Size-Based Microfluidic-Enriched Mesenchymal Stem Cell Subpopulations Enhance Articular Cartilage Repair.

BACKGROUND: The functional heterogeneity of culture-expanded mesenchymal stem cells (MSCs) has hindered the clinical application of MSCs. Previous studies have shown that MSC subpopulations with superior chondrogenic capacity can be isolated using a spiral microfluidic device based on the principle of inertial cell focusing. HYPOTHESIS: The delivery of microfluidic-enriched chondrogenic MSCs that are consistent in size and function will overcome the challenge of the functional heterogeneity of expanded MSCs and will significantly improve MSC-based cartilage repair. STUDY DESIGN: Controlled laboratory study. METHODS: A next-generation, fully automated multidimensional double spiral microfluidic device was designed to provide more refined and efficient isolation of MSC subpopulations based on size. Analysis of in vitro chondrogenic potential and RNA sequencing was performed on size-sorted MSC subpopulations. In vivo cartilage repair efficacy was demonstrated in an osteochondral injury model in 12-week-old rats. Defects were implanted with MSC subpopulations (n = 6 per group) and compared with those implanted with unsegregated MSCs (n = 6). Osteochondral repair was assessed at 6 and 12 weeks after surgery by histological, micro-computed tomography, and mechanical analysis. RESULTS: A chondrogenic MSC subpopulation was efficiently isolated using the multidimensional double spiral device. RNA sequencing revealed distinct transcriptomic profiles and identified differential gene expression between subpopulations. The delivery of a chondrogenic MSC subpopulation resulted in improved cartilage repair, as indicated by histological scoring, the compression modulus, and micro-computed tomography of the subchondral bone. CONCLUSION: We have established a rapid, label-free, and reliable microfluidic protocol for more efficient size-based enrichment of a chondrogenic MSC subpopulation. Our proof-of-concept in vivo study demonstrates the enhanced cartilage repair efficacy of these enriched chondrogenic MSCs. CLINICAL RELEVANCE: The delivery of microfluidic-enriched chondrogenic MSCs that are consistent in size and function can overcome the challenge of the functional heterogeneity of expanded MSCs, resulting in significant improvement in MSC-based cartilage repair. The availability of such rapid, label-free enriched chondrogenic MSCs can enable better cell therapy products for cartilage repair with improved treatment outcomes.

Secretive derived from hypoxia preconditioned mesenchymal stem cells promote cartilage regeneration and mitigate joint inflammation via extracellular vesicles.

Secretome derived from mesenchymal stem cells (MSCs) have profound effects on tissue regeneration, which could become the basis of future MSCs therapies. Hypoxia, as the physiologic environment of MSCs, has great potential to enhance MSCs paracrine therapeutic effect. In our study, the paracrine effects of secretome derived from MSCs preconditioned in normoxia and hypoxia was compared through both in vitro functional assays and an in vivo rat osteochondral defect model. Specifically, the paracrine effect of total EVs were compared to that of soluble factors to characterize the predominant active components in the hypoxic secretome. We demonstrated that hypoxia conditioned medium, as well as the corresponding EVs, at a relatively low dosage, were efficient in promoting the repair of critical-sized osteochondral defects and mitigated the joint inflammation in a rat osteochondral defect model, relative to their normoxia counterpart. In vitro functional test shows enhancement through chondrocyte proliferation, migration, and matrix deposition, while inhibit IL-1ß-induced chondrocytes senescence, inflammation, matrix degradation, and pro-inflammatory macrophage activity. Multiple functional proteins, as well as a change in EVs' size profile, with enrichment of specific EV-miRNAs were detected with hypoxia preconditioning, implicating complex molecular pathways involved in hypoxia pre-conditioned MSCs secretome generated cartilage regeneration.

Electrospun fibers enhanced the paracrine signaling of mesenchymal stem cells for cartilage regeneration.

BACKGROUND: Secretome profiles of mesenchymal stem cells (MSCs) are reflective of their local microenvironments. These biologically active factors exert an impact on the surrounding cells, eliciting regenerative responses that create an opportunity for exploiting MSCs towards a cell-free therapy for cartilage regeneration. The conventional method of culturing MSCs on a tissue culture plate (TCP) does not provide the physiological microenvironment for optimum secretome production. In this study, we explored the potential of electrospun fiber sheets with specific orientation in influencing the MSC secretome production and its therapeutic value in repairing cartilage. METHODS: Conditioned media (CM) were generated from MSCs cultured either on TCP or electrospun fiber sheets of distinct aligned or random fiber orientation. The paracrine potential of CM in affecting chondrogenic differentiation, migration, proliferation, inflammatory modulation, and survival of MSCs and chondrocytes was assessed. The involvement of FAK and ERK mechanotransduction pathways in modulating MSC secretome were also investigated. RESULTS: We showed that conditioned media of MSCs cultured on electrospun fiber sheets compared to that generated from TCP have improved secretome yield and profile, which enhanced the migration and proliferation of MSCs and chondrocytes, promoted MSC chondrogenesis, mitigated inflammation in both MSCs and chondrocytes, as well as protected chondrocytes from apoptosis. Amongst the fiber sheet-generated CM, aligned fiber-generated CM (ACM) was better at promoting cell proliferation and augmenting MSC chondrogenesis, while randomly oriented fiber-generated CM (RCM) was more efficient in mitigating the inflammation assault. FAK and ERK signalings were shown to participate in the modulation of MSC morphology and its secretome production. CONCLUSIONS: This study demonstrates topographical-dependent MSC paracrine activities and the potential of employing electrospun fiber sheets to improve the MSC secretome for cartilage regeneration.

Repair of Osteochondral Defects With Predifferentiated Mesenchymal Stem Cells of Distinct Phenotypic Character Derived From a Nanotopographic Platform.

BACKGROUND: Articular cartilage has a zonal architecture and biphasic mechanical properties. The recapitulation of surface lubrication properties with high compressibility of the deeper layers of articular cartilage during regeneration is essential in achieving long-term cartilage integrity. Current clinical approaches for cartilage repair, especially with the use of mesenchymal stem cells (MSCs), have yet to restore the hierarchically organized architecture of articular cartilage. HYPOTHESIS: MSCs predifferentiated on surfaces with specific nanotopographic patterns can provide phenotypically stable and defined chondrogenic cells and, when delivered as a bilayered stratified construct at the cartilage defect site, will facilitate the formation of functionally superior cartilage tissue in vivo. STUDY DESIGN: Controlled laboratory study. METHODS: MSCs were subjected to chondrogenic differentiation on specific nanopatterned surfaces. The phenotype of the differentiated cells was assessed by the expression of cartilage markers. The ability of the 2-dimensional nanopattern-generated chondrogenic cells to retain their phenotypic characteristics after removal from the patterned surface was tested by subjecting the enzymatically harvested cells to 3-dimensional fibrin hydrogel culture. The in vivo efficacy in cartilage repair was demonstrated in an osteochondral rabbit defect model. Repair by bilayered construct with specific nanopattern predifferentiated cells was compared with implantation with cell-free fibrin hydrogel, undifferentiated MSCs, and mixed-phenotype nanopattern predifferentiated MSCs. Cartilage repair was evaluated at 12 weeks after implantation. RESULTS: Three weeks of predifferentiation on 2-dimensional nanotopographic patterns was able to generate phenotypically stable chondrogenic cells. Implantation of nanopatterned differentiated MSCs as stratified bilayered hydrogel constructs improved the repair quality of cartilage defects, as indicated by histological scoring, mechanical properties, and polarized microscopy analysis. CONCLUSION: Our results indicate that with an appropriate period of differentiation, 2-dimensional nanotopographic patterns can be employed to generate phenotypically stable chondrogenic cells, which, when implanted as stratified bilayered hydrogel constructs, were able to form functionally superior cartilage tissue. CLINICAL RELEVANCE: Our approach provides a relatively straightforward method of obtaining large quantities of zone-specific chondrocytes from MSCs to engineer a stratified cartilage construct that could recapitulate the zonal architecture of hyaline cartilage, and it represents a significant improvement in current MSC-based cartilage regeneration.

Label-free separation of mesenchymal stem cell subpopulations with distinct differentiation potencies and paracrine effects.

Mesenchymal stem cells (MSCs) have the capability to differentiate into multiple cell lineages, and produce trophic factors to facilitate tissue repair and regeneration, and disease regression. However, the heterogeneity of MSCs, whether inherent or developed during culture expansion, has a significant impact on their therapeutic efficacy. Therefore, the ability to identify and select an efficacious subpopulation of MSCs targeting specific tissue damage or disease holds great clinical significance. In this study, we separated three subpopulations from culture expanded human bone marrow derived MSCs according to cell size, using a high-throughput label-free microfluidic cell sorting technology. The size-sorted MSC subpopulations varied in tri-lineage differentiation potencies. The large MSCs showed the strongest osteogenesis, medium-size MSCs were advantageous in chondrogenesis and adipogenesis, and the small MSCs showed the weakest tri-lineage differentiation. The size-sorted MSC subpopulations also exhibited different secretome profiles. The large MSC secretome possessed highest levels of osteogenic promotor proteins and senescence-associated factors, but lower levels of osteogenic inhibitor proteins compared to the medium-size MSC secretome. The medium-size MSC secretome had high levels of chondrogenic promotor proteins, and contained lower levels of chondrogenic inhibitor proteins compared to the large MSC secretome. The secretome of size-sorted MSC subpopulations showed differences in paracrine effects. We found that the secretome of large MSCs enhanced osteogenic and adipogenic potencies during MSC culture expansion, but also induced cell senescence; and the secretome of medium-size MSCs promoted chondrogenesis. This study demonstrates size-dependent differentiation potency and secretome profile of MSC subpopulations, and provides an effective and practical technology to isolate the respective subpopulations, which may be used for more targeted tissue repair and regeneration.

Ascorbate and Iron Are Required for the Specification and Long-Term Self-Renewal of Human Skeletal Mesenchymal Stromal Cells.

The effects of ascorbate on adult cell fate specification remain largely unknown. Using our stepwise and chemically defined system to derive lateral mesoderm progenitors from human pluripotent stem cells (hPSCs), we found that ascorbate increased the expression of mesenchymal stromal cell (MSC) markers, purity of MSCs, the long-term self-renewal and osteochondrogenic capacity of hPSC-MSCs in vitro. Moreover, ascorbate promoted MSC specification in an iron-dependent fashion, but not in a redox-dependent manner. Further studies revealed that iron synergized with ascorbate to regulate hPSC-MSC histone methylation, promote their long-term self-renewal, and increase their osteochondrogenic capacity. We found that one of the histone demethylases affected by ascorbate, KDM4B, was necessary to promote the specification of hPSC-MSCs. This mechanistic understanding led to the metabolic optimization of hPSC-MSCs with an extended lifespan in vitro and the ability to fully repair cartilage defects upon transplantation in vivo. Our results highlight the importance of ascorbate and iron metabolism in adult human cell fate specification.

Characterization and application of size-sorted zonal chondrocytes for articular cartilage regeneration.

Current clinical approaches for articular cartilage repair have not been able to restore the tissue with zonal architecture, and its biomechanical and functional properties. Mimicking the zonal organization of articular cartilage in neo-tissue by implanting zonal chondrocyte subpopulations in multilayer construct could enhance the functionality of the graft, engineering of stratified tissue has not yet been realized due to lack of efficient and specific zonal chondrocyte isolation protocol. We show that by using a spiral microchannel device, the superficial, middle and deep zone chondrocytes can be separated based on cell size, and enriched from full thickness porcine cartilage in a high-throughput, label-free manner. The size-sorted cells show zone-specific characteristics in RT-PCR analysis of zonal cartilage markers. Both freshly sorted and two-passage expanded zonal chondrocytes formed cartilage tissue in 3D hydrogel, bearing respective zonal characteristics, indicated by RT-PCR, histology, extracellular matrix proteins, and mechanical compression test. In the proof-of-concept in vivo study using a rodent cartilage defect model, the size-sorted zonal chondrocytes when delivered in bi-layered hydrogel construct, facilitated better cartilage repair with mechanically enhanced cartilage tissue, in comparison to conventional chondrocytes implantation. This study provides an effective approach to obtain large numbers of zonal chondrocytes, and demonstrates the translational potential of stratified zonal chondrocyte implantation for clinical repair of critical size cartilage defects.

Microfluidic label-free selection of mesenchymal stem cell subpopulation during culture expansion extends the chondrogenic potential in vitro.

Mesenchymal stem cells (MSCs) have been shown as potential candidates for cell-based therapies for a diverse range of tissue regenerative applications. Therapeutic use of MSCs usually requires culture expansion, which increases the heterogeneity of MSCs in vitro, thus affecting the potency of the MSCs for more specific indications. The capacity for identifying and isolating special subsets of MSCs for treatment of specific diseases therefore holds great clinical significance. An important therapeutic application of MSC is for the regeneration of cartilage tissue. We and others have previously developed label-free microfluidic means to isolate subpopulations of culture expanded MSCs based on distinct biophysical characteristics. Here we utilize a spiral micro-channel device to separate culture expanded MSCs into five subgroups according to cell size, and study their proliferation and chondrogenesis at early, middle and late passages. Results show that in all passages, the medium-size subpopulation (cell size of 17-21 µm), compared to other subpopulations, displays significantly higher proliferation rate and chondrogenic capacity in terms of cartilage extracellular matrix formation. Also, the small cell subpopulation (average cell size of 11-12 µm) shows lower viability, and large cell subpopulation (average cell size 23-25 µm) expresses higher level of senescence-associated ß-galactosidase. Finally, we show that repeated microfluidic exclusion of MSCs larger than 21 µm and smaller than 17 µm at every passage during continuous culture expansion result in selected MSCs with faster proliferation and better chondrogenic potential as compared to MSC derived from conventional expansion approach. This study demonstrates the significant merit and utility of size-based cell selection for the application of MSCs in cartilage regeneration.

The Combined Effect of Substrate Stiffness and Surface Topography on Chondrogenic Differentiation of Mesenchymal Stem Cells.

Stem cell differentiation is guided by contact with the physical microenvironment, influence by both topography and mechanical properties of the matrix. In this study, the combined effect of substratum nano-topography and mechanical stiffness in directing mesenchymal stem cell (MSC) chondrogenesis was investigated. Three polyesters of varying stiffness were thermally imprinted to create nano-grating or pillar patterns of the same dimension. The surface of the nano-patterned substrate was coated with chondroitin sulfate (CS) to provide an even surface chemistry, with cell-adhesive and chondro-inductive properties, across all polymeric substrates. The surface characteristic, mechanical modulus, and degradation of the CS-coated patterned polymeric substrates were analyzed. The cell morphology adopted on the nano-topographic surfaces were accounted by F-actin distribution, and correlated to the cell proliferation and chondrogenic differentiation outcomes. Results show that substratum stiffness and topographical cues affected MSC morphology and aggregation, and influenced the phenotypic development at the earlier stage of chondrogenic differentiation. Hyaline-like cartilage with middle/deep zone cartilage characteristics was generated on softer pillar surface, while on stiffer nano-pillar material MSCs showed potential to generate constituents of hyaline/fibro/hypertrophic cartilage. Fibro/superficial zone-like cartilage could be derived from nano-grating of softer stiffness, while stiffer nano-grating resulted in insignificant chondrogenesis. This study demonstrates the possibility of refining the phenotype of cartilage generated from MSCs by manipulating surface topography and material stiffness.

Temporal activation of ß-catenin signaling in the chondrogenic process of mesenchymal stem cells affects the phenotype of the cartilage generated.

Adult mesenchymal stem cells (MSCs) are an attractive cell source for cartilage tissue engineering. In vitro predifferentiation of MSCs has been explored as a means to enhance MSC-based articular cartilage repair. However, there remain challenges to control and prevent the premature progression of MSC-derived chondrocytes to the hypertrophy. This study investigated the temporal effect of transforming growth factor (TGF)-ß and ß-catenin signaling co-activation during MSC chondrogenic differentiation and evaluated the influence of these predifferentiation conditions to subsequent phenotypic development of the cartilage. MSCs were differentiated in chondrogenic medium that contained either TGFß alone, TGFß with transient ß-catenin coactivation, or TGFß with continuous ß-catenin coactivation. After in vitro differentiation, the pellets were transplanted into SCID mice. Both coactivation protocols resulted in the enhancement of chondrogenic differentiation of MSCs. Compared with TGFß activation, transient coactivation of TGFß-induction with ß-catenin activation resulted in heightened hypertrophy and formed highly ossified tissues with marrow-like hematopoietic tissue in vivo. The continuous coactivation of the 2 signaling pathways, however, resulted in inhibition of progression to hypertrophy, marked by the suppression of type X collagen, Runx2, and alkaline phosphatase expression, and did not result in ossified tissue in vivo. Chondrocytes of the continuous co-activation samples secreted significantly more parathyroid hormone-related protein (PTHrP) and expressed cyclin D1. Our results suggest that temporal co-activation of the TGFß signaling pathway with ß-catenin can yield cartilage of different phenotype, represents a potential MSC predifferentiation protocol before clinical implantation, and has potential applications for the engineering of cartilage tissue.

Antibacterial potential of antagonistic Streptomyces sp. isolated from marine sponge Dendrilla nigra.

The role of Streptomyces sp. (BTL7) in synthesis of antibacterial agents reported from the marine sponge Dendrilla nigra was evaluated. Selective isolation of actinomycetes was performed on the newly developed selective media, Sponge Agar (SA) 1 and SA 2. The growth rate and antibiotic production were increased on the media supplemented with sponge extract. The chosen isolate BTL7 showed inhibitory interaction with Micrococcus luteus and the extracellular products contained potent antibacterial agents. The minimum inhibitory concentration of BTL7 against M. luteus was 44 microg protein/ml and the minimum bactericidal concentration was 88 microg protein/ml. Peak antibacterial activity was observed at 72 h in batch culture. Based on the findings, it could be inferred that bacterial endosymbionts sponges could form a reliable source for bioprospecting of next generation pharmaceutical agents.