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 a rat model of rotator cuff muscle injury. The hydrogel formulations demonstrated similar release profiles , thus facilitating comparisons between delivery from heparin derivatives on the 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.

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

Unlabelled: 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 a rat model of rotator cuff muscle injury. The hydrogel formulations demonstrated similar release profiles , 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.

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).

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).

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.

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).

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.

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.

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.

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.

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.

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 β-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.

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.

Sequential, but not Concurrent, Incubation of Cathepsin K and L with Type I Collagen Results in Extended Proteolysis.

Degradation of extracellular matrix (ECM) during tendinopathy is, in part, mediated by the collagenolytic cathepsin K (catK) and cathepsin L (catL), with a temporal component to their activity. The objective of this study was to determine how catK and catL act in concert or in conflict to degrade collagen and tendon ECM during tissue degeneration. To do so, type I collagen gels or ECM extracted from apolipoprotein E deficient mouse Achilles tendons were incubated with catK and catL either concurrently or sequentially, incubating catK first, then catL after a delayed time period.

Full-thickness rotator cuff tear in rat results in distinct temporal expression of multiple proteases in tendon, muscle, and cartilage.

The etiology of joint tissue degeneration following rotator cuff tear remains unclear. Thus, the purpose of this study was to understand the timeline of protease activity in the soft tissues of the shoulder (tendon, muscle, and cartilage) that may lead to down-stream degeneration following rotator cuff tear. A well-established rat model involving suprascapular nerve denervation and supraspinatus/infraspinatus tendon transection was employed.

Dual Affinity Heparin-Based Hydrogels Achieve Pro-Regenerative Immunomodulation and Microvascular Remodeling.

The immune response to biomaterial implants critically regulates functional outcomes such as vascularization, transplant integration/survival, and fibrosis. To create "immunologically smart" materials, the host-material response may be engineered to optimize the recruitment of pro-regenerative leukocyte subsets which mature into corresponding wound-healing macrophages. We have recently identified a unique feature of pro-regenerative Ly6C monocytes that is a higher expression of both the bioactive lipid receptor sphingosine-1-phosphate receptor 3 (S1PR3) and the stromal derived factor-1α (SDF-1α) receptor CXCR4.

Intra-articular TSG-6 delivery from heparin-based microparticles reduces cartilage damage in a rat model of osteoarthritis.

As a potential treatment for osteoarthritis (OA), we have developed injectable and hydrolytically degradable heparin-based biomaterials with tunable sulfation for the intra-articular delivery of tumor necrosis factor-alpha stimulated gene-6 (TSG-6), a protein known to inhibit plasmin which may degrade extracellular matrix within OA joints. We first assessed the effect of heparin sulfation on TSG-6 anti-plasmin activity and found that while fully sulfated (Hep) and heparin desulfated at only the N position (Hep-N) significantly enhanced TSG-6 bioactivity in vitro, fully desulfated heparin (Hep-) had no effect, indicating that heparin sulfation plays a significant role in modulating TSG-6 bioactivity. Next, TSG-6 loaded, degradable 10 wt% Hep-N microparticles (MPs) were delivered via intra-articular injection into the knee at 1, 7, and 15 days following medial meniscal transection (MMT) injury in a rat model.

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.

Microparticle-mediated sequestration of cell-secreted proteins to modulate chondrocytic differentiation.

Unlabelled: Protein delivery is often used in tissue engineering applications to control differentiation processes, but is limited by protein instability and cost. An alternative approach is to control the cellular microenvironment through biomaterial-mediated sequestration of cell-secreted proteins important to differentiation. Thus, we utilized heparin-based microparticles to modulate cellular differentiation via protein sequestration in an in vitro model system of endochondral ossification.

Core-shell microparticles for protein sequestration and controlled release of a protein-laden core.

Unlabelled: Development of multifunctional biomaterials that sequester, isolate, and redeliver cell-secreted proteins at a specific timepoint may be required to achieve the level of temporal control needed to more fully regulate tissue regeneration and repair. In response, we fabricated core-shell heparin-poly(ethylene-glycol) (PEG) microparticles (MPs) with a degradable PEG-based shell that can temporally control delivery of protein-laden heparin MPs. Core-shell MPs were fabricated via a re-emulsification technique and the number of heparin MPs per PEG-based shell could be tuned by varying the mass of heparin MPs in the precursor PEG phase.

Supraspinatus tendon overuse results in degenerative changes to tendon insertion region and adjacent humeral cartilage in a rat model.

The etiology of rotator cuff tendon overuse injuries is still not well understood. Furthermore, how this overuse injury impacts other components of the glenohumeral joint, including nearby articular cartilage, is also unclear. Therefore, this study sought to better understand the time course of tendon protease activity in a rat model of supraspinatus overuse, as well as determine effects of 10 weeks of overuse on humeral head articular cartilage.

Hydrolysis and Sulfation Pattern Effects on Release of Bioactive Bone Morphogenetic Protein-2 from Heparin-Based Microparticles.

Glycosaminoglycans (GAGs) such as heparin are promising materials for growth factor delivery due to their ability to efficiently bind positively charged growth factors including bone morphogenetic protein-2 (BMP-2) through their negatively charged sulfate groups. Therefore, the goal of this study was to examine BMP-2 release from heparin-based microparticles (MPs) after first, incorporating a hydrolytically degradable crosslinker and varying heparin content within MPs to alter MP degradation and second, altering the sulfation pattern of heparin within MPs to vary BMP-2 binding and release. Using varied MP formulations, it was found that the time course of MP degradation for 1 wt% heparin MPs was ~4 days slower than 10 wt% heparin MPs, indicating that MP degradation was dependent on heparin content.

Heparin-based hydrogels with tunable sulfation & degradation for anti-inflammatory small molecule delivery.

Sustained release of anti-inflammatory agents remains challenging for small molecule drugs due to their low molecular weight and hydrophobicity. Therefore, the goal of this study was to control the release of a small molecule anti-inflammatory agent, crystal violet (CV), from hydrogels fabricated with heparin, a highly sulfated glycosaminoglycan capable of binding positively-charged molecules such as CV. In this system, both electrostatic interactions between heparin and CV and hydrogel degradation were tuned simultaneously by varying the level of heparin sulfation and varying the amount of dithiothreitol within hydrogels, respectively.

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.