The co-influence of hyaluronic acid and collagen on the development of an engineered annulus tissue model with bone marrow stromal cells.

Inveterbral disc degeneration is a significant musculoskeletal disease that brings huge burden of pain, disability, psychological and social consequences to the affected population worldwide with treatments that only alleviate the pain but does not address the underlying biological problems. For the past decades, tissue engineering of the disc has been investigated with annulus fibrosus (AF) been one of the complicated disc component to be engineered. With the limited source of annulus cells, bone marrow stromal cells (BMSCs) have been frequently investigated as a potental cell candidate to develop an AF-like tissue which often require a multi-disciplinary effort to achieve. The extracellular matrix of AF is largely make up of collagen and proteoglycan which is still unclear how these matrix proteins could influence the BMSCs towards constructing a AF-like tissue. In this study, we adopted a coiled hydrogel microfiber that resembles the micro-architecture of the native AF tissue to encapsulate BMSCs and incorporated collagen type 1 and hyaluronic acid which later demonstrated that the co-presence of hyaluronic acid and collagen could potentially regulated AF-associated biomarkers and protease expression which are critical for later development of an engineered AF tissue construct.

A Facile and Scalable Hydrogel Patterning Method for Microfluidic 3D Cell Culture and Spheroid-in-Gel Culture Array.

Incorporation of extracellular matrix (ECM) and hydrogel in microfluidic 3D cell culture platforms is important to create a physiological microenvironment for cell morphogenesis and to establish 3D co-culture models by hydrogel compartmentalization. Here, we describe a simple and scalable ECM patterning method for microfluidic cell cultures by achieving hydrogel confinement due to the geometrical expansion of channel heights (stepped height features) and capillary burst valve (CBV) effects. We first demonstrate a sequential "pillar-free" hydrogel patterning to form adjacent hydrogel lanes in enclosed microfluidic devices, which can be further multiplexed with one to two stepped height features. Next, we developed a novel "spheroid-in-gel" culture device that integrates (1) an on-chip hanging drop spheroid culture and (2) a single "press-on" hydrogel confinement step for rapid ECM patterning in an open-channel microarray format. The initial formation of breast cancer (MCF-7) spheroids was achieved by hanging a drop culture on a patterned polydimethylsiloxane (PDMS) substrate. Single spheroids were then directly encapsulated on-chip in individual hydrogel islands at the same positions, thus, eliminating any manual spheroid handling and transferring steps. As a proof-of-concept to perform a spheroid co-culture, endothelial cell layer (HUVEC) was formed surrounding the spheroid-containing ECM region for drug testing studies. Overall, this developed stepped height-based hydrogel patterning method is simple to use in either enclosed microchannels or open surfaces and can be readily adapted for in-gel cultures of larger 3D cellular spheroids or microtissues.

Surface Creasing-Induced Micropatterned GelMA Using Heating-Hydration Fabrication for Effective Vascularization.

BACKGROUND: Surface modification is used to modify the biomaterials for the regulation of cell culture using different approaches, such as chemical graft and mechanical treatment. However, those conventional methodologies often require precise fabrication in a high resolution involving either high cost or laborious steps to remove chemical residues that are toxic to the cells. METHODS: A novel and simple method was proposed and evaluated to rapidly generate surface ceases on the gelatin methacrylate (gelMA) surface using the heating-hydration process. Human umbilical vein endothelial cells (HUVECs) were cultured on the gelMA surface. The surface binding was characterized using the RGD (Arg-Gly-Asp) antibodies and cell adhesion pattern captured by scanning electron microscopy. The effect of the heating-hydration parameters on the creasing formation was investigated. The morphology of HUVECs cultured on such micropatterned gelMA was characterized and compared. RESULTS: It is found that the hydration solution, gelMA mixture, and hydration rate are the major factors that influence the cracking sizes in the range from 20 to 120 µm which resulted in capillary-like patterns on the gelMA surface. Low concentration of gelMA, high water concentration of cooling agent, and slow hydration rate result in the long creases, and heating of at least 60 min is required for complete dehydration. Strong fluorescence was around the creases with RGD-staining. Consequently, micropatterned gelMA demonstrated good biocompatibility with endothelial cells with more than 95% cell viability and continuous cell proliferation throughout 2 weeks as well as a good trace of neovascular formation. In comparison, normal gelMA surface did not exhibit RGD-fluorescent signals, and the cultured HUVECs on it were rounded with no spreading for network formation. CONCLUSION: The heating-hydration approach can successfully and easily produce the micropatterned gelMA that allows rapid and effective vascularization to potentially improve the functionalities of the tissue-engineered construct.

Surface modifications to polydimethylsiloxane substrate for stabilizing prolonged bone marrow stromal cell culture.

Polydimethylsiloxane (PDMS) has been extensively used as a supporting material for studies of cell mechanobiology, cell micropatterning and microscale-cell analysis in microfluidic chips due to its numerous advantages, such as low cytotoxicity, ease of modification, inexpensive costs and biocompatibility. However, the innate hydrophobicity of PDMS often poses a problem for stable cell adhesion, seriously limiting its applicability for prolonged cell culture. UV exposure and protein coating are suboptimal solutions, while chemical surface functionalization is often associated with laborious procedures and producing environmental toxics. Plasma treatment can render a hydrophilic substrate by altering the surface chemistry, but such effect is often short-lived due to its tendency to hydrophobic recovery. Variation of physical properties of the substratum are known to influence cell behaviour. Nevertheless, the combination of varying PDMS substratum properties via base:curing agent ratio and plasma treatment to stabilize the long-term culture of bone marrow derived stromal cells (BMSCs) still remain poorly understood. In this study, we developed a protocol to maintain the hydrophilicity of the plasma-treated PDMS over a range of substratum properties. This study demonstrated that varying the substratum properties of PDMS can enhance the stability of BMSC culture for at least three weeks, while plasma treatment with or without additional collagen coating further enhanced such effect. The changes in the physical properties of PDMS have rendered difference in BMSCs adhesion, proliferation and in-vitro plasticity, thereby offering a simple and effective strategy for PDMS surface modification to enable long term cell analysis in PDMS-based culture platform.

Decellularized tissue engineered hyaline cartilage graft for articular cartilage repair.

Articular cartilage repair has been a long-standing challenge in orthopaedic medicine due to the limited self-regenerative capability of cartilage tissue. Currently, cartilage lesions are often treated by microfracture or autologous chondrocyte implantation (ACI). However, these treatments are frequently reported to result in a mixture of the desired hyaline cartilage and mechanically inferior fibrocartilage. In this study, by combining the advantages of cartilage tissue engineering and decellularization technology, we developed a decellularized allogeneic hyaline cartilage graft, named dLhCG, which achieved superior efficacy in articular cartilage repair and surpassed living autologous chondrocyte-based cartilaginous engraftment and ACI. By the 6-month time point after implantation in porcine knee joints, the fine morphology, composition, phenotype, microstructure and mechanical properties of the regenerated hyaline-like cartilaginous neo-tissue have been demonstrated via histology, biochemical assays, DNA microarrays and mechanical tests. The articular cartilaginous engraftment with allogeneic dLhCG was indicated to be well consistent, compatible and integrated with the native cartilage of the host. The successful repair of articular chondral defects in large animal models suggests the readiness of allogeneic dLhCG for clinical trials.

Scaffold-Free tissue engineering with aligned bone marrow stromal cell sheets to recapitulate the microstructural and biochemical composition of annulus fibrosus.

Current tissue engineering strategies through scaffold-based approaches fail to recapitulate the complex three-dimensional microarchitecture and biochemical composition of the native Annulus Fibrosus tissue. Considering limited access to healthy annulus fibrosus cells from patients, this study explored the potential of bone marrow stromal cells (BMSC) to fabricate a scaffold-free multilamellar annulus fibrosus-like tissue by integrating micropatterning technologies into multi-layered BMSC engineering. BMSC sheet with cells and collagen fibres aligned at ~30° with respect to their longitudinal dimension were developed on a microgroove-patterned PDMS substrate. Two sheets were then stacked together in alternating directions to form an angle-ply bilayer tissue, which was rolled up, sliced to form a multi-lamellar angle-ply tissue and cultured in a customized medium. The development of the annulus fibrosus-like tissue was further characterized by histological, gene expression and microscopic and mechanical analysis. We demonstrated that the engineered annulus fibrosus-like tissue with aligned BMSC sheet showed parallel collagen fibrils, biochemical composition and microstructures that resemble the native disk. Furthermore, aligned cell sheet showed enhanced expression of annulus fibrosus associated extracellular matrix markers and higher mechanical strength than that of the non-aligned cell sheet. The present study provides a new strategy in annulus fibrosus tissue engineering methodology to develop a scaffold-free annulus fibrosus-like tissue that resembles the microarchitecture and biochemical attributes of a native tissue. This can potentially lead to a promising avenue for advancing BMSC-mediated annulus fibrosus regeneration towards future clinical applications.

Full-Scale Osteochondral Regeneration by Sole Graft of Tissue-Engineered Hyaline Cartilage without Co-Engraftment of Subchondral Bone Substitute.

In this study, full-scale osteochondral defects are hypothesized, which penetrate the articular cartilage layer and invade into subchondral bones, and can be fixed by sole graft of tissue-engineered hyaline cartilage without co-engraftment of any subchondral bone substitute. It is hypothesized that given a finely regenerated articular cartilage shielding on top, the restoration of subchondral bones can be fulfilled via spontaneous self-remodeling in situ. Hence, the key challenge of osteochondral regeneration lies in restoration of the non-self-regenerative articular cartilage. Here, traumatic osteochondral lesions to be repaired in rabbit knee models are endeavored using novel tissue-engineered hyaline-like cartilage grafts that are produced by 3D cultured porcine chondrocytes in vitro. Comparative trials are conducted in animal models that are implanted with living hyaline cartilage grafts (LhCG) and decellularized LhCG (dLhCG). Sound osteochondral regeneration is gradually revealed from both LhCG and dLhCG-implanted samples 50-100 d after implantation. Quality regeneration in both zones of articular cartilage and subchondral bones are validated by the restored osteochondral composition, structure, phenotype, and mechanical property, which validate the hypothesis of this study.

Engineering a multiphasic, integrated graft with a biologically developed cartilage-bone interface for osteochondral defect repair.

Tissue engineering is a promising approach to repair osteochondral defects, yet successful reconstruction of different layers in an integrated graft, especially the interface remains challenging. The multiphasic, functionally integrated tissue engineering graft described herein mimics the entire osteochondral tissue in terms of structure and composition at the cartilage, bone and cartilage-bone interface layer to repair osteochondral defects. In this manuscript, we report the fabrication of a multiphasic graft via bonding of a cartilaginous hydrogel and a sintered poly(lactic-co-glycolic acid) microsphere scaffold by an endogenous fibrotic cartilaginous extracellular matrix. We demonstrated that culturing chondrocytes within the alginate hydrogel conjugated to the poly(lactic-co-glycolic acid) scaffold allows for (i) gradient transition and integration from the cartilage layer to the subchondral bone layer as assessed by scanning electron microscopy, histology and biochemistry, and (ii) superior tissue repair efficacy in a rabbit knee defect model. Industrialization of the graft remains an unsolved challenge as after decellularization the tissue repair efficacy of the graft decreased. Taken together, the multiphasic osteochondral graft repaired the osteochondral defects successfully and has the potential to be applied clinically as an implant in orthopaedic surgery.

Co-culture of human umbilical vein endothelial cells and human bone marrow stromal cells into a micro-cavitary gelatin-methacrylate hydrogel system to enhance angiogenesis.

Vascular tissue engineering seeks to develop functional blood vessels that comprise of both endothelial cells and pericytes for translational medicine and is often faced with numerous challenges such as nutrients and wastes diffusion problem in the centre of the scaffolds. Various strategies have been adopted to solve the diffusion problem in thick engineered scaffolds. Typically, microchannels or dissolvable microspheres are introduced into three-dimensional (3D) scaffolds as an alternative way to improve the infiltration of scaffolds and endothelial cells are usually incorporated into the biomaterials. While some research groups now focus on finding supporting cells to build further vascularized structures in the scaffolds. In this study, a bioinspired 3D gelatin-methacrylate (Gel-MA) hydrogel with dissolvable microspheres was created to encapsulate human bone marrow stromal cells (HMSCs) and human umbilical vein endothelial cells (HUVECs) which was used to investigate whether HMSCs could play a pericytes-like role and enhance vascularization within the engineered scaffolds. The results showed co-culture of HMSCs and HUVECs demonstrated significantly improved vascularization when compared to either HUVECs or HMSCs monoculture. Angiogenic genes were expressed significantly higher in co-culture group. Moreover, when implanting the pre-vascularized scaffolds in vivo, co-culture system integrated more successfully with host tissue and showed higher host tissue invasion than any other groups. More importantly, both the qPCR and immunofluorescence results indicated MSCs differentiated towards pericytes to enhance vascularization in this study. This paper highlights the enhanced capability of 3D micro-cavitary Gel-MA hydrogel for co-culturing HUVECs and HMSCs to promote vascularization which presents a potential strategy for future tissue repair and regeneration.

Albumin conjugates and assemblies as versatile bio-functional additives and carriers for biomedical applications.

As the most abundant plasma protein, serum albumin has been extensively studied and employed for therapeutic applications. Despite its direct clinical use for the maintenance of blood homeostasis in various medical conditions, this review exclusively summarizes and discusses albumin-based bio-conjugates and assemblies as versatile bio-functional additives and carriers in biomedical applications. As one of the smallest-sized proteins in the human body, albumin is physiochemically stable and biochemically inert. Moreover, albumin is also endowed with abundant specific binding sites for numerous therapeutic compounds, which also endow it with superior bioactivities. Firstly, due to its small size and binding specificity, albumin alone or its derived assemblies can be utilized as competent drug carriers, which can deliver drugs through the enhanced permeability and retention (EPR) effect or actively target lesion sites through binding with gp60 and secreted protein acidic and rich in cysteine (SPARC) in tumor sites. Furthermore, its biochemical stability and inertness make it a safe and biocompatible coating material for use in biomedical applications. Albumin-based surface modifying additives can be used to functionalize both macro substrates (e.g. surfaces of medical devices or implants) and nanoparticle surfaces (e.g. drug carriers and imaging contrast agents). In this review, we elaborate on the synthesis and applications of albumin-based bio-functional coatings and drug carriers, respectively.

Bioadhesives for internal medical applications: A review.

Bioadhesives such as tissue adhesives, hemostatic agents, and tissue sealants have gained increasing popularity in different areas of clinical operations during the last three decades. Bioadhesives can be categorized into internal and external ones according to their application conditions. External bioadhesives are generally applied in topical medications such as wound closure and epidermal grafting. Internal bioadhesives are mainly used in intracorporal conditions with direct contact to internal environment including tissues, organs and body fluids, such as chronic organ leak repair and bleeding complication reduction. This review focuses on internal bioadhesives that, in contrast with external bioadhesives, emphasize much more on biocompatibility and adhesive ability to wet surfaces rather than on gluing time and intensity. The crosslinking mechanisms of present internal bioadhesives can be generally classified as follows: 1) chemical conjugation between reactive groups; 2) free radical polymerization by light or redox initiation; 3) biological or biochemical coupling with specificity; and 4) biomimetic adhesion inspired from natural phenomena. In this review, bioadhesive products of each class are summarized and discussed by comparing their designs, features, and applications as well as their prospects for future development. STATEMENT OF SIGNIFICANCE: Despite the emergence of numerous novel bioadhesive formulations in recent years, thus far, the classification of internal and external bioadhesives has not been well defined and universally acknowledged. Many of the formulations have been proposed for treatment of several diseases even though they are not applicable for such conditions. This is because of the lack of a systematic standard or evaluation protocol during the development of a new adhesive product. In this review, the definition of internal and external bioadhesives is given for the first time, and with a focus on internal bioadhesives, the criteria of an ideal internal bioadhesive are adequately discussed; this is followed by the review of recently developed internal bioadhesives based on different gluing mechanisms.

Multidrug-eluting bi-layered microparticle-mesh scaffolds for musculoskeletal tissue regeneration.

Stem cell-based tissue engineering necessitates the development of a biocompatible scaffold, as a structural support, that provides a continuous supply of bioactive molecules for specific lineage differentiation. While incorporating bioactive molecules within a scaffold to improve stem cell differentiation has been reported in the literature, there is minimal evidence of any scaffold that can deliver a customized concoction of both hydrophobic and hydrophilic bioactive molecules to induce in situ lineage differentiation without any external supplements. In this study, we established a bioactive, drug-eluting bi-layered microparticle-mesh scaffold (BMMS) using the electrospinning technique. This BMMS was co-encapsulated with hydrophobic dexamethasone (in the mesh), hydrophilic ascorbic acid and ß-glycerophosphate or proline (in the microparticles). We hypothesized that a sustained-releasing BMMS can direct in situ specific lineage differentiation of MSCs (e.g. osteogenic and chondrogenic) in a minimally supplemented culture environment into musculoskeletal tissues. The characterization of this BMMS revealed good encapsulation efficiencies of the bioactive molecules with sustained-releasing capabilities. The release kinetics of each drug was further analyzed using mathematical drug-releasing models. These scaffolds were subsequently shown to have potential for osteogenic or chondrogenic lineage differentiation from mesenchymal stem cells (MSCs) in a minimally supplemented culture medium.

Respective Effects of Gelatin-Coated Polydimethylsiloxane (PDMS) Substrates on Self-renewal and Cardiac Differentiation of Induced Pluripotent Stem Cells (iPSCs).

The effects of substrate stiffness on the development of cardiomyocytes have been investigated extensively. Polydimethylsiloxane (PDMS) elastomer is one of biomaterials that are commonly used to explore the effects of substrate compliance on stem cell differentiation. Although the effects of substrate stiffness on cardiac differentiation of pluripotent stem cells, such as embryonic stem cells (ESCs) and induced pluripotent stem cells (iPSCs), have been reported, whether the stiffness of PDMS-based substrates could enhance differentiation of iPSCs toward cardiomyocyte lineage or not remains unknown. In this study, we found that a denser gelatin distribution and a higher gelatin adsorption on the stiffer PDMS. In addition, nanotopographies on PDMS substrates with different stiffness were distinct. iPSCs on the stiffer PDMS substrates showed higher pluripotency marker but lower cardiac gene expressions. In contrast, iPSCs on the softer PDMS substrates revealed lower pluripotency marker but higher cardiac gene expressions. These results indicate that stiffer PDMS substrates with gelatin coating could be used to support iPSC self-renewal and softer PDMS substrates coated with gelatin could be used for enhanced cardiac differentiation of iPSCs.

Yolk shell nanocomposite particles as bioactive bone fillers and growth factor carriers.

The efficient delivery of bioactive molecules via rationally designed nanoparticles is an important focus in regenerative medicine. The yolk shell nanocomposite particles described herein are composed of silk fibroin movable cores formed within voided calcium carbonate shells to load and control the release of labile cytokines. These particles are excellent carrier vehicles of potent molecules as they sustained the release of bioactive Bone Morphogenetic Protein 2 (BMP-2) for more than 28 days in vitro. Implantation into bone defects in rabbits corroborates the in vitro results and also reveals that upon contact with phosphate containing body fluids, implanted yolk shell particles agglomerate and transform into a filler that adapts to defect contour to further act as an absorbable hemostatic agent. Taken together, the fabrication of these yolk shell particle-based "bone fillers" could expand the horizon for the development of newer generations of advanced bioactive materials in tissue regeneration applications.

Combined effects of multi-scale topographical cues on stable cell sheet formation and differentiation of mesenchymal stem cells.

To decipher specific cell responses to diverse and complex in vivo signals, it is essential to emulate specific surface chemicals, extra cellular matrix (ECM) components and topographical signals through reliable and easily reproducible in vitro systems. However, the effect of multiple cues such as micro-hole/pillar architectures under a common and easily tunable platform remains unexplored. Recently we have demonstrated the positive influence of surface chemical modification of polydimethylsiloxane (PDMS) surfaces on directing long-term adhesion, viability and potency of hMSCs. In this study, we include biophysical signals from diverse surface topographical elements along with biochemical influences to develop a holistic understanding of hMSC responses in complex tissue-like niches. We report the influence of chemically modified PDMS structures encompassing hole-, pillar- and groove-based multi-scale architectures on hMSC morphology, adhesion, proliferation and differentiation. The inclusion of hole and pillar features resulted in enhanced adhesion and proliferation of hMSCs. These effects were more pronounced with the inclusion of grooves, which resulted in the highest osteogenic differentiation among other substrates. Our study provides an additional basis for the chemical/physical regulation of hMSC behavior within controlled biomimetic architectures with an aim to foster efficient tissue regeneration strategies.

Optimization of a polydopamine (PD)-based coating method and polydimethylsiloxane (PDMS) substrates for improved mouse embryonic stem cell (ESC) pluripotency maintenance and cardiac differentiation.

Myocardiocyte derived from pluripotent stem cells, such as induced pluripotent stem cells (iPSCs) and embryonic stem cells (ESCs), is a promising cell source for cardiac tissue engineering. Combined with microfluidic technologies, a heart-on-a-chip is very likely to be developed and function as a platform for high throughput drug screening. Polydimethylsiloxane (PDMS) silicone elastomer is a widely-used biomaterial for the investigation of cell-substrate interactions and biochip fabrication. However, the intrinsic PDMS surface hydrophobicity inhibits cell adhesion on the PDMS surface, and PDMS surface modification is required for effective cell adhesion. Meanwhile, the formulation of PDMS also affects the behaviors of the cells. To fabricate PDMS-based biochips for ESC pluripotency maintenance and cardiac differentiation, PDMS surface modification and formulation were optimized in this study. We found that a polydopamine (PD) with gelatin coating greatly improved the ESC adhesion, proliferation and cardiac differentiation on its surface. In addition, different PDMS substrates varied in their surface properties, which had different impacts on ESCs, with the 40 : 1 PDMS substrate being more favorable for ESC adhesion and proliferation as well as embryoid body (EB) attachment than the other PDMS substrates. Moreover, the ESC pluripotency was best maintained on the 5 : 1 PDMS substrate, while the cardiac differentiation of the ESCs was optimal on the 40 : 1 PDMS substrate. Based on the optimized coating method and PDMS formulation, biochips with two different designs were fabricated and evaluated. Compared to the single channels, the multiple channels on the biochips could provide larger areas and accommodate more nutrients to support improved ESC pluripotency maintenance and cardiac differentiation. These results may contribute to the development of a real heart-on-a-chip for high-throughput drug screening in the future.

Hydrogel based cartilaginous tissue regeneration: recent insights and technologies.

Hydrogels have been extensively employed as an attractive biomaterial to address numerous existing challenges in the fields of regenerative medicine and research because of their unique properties such as the capability to encapsulate cells, high water content, ease of modification, low toxicity, injectability, in situ spatial fit and biocompatibility. These inherent properties have created many opportunities for hydrogels as a scaffold or a cell/drug carrier in tissue regeneration, especially in the field of cartilaginous tissue such as articular cartilage and intervertebral discs. A concise overview of the anatomy/physiology of these cartilaginous tissues and their pathophysiology, epidemiology and existing clinical treatments will be briefly described. This review article will discuss the current state-of-the-art of various polymers and developing strategies that are explored in establishing different technologies for cartilaginous tissue regeneration. In particular, an innovative approach to generate scaffold-free cartilaginous tissue via a transient hydrogel scaffolding system for disease modeling to pre-clinical trials will be examined. Following that, the article reviews numerous hydrogel-based medical implants used in clinical treatment of osteoarthritis and degenerated discs. Last but not least, the challenges and future directions of hydrogel based medical implants in the regeneration of cartilaginous tissue are also discussed.

Noninvasive Monitoring of Three-Dimensional Chondrogenic Constructs Using Molecular Beacon Nanosensors.

Chondrogenic differentiation of human mesenchymal stem cells (MSCs) in three-dimensional hydrogel holds promise as a method for repairing injured articular cartilage. Given MSC plasticity (its potential to mature into alternative lineages), nondestructive monitoring is critical for the optimization of chondrogenic differentiation conditions and the evaluation of the final product. However, conventional validation/assessments of the differentiation process (i.e., quantitative reverse transcription polymerase chain reaction [qRT-PCR] and histology) are end-point assays requiring disruption of the sample. This report introduces molecular beacon (MB)-based nanosensors to achieve noninvasive monitoring of chondrogenic differentiation. These nanosensors consist of biodegradable poly(lactic-co-glycolic acid) nanoparticles (PLGA NPs) encapsulating MBs to detect Type II Collagen (Col2) and glyceraldehyde-3-phosphate dehydrogenase (GAPDH) mRNAs that serve as lineage-specific and housekeeping biomarkers, respectively. The sustainable release of MBs from MB-NPs allows longitudinal monitoring of MSCs undergoing chondrogenic differentiation over a period of 28 days. Dual-colored MB loading ensures accurate assessment of Col2 mRNA expression level, where potential heterogeneity in nanosensor uptake and retention by MSCs are taken into account. When normalized nanosensor signal was compared against qRT-PCR result, a tight correlation was observed (R2 = 0.9301). Finally, nanosensor usage was compatible with MSC potency with minimal influence on chondrogenic, adipogenic, and osteogenic differentiation.