in stroke treatment based on stem cell transplantation.     

Injectable Self‐Healing Antibacterial Bioactive Polypeptide‐Based Hybrid Nanosystems for Efficiently Treating Multidrug Resistant Infection,Skin‐Tumor Therapy,and Enhancing Wound Healing

The surgical procedure in skin‐tumor therapy usually results in cutaneous defects, and multidrug‐resistant bacterial infection could cause chronic wounds. Here, for the first time, an injectable self‐healing antibacterial bioactive polypeptide‐based hybrid nanosystem is developed for treating multidrug resistant infection, skin‐tumor therapy, and wound healing. The multifunctional hydrogel is successfully prepared through incorporating monodispersed polydopamine functionalized bioactive glass nanoparticles (BGN@PDA) into an antibacterial F127‐ε‐Poly‐L‐lysine hydrogel. The nanocomposites hydrogel displays excellent self‐healing and injectable ability, as well as robust antibacterial activity, especially against multidrug‐resistant bacteria in vitro and in vivo. The nanocomposites hydrogel also demonstrates outstanding photothermal performance with (near‐infrared laser irradiation) NIR irradiation, which could effectively kill the tumor cell (>90%) and inhibit tumor growth (inhibition rate up to 94%) in a subcutaneous skin‐tumor model. In addition, the nanocomposites hydrogel effectively accelerates wound healing in vivo. These results suggest that the BGN‐based nanocomposite hydrogel is a promising candidate for skin‐tumor therapy, wound healing, and anti‐infection. This work may offer a facile strategy to prepare multifunctional bioactive hydrogels for simultaneous tumor therapy, tissue regeneration, and anti‐infection.     

Multimodal Magneto‐Plasmonic Nanoclusters for Biomedical Applications

Multimodal nanostructures can help solve many problems in the biomedical field including sensitive molecular imaging, highly specific therapy, and early cancer detection. However, the synthesis of densely packed, multicomponent nanostructures with multimodal functionality represents a significant challenge. Here, a new type of hybrid magneto‐plasmonic nanoparticles is developed using an oil‐in‐water microemulsion method. The nanostructures are synthetized by self‐assembly of primary 6 nm iron oxide core‐gold shell particles resulting into densely packed spherical nanoclusters. The dense packing of primary particles does not change their superparamagnetic behavior; however, the close proximity of the constituent particles in the nanocluster leads to strong near‐infrared (NIR) plasmon resonances. The synthesis is optimized to eliminate nanocluster cytotoxicity. Immunotargeted nanoclusters are also developed using directional conjugation chemistry through the Fc antibody moiety, leaving the Fab antigen recognizing region available for targeting. Cancer cells labeled with immunotargeted nanoclusters produce a strong photoacoustic signal in the NIR that is optimum for tissue imaging. Furthermore, the labeled cells can be efficiently captured using an external magnetic field. The biocompatible magneto‐plasmonic nanoparticles can make a significant impact in development of point‐of‐care assays for detection of circulating tumor cells, as well as in cell therapy with magnetic cell guidance and imaging monitoring.     

Directing Osteogenesis of Stem Cells with Drug‐Laden,Polymer‐Microsphere‐Based Micropatterns Generated by Teflon Microfluidic Chips

Human bone tissue is built in a hierarchical way by assembling various cells of specific functions; the behaviors of these cells in vivo are sophisticatedly regulated. However, the cells in an injured bone caused by tumor or other bone‐related diseases cannot properly perform self‐regulation behaviors, such as specialized differentiation. To address this challenge, a simple one‐step strategy for patterning drug‐laden poly(lactic‐co‐glycolic acid) (PLGA) microspheres into grooves by Teflon chips is developed to direct cellular alignment and osteogenic commitment of adipose‐derived stem cells (ADSCs) for bone regeneration. A hydrophilic model protein and a hydrophobic model drug are encapsulated into microsphere‐based grooved micropatterns to investigate the release of the molecules from the PLGA matrix. Both types of molecules show a sustained release with a small initial burst during the first couple of days. Osteogenic differentiated factors are also encapsulated in the micropatterns and the effect of these factors on inducing the osteogenic differentiation of ADSCs is studied. The ADSCs on the drug‐laden micropatterns show stronger osteogenic commitment in culture than those on flat PLGA film or on drug‐free grooved micropatterns cultured under the same conditions. The results demonstrate that a combination of chemical and topographical cues is more effective to direct the osteogenic commitment of stem cells than either is alone. The microsphere‐based groove micropatterns show potential for stem cell research and bone regenerative therapies.