Growth Factor Immobilization Strategies for Musculoskeletal Disorders.

PURPOSE OF REVIEW: Tissue regenerative solutions for musculoskeletal disorders have become increasingly important with a growing aged population. Current growth factor treatments often require high dosages with the potential for off-target effects. Growth factor immobilization strategies offer approaches towards alleviating these concerns. This review summarizes current growth factor immobilization techniques (encapsulation, affinity interactions, and covalent binding) and the effects of immobilization on growth factor loading, release, and bioactivity. RECENT FINDINGS: The breadth of immobilization techniques based on encapsulation, affinity, and covalent binding offer multiple methods to improve the therapeutic efficacy of growth factors by controlling bioactivity and release. Growth factor immobilization strategies have evolved to more complex systems with the capacity to load and release multiple growth factors with spatiotemporal control. The advancements in immobilization strategies allow for development of new, complex musculoskeletal tissue treatment strategies with improved spatiotemporal control of loading, release, and bioactivity.

Multiomics characterization of mesenchymal stromal cells cultured in monolayer and as aggregates.

Mesenchymal stromal cells (MSCs) have failed to consistently demonstrate their therapeutic efficacy in clinical trials, due in part to variability in culture conditions used for their production. Of various culture conditions used for MSC production, aggregate culture has been shown to improve secretory capacity (a putative mechanism of action in vivo) compared with standard monolayer culture. The purpose of this study was to perform multiomics characterization of MSCs cultured in monolayer and as aggregates to identify aspects of cell physiology that differ between these culture conditions to begin to understand cellular-level changes that might be related to secretory capacity. Targeted secretome characterization was performed on multiple batches of MSC-conditioned media, while nontargeted proteome and metabolome characterization was performed and integrated to identify cellular processes differentially regulated between culture conditions. Secretome characterization revealed a reduction in MSC batch variability when cultured as aggregates. Proteome and metabolome characterization showed upregulation of multiple protein and lipid metabolic pathways, downregulation of several cytoskeletal processes, and differential regulation of extracellular matrix synthesis. Integration of proteome and metabolome characterization revealed individual lipid metabolites and vesicle-trafficking proteins as key features for discriminating between culture conditions. Overall, this study identifies several aspects of MSC physiology that are altered by aggregate culture. Further exploration of these processes and pathways is needed to determine their potential role in regulating cell secretory capacity.

Combination of Heparin Binding Peptide and Heparin Cell Surface Coatings for Mesenchymal Stem Cell Spheroid Assembly.

Microtissues containing multiple cell types have been used in both in vitro models and in vivo tissue repair applications. However, to improve throughput, there is a need to develop a platform that supports self-assembly of a large number of 3D microtissues containing multiple cell types in a dynamic suspension system. Thus, the objective of this study was to exploit the binding interaction between the negatively charged glycosaminoglycan, heparin, and a known heparin binding peptide to establish a method that promotes assembly of mesenchymal stem cell (MSC) spheroids into larger aggregates. We characterized heparin binding peptide (HEPpep) and heparin coatings on cell surfaces and determined the specificity of these coatings in promoting assembly of MSC spheroids in dynamic culture. Overall, combining spheroids with both coatings promoted up to 70 ± 11% of spheroids to assemble into multiaggregate structures, as compared to only 10 ± 4% assembly when cells having the heparin coating were cultured with cells coated with a scrambled peptide. These results suggest that this self-assembly method represents an exciting approach that may be applicable for a wide range of applications in which cell aggregation is desired.

Cell Surface Access Is Modulated by Tethered Bottlebrush Proteoglycans.

The hyaluronan-rich pericellular matrix (PCM) plays physical and chemical roles in biological processes ranging from brain plasticity, to adhesion-dependent phenomena such as cell migration, to the onset of cancer. This study investigates how the spatial distribution of the large negatively charged bottlebrush proteoglycan, aggrecan, impacts PCM morphology and cell surface access. The highly localized pericellular milieu limits transport of nanoparticles in a size-dependent fashion and sequesters positively charged molecules on the highly sulfated side chains of aggrecan. Both rat chondrocyte and human mesenchymal stem cell PCMs possess many unused binding sites for aggrecan, showing a 2.5x increase in PCM thickness from ∼7 to ∼18 µm when provided exogenous aggrecan. Yet, full extension of the PCM occurs well below aggrecan saturation. Hence, cells equipped with hyaluronan-rich PCM can in principle manipulate surface accessibility or sequestration of molecules by tuning the bottlebrush proteoglycan content to alter PCM porosity and the number of electrostatic binding sites.

Effect of selective heparin desulfation on preservation of bone morphogenetic protein-2 bioactivity after thermal stress.

Bone morphogenetic protein-2 (BMP-2) plays an important role in bone and cartilage formation and is of interest in regenerative medicine. Heparin can interact electrostatically with BMP-2 and thus has been explored for controlled release and potential stabilization of this growth factor in vivo. However, in its natively sulfated state, heparin has potent anticoagulant properties that may limit its use. Desulfation reduces anticoagulant properties, but may impact heparin's ability to interact and protect BMP-2 from denaturation. The goal of this study was to characterize three selectively desulfated heparin species (N-desulfated (Hep(-N)), 6-O,N-desulfated (Hep(-N,-6O)), and completely desulfated heparin (Hep(-))) and determine if the sulfation level of heparin affected the level of BMP-2 bioactivity after heat treatment at 65 °C. BMP-2 bioactivity was evaluated using the established C2C12 cell assay. The resulting alkaline phosphatase activity data demonstrated that native heparin maintained a significant amount of BMP-2 bioactivity and the effect appeared to be heparin concentration dependent. Although all three had the same molecular charge as determined by zeta potential measurements, desulfated heparin derivatives Hep(-N) and Hep(-N,-6O) were not as effective as native heparin in maintaining BMP-2 bioactivity (only ~35% of original activity remained in both cases). These findings can be used to better select desulfated heparin species that exhibit low anticoagulant activity while extending the half-life of BMP-2 in solution and in delivery systems.

Chondroitin sulfate microparticles modulate transforming growth factor-ß1-induced chondrogenesis of human mesenchymal stem cell spheroids.

Mesenchymal stem cells (MSCs) have been previously explored as a part of cell-based therapies for the repair of damaged cartilage. Current MSC chondrogenic differentiation strategies employ large pellets; however, we have developed a technique to form small MSC aggregates (500-1,000 cells) that can reduce transport barriers while maintaining a multicellular structure analogous to cartilaginous condensations. The objective of this study was to examine the effects of incorporating chondroitin sulfate methacrylate (CSMA) microparticles (MPs) within small MSC spheroids cultured in the presence of transforming growth factor (TGF)-ß1 on chondrogenesis. Spheroids with MPs induced earlier increases in collagen II and aggrecan gene expression (chondrogenic markers) than spheroids without MPs, although no large differences in immunostaining for these matrix molecules were observed by day 21 between these groups. Collagen I and X were also detected in the extracellular matrix (ECM) of all spheroids by immunostaining. Interestingly, histology revealed that CSMA MPs clustered together near the center of the MSC spheroids and induced circumferential alignment of cells and ECM around the material core. This study demonstrates the use of CSMA materials to further examine the effects of matrix molecules on MSC phenotype as well as potentially direct differentiation in a more spatially controlled manner that better mimics the architecture of specific musculoskeletal tissues.

Effects of Release of TSG-6 from Heparin Hydrogels on Supraspinatus Muscle Regeneration.

Muscle degeneration after rotator cuff tendon tear is a significant clinical problem. In these experiments, we developed a poly(ethylene glycol)-based injectable granular hydrogel containing two heparin derivatives (fully sulfated (Hep) and fully desulfated (Hep-)) as well as a matrix metalloproteinase-sensitive peptide to promote sustained release of Tumor Necrosis Factor Stimulated Gene 6 (TSG-6) over 14+ days in vivo in a rat model of rotator cuff muscle injury. The hydrogel formulations demonstrated similar release profiles in vivo , thus facilitating comparisons between delivery from heparin derivatives on level of tissue repair in two different areas of muscle (near the myotendious junction (MTJ) and in the muscle belly (MB)) that have been shown previously to have differing responses to rotator cuff tendon injury. We hypothesized that sustained delivery of TSG-6 would enhance the anti-inflammatory response following rotator cuff injury through macrophage polarization, and that release from a fully sulfated heparin derivative (Hep) would potentiate this effect throughout the muscle. Inflammatory/immune cells, satellite cells, and fibroadipogenic progenitor cells, were analyzed by flow cytometery 3 and 7 days after injury and hydrogel injection, while metrics of muscle healing were examined via immunohistochemistry up to Day 14. Results showed controlled delivery of TSG-6 from Hep caused heightened macrophage response (Day 14 macrophages, 4.00 ± 1.85% single cells, M2a, 3.27 ± 1.95% single cells) and increased markers of early muscle regeneration (embryonic heavy chain staining) by Day 7, particularly in the MTJ region of the muscle, compared to release from desulfated heparin hydrogels. This work provides a novel strategy for localized, controlled delivery of TSG-6 to enhance muscle healing after rotator cuff tear. IMPACT STATEMENT: Rotator cuff tear is a significant problem that can cause muscle degeneration. In this study, a hydrogel particle system was developed for sustained release of an anti-inflammatory protein, Tumor Necrosis Factor Stimulated Gene 6 (TSG-6), to injured muscle. Release of the protein from a fully sulfated heparin hydrogel-based carrier demonstrated greater changes in amount inflammatory cells and more early regenerative effects than a less-sulfated carrier. Thus, this work provides a novel strategy for localized, controlled delivery of an anti-inflammatory protein to enhance muscle healing after rotator cuff tear.

Development and Characterization of Heparin-Containing Hydrogel/3D-Printed Scaffold Composites for Craniofacial Reconstruction.

Regeneration of cartilage and bone tissues remains challenging in tissue engineering due to their complex structures, and the need for both mechanical support and delivery of biological repair stimuli. Therefore, the goal of this study was to develop a composite scaffold platform for anatomic chondral and osteochondral repair using heparin-based hydrogels to deliver small molecules within 3D-printed porous scaffolds that provide structure, stiffness, and controlled biologic delivery. We designed a mold-injection system to combine hydrolytically degradable hydrogels and 3D-printed scaffolds that could be employed rapidly (< 30 min) in operating room settings (~23 °C). Micro-CT analysis demonstrated the effectiveness of our injection system through homogeneously distributed hydrogel within the pores of the scaffolds. Hydrogels and composite scaffolds exhibited efficient loading (~94%) of a small positively charged heparin-binding molecule (crystal violet) with sustained release over 14 days and showed high viability of encapsulated porcine chondrocytes over 7 days. Compression testing demonstrated nonlinear viscoelastic behavior where tangent stiffness decreased with scaffold porosity (porous scaffold tangent stiffness: 70%: 4.9 MPa, 80%: 1.5 MPa, and 90%: 0.20 MPa) but relaxation was not affected. Lower-porosity scaffolds (70%) showed stiffness similar to lower ranges of trabecular bone (4-8 MPa) while higher-porosity scaffolds (80% and 90%) showed stiffness similar to auricular cartilage (0.16-2 MPa). Ultimately, this rapid composite scaffold fabrication method may be employed in the operating room and utilized to control biologic delivery within load-bearing scaffolds.

Bone Marrow Mobilization and Local Stromal Cell-Derived Factor-1α Delivery Enhances Nascent Supraspinatus Muscle Fiber Growth.

Rotator cuff tear is a significant problem that leads to poor clinical outcomes due to muscle degeneration after injury. The objective of this study was to synergistically increase the number of proregenerative cells recruited to injure rotator cuff muscle through a novel dual treatment system, consisting of a bone marrow mobilizing agent (VPC01091), hypothesized to "push" prohealing cells into the blood, and localized delivery of stromal cell-derived factor-1α (SDF-1α), to "pull" the cells to the injury site. Immediately after rotator cuff tendon injury in rat, the mobilizing agent was delivered systemically, and SDF-1α-loaded heparin-based microparticles were injected into the supraspinatus muscle. Regenerative and degenerative changes to supraspinatus muscle and the presence of inflammatory/immune cells, mesenchymal stem cells (MSCs), and satellite cells were assessed via flow cytometry and histology for up to 21 days. After dual treatment, significantly more MSCs (31.9 ± 8.0% single cells) and T lymphocytes (6.7 ± 4.3 per 20 × field of view) were observed in supraspinatus muscle 7 days after injury and treatment compared to injury alone (14.4 ± 6.5% single cells, 1.2 ± 0.7 per 20 × field of view), in addition to an elevated M2:M1 macrophage ratio (3.0 ± 0.5), an indicator of a proregenerative environment. These proregenerative cellular changes were accompanied by increased nascent fiber formation (indicated by embryonic myosin heavy chain staining) at day 7 compared to SDF-1α treatment alone, suggesting that this method may be a promising strategy to influence the early cellular response in muscle and promote a proregenerative microenvironment to increase muscle healing after severe rotator cuff tear.

Biological properties of dehydrated human amnion/chorion composite graft: implications for chronic wound healing.

Human amnion/chorion tissue derived from the placenta is rich in cytokines and growth factors known to promote wound healing; however, preservation of the biological activities of therapeutic allografts during processing remains a challenge. In this study, PURION® (MiMedx, Marietta, GA) processed dehydrated human amnion/chorion tissue allografts (dHACM, EpiFix®, MiMedx) were evaluated for the presence of growth factors, interleukins (ILs) and tissue inhibitors of metalloproteinases (TIMPs). Enzyme-linked immunosorbent assays (ELISA) were performed on samples of dHACM and showed quantifiable levels of the following growth factors: platelet-derived growth factor-AA (PDGF-AA), PDGF-BB, transforming growth factor α (TGFα), TGFß1, basic fibroblast growth factor (bFGF), epidermal growth factor (EGF), placental growth factor (PLGF) and granulocyte colony-stimulating factor (GCSF). The ELISA assays also confirmed the presence of IL-4, 6, 8 and 10, and TIMP 1, 2 and 4. Moreover, the relative elution of growth factors into saline from the allograft ranged from 4% to 62%, indicating that there are bound and unbound fractions of these compounds within the allograft. dHACM retained biological activities that cause human dermal fibroblast proliferation and migration of human mesenchymal stem cells (MSCs) in vitro. An in vivo mouse model showed that dHACM when tested in a skin flap model caused mesenchymal progenitor cell recruitment to the site of implantation. The results from both the in vitro and in vivo experiments clearly established that dHACM contains one or more soluble factors capable of stimulating MSC migration and recruitment. In summary, PURION® processed dHACM retains its biological activities related to wound healing, including the potential to positively affect four distinct and pivotal physiological processes intimately involved in wound healing: cell proliferation, inflammation, metalloproteinase activity and recruitment of progenitor cells. This suggests a paracrine mechanism of action for dHACM when used for wound healing applications.

Poly(ethylene glycol)-Based Hydrogel Microcarriers Alter Secretory Activity of Genetically Modified Mesenchymal Stromal Cells.

In order to scale up culture therapeutic cells, such as mesenchymal stromal cells (MSCs), culture in suspension bioreactors using microcarriers (µCs) is preferred. However, the impact of microcarrier type on the resulting MSC secretory activity has not been investigated. In this study, two poly(ethylene glycol) hydrogel formulations with different swelling ratios (named "stiffer" and "softer") were fabricated as µC substrates to culture MSCs and MSCs genetically modified to express the interleukin-1 receptor antagonist (IL-1Ra-MSCs). Changes in cell number, secretory and angiogenic activity, and changes in MAPK signaling were evaluated when cultured on hydrogel µCs, as well as on tissue culture plastic-based Synthemax µCs. We demonstrated that culture on stiffer µCs increased secretion of IL-1Ra compared to culture on Synthemax µCs by IL-1Ra-MSCs by 1.2- to 1.6-fold, as well as their in vitro angiogenic activity, compared to culture on Synthemax µCs, while culture on both stiffer and softer µCs altered the secretion of several other factors compared to culture on Synthemax µCs. Changes in angiogenic activity corresponded with increased gene expression and secretion of hepatocyte growth factor by MSCs cultured on softer µCs by 2.5- to 6-fold compared to MSCs cultured on Synthemax µCs. Quantification of phosphoprotein signaling with the MAPK pathway revealed broad reduction of pathway activation by IL-1Ra-MSCs cultured on both stiffer and softer µCs compared to Synthemax, where phosphorylated c-Jun, ATF2, and MEK1 were reduced specifically on softer µCs. Overall, this study showed that µC surfaces can influence the secretory activity of genetically modified MSCs and identified associated changes in MAPK pathway signaling, which is a known central regulator of cytokine secretion.

Development and characterization of Factor Xa-responsive materials for applications in cell culture and biologics delivery.

Stimuli-responsive biomaterials may be used to better control the release of bioactive molecules or cells for applications involving drug delivery and controlled cell release. In this study, we developed a Factor Xa (FXa)-responsive biomaterial capable of controlled release of pharmaceutical agents and cells from in vitro culture. FXa-cleavable substrates were formed as hydrogels that degraded in response to FXa enzyme over several hours. Hydrogels were shown to release both heparin and a model protein in response to FXa. Additionally, RGD-functionalized FXa-degradable hydrogels were used to culture mesenchymal stromal cells (MSCs), enabling FXa-mediated cell dissociation from hydrogels in a manner that preserved multicellular structures. Harvesting MSCs using FXa-mediated dissociation did not influence their differentiation capacity or indoleamine 2,3-dioxygenase (IDO) activity (a measure of immunomodulatory capacity). In all, this FXa-degradable hydrogel is a novel responsive biomaterial system that may be used for on-demand drug delivery, as well as for improving processes for in vitro culture of therapeutic cells.

Microfluidic Platform for Microparticle Fabrication and Release of a Cathepsin Inhibitor.

Cathepsins are a family of cysteine proteases responsible for a variety of homeostatic functions throughout the body, including extracellular matrix remodeling, and have been implicated in a variety of degenerative diseases. However, clinical trials using systemic administration of cathepsin inhibitors have been abandoned due to side effects, so local delivery of cathepsin inhibitors may be advantageous. In these experiments, a novel microfluidic device platform was developed that can synthesize uniform, hydrolytically degradable microparticles from a combination of poly(ethylene glycol) diacrylate (PEGDA) and dithiothreitol (DTT). Of the formulations examined, the 10-polymer weight percentage 10 mM DTT formulation degraded after 77 days in vitro. A modified assay using the DQ Gelatin Fluorogenic Substrate was used to demonstrate sustained release and bioactivity of a cathepsin inhibitor (E-64) released from hydrogel microparticles over 2 weeks in vitro (up to ∼13 µg/mL released with up to ∼40% original level of inhibition remaining at day 14). Altogether, the technologies developed in this study will allow a small-molecule, broad cathepsin inhibitor E-64 to be released in a sustained manner for localized inhibition of cathepsins for a wide variety of diseases.

Differentiation of mesenchymal stem cells in heparin-containing hydrogels via coculture with osteoblasts.

The therapeutic potency of delivered mesenchymal stem cells (MSCs) in tissue engineering applications may be improved by priming cells toward a differentiated state via coculture with native, differentiated cells prior to implantation; however, there is a lack of understanding in what may be the most efficacious method. The objective of this study was to investigate the role of negatively-charged heparin in priming hydrogel-encapsulated MSCs toward the osteoblastic lineage during coculture with a monolayer of osteoblasts in the absence of dexamethasone. MSCs encapsulated with higher amounts of heparin and cocultured with osteoblasts exhibited an over 36-fold increase in alkaline phosphatase activity and 13-fold increase in calcium accumulation by day 21, compared to MSCs cocultured with MSCs at the same heparin content. Moreover, hydrogels with higher amounts of heparin and cocultured with osteoblasts exhibited enhanced mineralization on the edges, suggesting that heparin may be important in sequestering osteoblast-secreted soluble factors, particularly on the surfaces of hydrogels. The ability of heparin to selectively interact with soluble positively-charged proteins from the surroundings was confirmed through protein labeling and microscopy. These results suggest that heparin-containing hydrogels as part of a coculture system can be utilized as a versatile platform to study and enhance priming of MSCs toward various cell types for a wide variety of regenerative medicine-based therapies.

Aggregation of bovine anterior cruciate ligament fibroblasts or marrow stromal cells promotes aggrecan production.

The development of a tissue-engineered alternative for current ligament grafts requires the creation of a fibrocartilaginous interface between the engineered ligament midsubstance and bone tissue. Therefore, the focus of this study was to examine the potential for cartilaginous extracellular matrix (ECM) formation by altering culture parameters for bovine anterior cruciate ligament (ACL) fibroblasts and marrow stromal cells (MSCs). Specifically, cells were cultured without chondrogenic media supplements on aggrecan-coated surfaces, tissue culture-treated control surfaces, and nonadhesive surfaces that promoted cell aggregation, and examined over 14 days. Aggrecan-coated surfaces promoted the aggregation of ACL fibroblasts and MSCs within 24 h after seeding. Aggrecan gene expression was significantly upregulated in cell aggregates, regardless of how cell clustering was induced, with as much as 10.9 ± 1.2-fold upregulation in ACL fibroblasts and 9.7 ± 1.1-fold in MSCs after 3 days, compared to control surfaces. Dimethylmethylene blue (DMMB) results and immunostaining verified the presence of aggrecan in ACL fibroblast and MSC aggregates throughout the culture period. Results indicate that ACL fibroblasts retained the ability to alter their gene expression and produce aggrecan, though MSCs, in general, had a more consistent response to aggregation. These findings support the use of aggregate-inducing materials to encourage production of aggrecan and suggest that altering the degree of clustering could produce a range of phenotypes from a single cell source. As such, this represents a first step which may inform future approaches to producing tissue-engineered ligament grafts.

Culture Substrates for Improved Manufacture of Mesenchymal Stromal Cell Therapies.

Recent developments in mesenchymal stromal cell (MSC) therapies have increased the demand for tools to improve their manufacture, including the selection of optimal culture substrate materials. While many clinical manufacturers use planar tissue culture plastic (TCP) surfaces for MSC production, others have begun exploring the use of alternative culture substrates that present a variety of spatial, mechanical, and biochemical cues that influence cell expansion and resulting cell quality. In this review, the effects of culture and material properties distinct from traditional planar TCP surfaces on MSC proliferation, surface marker expression, and commonly used indications for therapeutic potency are examined. The different properties summarized include the use of alternative culture formats such as cellular aggregates or 3D scaffolds, as well as the effects of culture substrate stiffness and presentation of specific adhesive ligands and topographical cues. Specific substrate properties can be related to greater cell expansion and improvement in specific therapeutic functionalities, demonstrating the utility of culture materials in further improving the clinical-scale manufacture of highly secretory MSC products.

Injection of Micronized Human Amnion/Chorion Membrane Results in Increased Early Supraspinatus Muscle Regeneration in a Chronic Model of Rotator Cuff Tear.

Surgical repair of severe rotator cuff tear often results in retear due to unaddressed muscle degeneration. The objective of this study was to test the regenerative potential of micronized dehydrated Human Amnion/Chorion Membrane (dHACM), in a clinically relevant delayed reattachment model of rotator cuff repair. Micronized dHACM was injected into rat supraspinatus muscle during tendon re-attachment surgery, three weeks after original tendon injury. One week after material injection, inflammatory and mesenchymal stem cell infiltration into supraspinatus muscles was assessed via flow cytometry. Histological methods were utilized to assess structural and regenerative changes in muscle one and three weeks after material injection. Micronized dHACM injection resulted in increased M1-like macrophages (17.1 [Formula: see text] fold change over contralateral controls) and regenerating muscle fibers (4.3% vs 1.7% in saline treated muscles) one week after injection compared to saline treated muscles. Tendon reattachment itself exhibited intrinsic healing in this model, demonstrated by a general return of muscle weight and reduced fibrosis. Our results indicate that injection of micronized dHACM may initiate an inflammatory response in degenerated muscle that promotes early muscle regeneration, and that our animal model may be a suitable platform for studying treatments in muscle at early timepoints, before intrinsic healing occurs.

Hydrogel Culture Surface Stiffness Modulates Mesenchymal Stromal Cell Secretome and Alters Senescence.

Current cell culture surfaces used for the expansion and production of mesenchymal stromal cells (MSCs) are not optimized for the production of highly secretory and nonsenescent cells. In this study, we used poly (ethylene glycol) hydrogel substrates with tunable mechanical and biochemical properties to screen the effect of culture surfaces on pro-regenerative secretome by multiplex enzyme-linked immunosorbent assay, proliferation by PicoGreen DNA analysis, and senescence by senescence-associated ß-galactosidase activity. We demonstrate that MSCs cultured on 30 kPa hydrogels, regardless of biochemical functionalization, broadly enhanced the secretion of immunomodulatory and regenerative factors versus stiffer 100 kPa or tissue culture plastic surfaces, but did not support robust proliferation. In contrast, culture on 100 kPa hydrogel surfaces promoted proliferation at a similar level and did not substantially alter the amount of secreted factors as compared with tissue culture plastic. Culture on integrin-engaging, cadherin-engaging, and hyaluronic acid-containing 30 kPa substrates enhanced MSC-conditioned media (CM) angiogenic activity in a human umbilical vein endothelial cell tube formation assay and human THP-1 monocyte chemoattraction in a transwell assay. However, 30 kPa substrate culture did not impact the myogenic activity of MSC CM in a C2C12 myoblast tube formation assay. Culture on selected 100 kPa surfaces enhanced CM angiogenic activity and monocyte chemotaxis, but not myogenic activity. Serial culture on 100 kPa RGD hydrogel surfaces significantly reduced senescence in MSCs versus tissue culture plastic, while maintaining the capacity of the cells to enhance their secretome in response to 30 kPa surfaces. Thus, hydrogel substrates that exhibit stiffness orders of magnitude lower than standard tissue culture plastic can serve as novel surfaces for the production of MSCs with an improved therapeutic secretory capacity and reduced senescence. Impact statement The success of mesenchymal stromal cell (MSC)-based therapies is dependent on the manufacture of a large number of cells with high therapeutic potency. Among the culture surfaces tested in this study, we demonstrate that substrate stiffness rather than biochemical functionalization predominantly guides changes in MSC proliferation and secretory capacity. We have identified substrate parameters to support MSC proliferation, enhance secretion of paracrine factors, and to reduce replicative senescence. By maximizing secretory capacity and reducing senescence through the choice of hydrogel culture materials, these findings have great potential to improve the large-scale production of therapeutic MSCs.

Sub-nanoliter metabolomics via mass spectrometry to characterize volume-limited samples.

The human metabolome provides a window into the mechanisms and biomarkers of various diseases. However, because of limited availability, many sample types are still difficult to study by metabolomic analyses. Here, we present a mass spectrometry (MS)-based metabolomics strategy that only consumes sub-nanoliter sample volumes. The approach consists of combining a customized metabolomics workflow with a pulsed MS ion generation method, known as triboelectric nanogenerator inductive nanoelectrospray ionization (TENGi nanoESI) MS. Samples tested with this approach include exhaled breath condensate collected from cystic fibrosis patients as well as in vitro-cultured human mesenchymal stromal cells. Both test samples are only available in minimum amounts. Experiments show that picoliter-volume spray pulses suffice to generate high-quality spectral fingerprints, which increase the information density produced per unit sample volume. This TENGi nanoESI strategy has the potential to fill in the gap in metabolomics where liquid chromatography-MS-based analyses cannot be applied. Our method opens up avenues for future investigations into understanding metabolic changes caused by diseases or external stimuli.

Effect of swelling ratio of injectable hydrogel composites on chondrogenic differentiation of encapsulated rabbit marrow mesenchymal stem cells in vitro.

An injectable, biodegradable hydrogel composite of oligo(poly(ethylene glycol) fumarate) (OPF) and gelatin microparticles (MPs) has been investigated as a cell and growth factor carrier for cartilage tissue engineering applications. In this study, hydrogel composites with different swelling ratios were prepared by cross-linking OPF macromers with poly(ethylene glycol) (PEG) repeating units of varying molecular weights from 1000 approximately 35000. Rabbit marrow mesenchymal stem cells (MSCs) and MPs loaded with transforming growth factor-beta1 (TGF-beta1) were encapsulated in the hydrogel composites to examine the effect of the swelling ratio of the hydrogel composites on the chondrogenic differentiation of encapsulated rabbit marrow MSCs both in the presence and in the absence of TGF-beta1. The swelling ratio of the hydrogel composites increased as the PEG molecular weight in the OPF macromers increased. Chondrocyte-specific genes were expressed at higher levels in groups containing TGF-beta1-loaded MPs and varied with the swelling ratio of the hydrogel composites. OPF hydrogel composites with PEG repeating units of molecular weight 35000 and 10000 with TGF-beta1-loaded MPs exhibited a 159 +/- 95- and a 89 +/- 31-fold increase in type II collagen gene expression at day 28, respectively, while OPF hydrogel composites with PEG repeating units of molecular weight 3000 and 1000 with TGF-beta1-loaded MPs showed a 27 +/- 10- and a 17 +/- 7-fold increase in type II collagen gene expression, respectively, as compared to the composites with blank MPs at day 0. The results indicate that chondrogenic differentiation of encapsulated rabbit marrow MSCs within OPF hydrogel composites could be affected by their swelling ratio, thus suggesting the potential of OPF composite hydrogels as part of a novel strategy for controlling the differentiation of stem cells.