Heparin Hydrogel Research Articles

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 invivo in a rat model of rotator cuff muscle injury. The hydrogel formulations demonstrated similar release profiles invivo, 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. 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 Hep would potentiate this effect throughout the muscle. Inflammatory/immune cells, satellite cells, and fibroadipogenic progenitor cells were analyzed by flow cytometry 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 7 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. This work provides a novel strategy for localized, controlled delivery of TSG-6 to enhance muscle healing after rotator cuff tear.

Interleukin-4-Loaded Heparin Hydrogel Regulates Macrophage Polarization to Promote Osteogenic Differentiation.

In bone tissue engineering, biological scaffolds are designed with structural and functional properties that closely resemble the extracellular environment, aiming to establish a microenvironment conducive to osteogenesis. Macrophages hold significant potential for promoting osteogenesis and modulating the biological behavior of tumor cells. Multiple coculture experiments of macrophages and osteoblasts have demonstrated that macrophage polarization significantly impacts osteogenesis. Therefore, exploring bone biomaterials that can modulate macrophage polarization holds great clinical significance. In this study, heparin was modified with maleimide and was used as a raw material to form a hydrogel with 4-am-PEG-SH. The compound was used to polarize macrophages and promote osteogenesis after combining with interleukin 4 (IL-4) by taking advantage of the electronegativity of heparin. The results revealed overexpressed M2 macrophage-related phenotypic genes and cocultivation with MC3T3-E1 cells demonstrated the osteogenesis-promoting effect of the loaded IL-4 heparin hydrogel. Previous research reported that hydrogel loaded with IL-4 can be used as a biomaterial for osteogenesis promotion. Heparin materials used in this paper are derived from clinically anticoagulant drugs and feature a simple operation. The synthesized hydrogel effectively binds cytokines, regulates macrophages to induce osteogenesis and has many potential clinical applications.

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

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.

Enhancing thromboresistance of neurovascular nickel-titanium devices with responsive heparin hydrogel coatings

BackgroundNeurointerventional devices, particularly laser-cut thin-strut stents made of self-expanding nickel-titanium alloy, are increasingly utilized for endovascular applications in intracranial arteries and dural venous sinuses. Preventing thrombosis and stroke necessitates systemic...

A Tumor Microenvironment Model of Pancreatic Cancer to Elucidate Responses toward Immunotherapy (Adv. Healthcare Mater. 14/2023)

3D Cancer Models In article 2201907 by Daniela Loessner and co-workers, the tumor microenvironment of pancreatic cancer is engineered using star poly(ethylene glycol) (PEG)–heparin hydrogels, that are functionalized with integrin-binding and protease-responsive peptides to stimulate cell proliferation and migration. The coculture of cancer cells and patient-derived stromal cells mimics the multicellular composition of tumor tissues and cancer–immune interactions and allows the testing of immunomodulatory treatments in combination with chemotherapy.

Function of hepatocyte spheroids in bioactive microcapsules is enhanced by endogenous and exogenous hepatocyte growth factor

The ability to maintain functional hepatocytes has important implications for bioartificial liver development, cell-based therapies, drug screening, and tissue engineering. Several approaches can be used to restore hepatocyte function in vitro, including coating a culture substrate with extracellular matrix (ECM), encapsulating cells within biomimetic gels (Collagen- or Matrigel-based), or co-cultivation with other cells. This paper describes the use of bioactive heparin-based core-shell microcapsules to form and cultivate hepatocyte spheroids. These microcapsules are comprised of an aqueous core that facilitates hepatocyte aggregation into spheroids and a heparin hydrogel shell that binds and releases growth factors. We demonstrate that bioactive microcapsules retain and release endogenous signals thus enhancing the function of encapsulated hepatocytes. We also demonstrate that hepatic function may be further enhanced by loading exogenous hepatocyte growth factor (HGF) into microcapsules and inhibiting transforming growth factor (TGF)-β1 signaling. Overall, bioactive microcapsules described here represent a promising new strategy for the encapsulation and maintenance of primary hepatocytes and will be beneficial for liver tissue engineering, regenerative medicine, and drug testing applications.

Exploring the Potential of PEG-Heparin Hydrogels to Support Long-Term Ex Vivo Culture of Patient-Derived Breast Explant Tissues.

Breast cancer is a complex, highly heterogenous, and dynamic disease and the leading cause of cancer-related death in women worldwide. Evaluation of the heterogeneity of breast cancer and its various subtypes is crucial to identify novel treatment strategies that can overcome the limitations of currently available options. Explant cultures of human mammary tissue have been known to provide important insights for the study of breast cancer structure and phenotype as they include the context of the surrounding microenvironment, allowing for the comprehensive exploration of patient heterogeneity. However, the major limitation of currently available techniques remains the short-term viability of the tissue owing to loss of structural integrity. Here, an ex vivo culture model using star-shaped poly(ethylene glycol) and maleimide-functionalized heparin (PEG-HM) hydrogels to provide structural support to the explant cultures is presented. The mechanical support allows the culture of the human mammary tissue for up to 3 weeks and prevent disintegration of the cellular structures including the epithelium and surrounding stromal tissue. Further, maintenance of epithelial phenotype and hormonal receptors is observed for up to 2 weeks of culture which makes them relevant for testing therapeutic interventions. Through this study, the importance of donor-to-donor variability and intra-patient tissue heterogeneity is reiterated.

A Tumor Microenvironment Model of Pancreatic Cancer to Elucidate Responses toward Immunotherapy.

Pancreatic cancer is a devastating malignancy with minimal treatment options. Standard-of-care therapy, including surgery and chemotherapy, is unsatisfactory, and therapies harnessing the immune system have been unsuccessful in clinical trials. Resistance to therapy and disease progression are mediated by the tumor microenvironment, which contains excessive amounts of extracellular matrix and stromal cells, acting as a barrier to drug delivery. There is a lack of preclinical pancreatic cancer models that reconstruct the extracellular, cellular, and biomechanical elements of tumor tissues to assess responses toward immunotherapy. To address this limitation and explore the effects of immunotherapy in combination with chemotherapy, a multicellular 3D cancer model using a star-shaped poly(ethylene glycol)-heparin hydrogel matrix is developed. Human pancreatic cancer cells, cancer-associated fibroblasts, and myeloid cells are grown encapsulated in hydrogels to mimic key components of tumor tissues, and cell responses toward treatment are assessed. Combining the CD11b agonist ADH-503 with anti-PD-1 immunotherapy and chemotherapy leads to a significant reduction in tumor cell viability, proliferation, metabolic activity, immunomodulation, and secretion of immunosuppressive and tumor growth-promoting cytokines.

Inflammation Self-Limiting Electrospun Fibrous Tape via Regional Immunity for Deep Soft Tissue Repair.

Overexpression of inflammatory cytokines and chemokines occurs at deep soft tissue injury sites impeding the inflammation self-limiting and impairing the tissue remodeling process. Inspired by the electrostatically extracellular matrix (ECM) binding property of the inflammatory signals, an inflammation self-limiting fibrous tape is designed by covalently modifying the thermosensitive methacrylated gelatin (GelMA) and negatively charged methacrylated heparin (HepMA) hydrogel mixture with proper ratio onto the electrospun fibrous membrane by mild alkali hydrolysis and carboxyl-amino condensation reaction to restore inflammation self-limiting and promote tissue repair via regional immunity regulation. While the GelMA guarantees cell compatibility, the negatively charged HepMA successfully adsorbs the inflammatory cytokines and chemokines by electrostatic interactions and inhibits immune cell migration in vitro. Furthermore, in vivo inflammation self-limiting and regional immunity regulation efficacy is evaluated in a rat abdominal hernia model. Reduced local inflammatory cytokines and chemokines in the early stage and increased angiogenesis and ECM remodeling in the later phase confirm that the tape is an approach to maintain an optimal regional immune activation level after soft tissue injury. Overall, the reported electrospun fibrous tape will find its way into clinical transformation and solve the challenges of deep soft tissue injury.

StarPEG–heparin biosensors for rapid and portable diagnostics in complex biofluids

We demonstrate impedance chips modified with starPEG–heparin hydrogels as hemocompatible portable biosensors in portable format.

Mapping Tumor Spheroid Mechanics in Dependence of 3D Microenvironment Stiffness and Degradability by Brillouin Microscopy.

Simple SummaryLittle is known about how cancer cells adapt their mechanical properties in complex 3D microenvironments. Here we generated different types of tumor spheroids within compliant or stiff hydrogels. We then quantitatively mapped the mechanical properties of these spheroids in situ using Brillouin microscopy. Maps acquired for tumor spheroids grown within stiffer hydrogels showed elevated Brillouin shifts, hence spheroids became “stiffer” compared to the ones cultured within compliant gels. The spheroid’s mechanical properties were modulated by various microenvironment properties including matrix stiffness and degradability and the resultant compressive stress but also depending on whether single cells or cell aggregates were analyzed. Moreover, spheroids generated from a panel of invasive breast, prostate and pancreatic cancer cell lines within degradable stiff hydrogels became stiffer and at the same time, less invasive compared to those in compliant hydrogels. Taken together, our findings contribute to a better understanding of the interplay between cancer cells and their microenvironment, which is relevant to better understand cancer progression.Altered biophysical properties of cancer cells and of their microenvironment contribute to cancer progression. While the relationship between microenvironmental stiffness and cancer cell mechanical properties and responses has been previously studied using two-dimensional (2D) systems, much less is known about it in a physiologically more relevant 3D context and in particular for multicellular systems. To investigate the influence of microenvironment stiffness on tumor spheroid mechanics, we first generated MCF-7 tumor spheroids within matrix metalloproteinase (MMP)-degradable 3D polyethylene glycol (PEG)-heparin hydrogels, where spheroids showed reduced growth in stiffer hydrogels. We then quantitatively mapped the mechanical properties of tumor spheroids in situ using Brillouin microscopy. Maps acquired for tumor spheroids grown within stiff hydrogels showed elevated Brillouin frequency shifts (hence increased longitudinal elastic moduli) with increasing hydrogel stiffness. Maps furthermore revealed spatial variations of the mechanical properties across the spheroids’ cross-sections. When hydrogel degradability was blocked, comparable Brillouin frequency shifts of the MCF-7 spheroids were found in both compliant and stiff hydrogels, along with similar levels of growth-induced compressive stress. Under low compressive stress, single cells or free multicellular aggregates showed consistently lower Brillouin frequency shifts compared to spheroids growing within hydrogels. Thus, the spheroids’ mechanical properties were modulated by matrix stiffness and degradability as well as multicellularity, and also to the associated level of compressive stress felt by tumor spheroids. Spheroids generated from a panel of invasive breast, prostate and pancreatic cancer cell lines within degradable stiff hydrogels, showed higher Brillouin frequency shifts and less cell invasion compared to those in compliant hydrogels. Taken together, our findings contribute to a better understanding of the interplay between cancer cells and microenvironment mechanics and degradability, which is relevant to better understand cancer progression.

Injectable, antioxidative, and neurotrophic factor-deliverable hydrogel for peripheral nerve regeneration and neuropathic pain relief

Although peripheral nerve system has intrinsic regeneration potential, recovery from peripheral nerve injury (PNI) is often far from optimal, leading to partial or complete loss of sensorimotor function and neuropathic pain. The failure for PNI repair can be due to multiple reasons. Excessively elevated oxidative stress in injured tissue is one of primary causes for poor nerve regeneration and pain by damaging neurons and myelin structure. In addition, deprivation in various neurotrophic factors (NTFs) in chronic nerve denervation will lose support for nerve regrowth. Here, we developed a multifunctional hyaluronic acid-phenylboronic acid-poly (vinyl alcohol) -heparin (HA-PVA-Hep) hydrogel by mixing cysteamine and phenylboronic acid modified hyaluronic acid, commercially available poly (vinyl alcohol) and maleimide functionalized heparin. This hydrogel is biocompatible, stable, injectable, antioxidative, anti-inflammatory, and capable of sustainably releasing NTFs (i.e. glial cell derived neurotrophic factor-GDNF in this study). The therapeutic effect of HA-PVA-Hep hydrogel to repair acute PNI was tested by using a mouse sciatic nerve crush model. We found the hydrogel effectively improved sensorimotor function during the recovery period and significantly prevented muscular atrophy, protected neurons from injury and promoted nerve regeneration as well as remyelination at 28 days post injury. In addition, the hydrogel could also alleviate PNI-induced pain as hydrogel treatment reversed pain-related changes induced by PNI, including increased expression of a proinflammatory modulator, TNF-α, at injured nerve, activation of microglia and upregulation of pain-sensor receptor, TRPA1 at ipsilateral spinal dorsal horn. Nevertheless, supplementing the hydrogel with GDNF interrupted the PNI recovery process and probably counteracted the pain alleviation effect of the hydrogel. Thus, therapeutic effect of GDNF through local administration on PNI treatment needs to be further determined. In summary, our work provides a hydrogel delivery system with inherent therapeutic effect for PNI therapy.

Techniques for RNA extraction from cells cultured in starPEG-heparin hydrogels.

Three-dimensional (3D) cell culture models that provide a biologically relevant microenvironment are imperative to investigate cell-cell and cell-matrix interactions in vitro. Semi-synthetic star-shaped poly(ethylene glycol) (starPEG)-heparin hydrogels are widely used for 3D cell culture due to their highly tuneable biochemical and biomechanical properties. Changes in gene expression levels are commonly used as a measure of cellular responses. However, the isolation of high-quality RNA presents a challenge as contamination of the RNA with hydrogel residue, such as polymer or glycosaminoglycan fragments, can impact template quality and quantity, limiting effective gene expression analyses. Here, we compare two protocols for the extraction of high-quality RNA from starPEG-heparin hydrogels and assess three subsequent purification techniques. Removal of hydrogel residue by centrifugation was found to be essential for obtaining high-quality RNA in both isolation methods. However, purification of the RNA did not result in further improvements in RNA quality. Furthermore, we show the suitability of the extracted RNA for cDNA synthesis of three endogenous control genes confirmed via quantitative polymerase chain reaction (qPCR). The methods and techniques shown can be tailored for other hydrogel models based on natural or semi-synthetic materials to provide robust templates for all gene expression analyses.

The biofunctionalization of titanium nanotube with chitosan/genipin heparin hydrogel and the controlled release of IL-4 for anti-coagulation and anti-thrombus through accelerating endothelialization.

The valve replacement is the main treatment of heart valve disease. However, thrombus formation following valve replacement has always been a major clinical drawback. Accelerating the endothelialization of cardiac valve prosthesis is the main approach to reduce thrombus. In the current study, a titanium nanotube was biofunctionalized with a chitosan/genipin heparin hydrogel and the controlled release of interleukin-4 (IL-4), and its regulation of macrophages was investigated to see if it could influence endothelial cells to eventually accelerate endothelialization. TNT60 (titanium dioxide nanotubes, 60 V) with nanoarray was obtained by anodic oxidation of 60 V, and IL-4 was loaded into the nanotube by vacuum drying. The hydrogel (chitosan : genipin = 4 : 1) was applied to the surface of the nanotubes following drying, and the heparin drops were placed on the hydrogel surface with chitosan as the polycation and heparin as the polyanion. A TNT/IL-4/G (G = gel, chitosan/genipin heparin) delivery system was prepared. Our results demonstrated that the biofunctionalization of titanium nanotube with chitosan/genipin heparin hydrogel and the controlled release of IL-4 had a significant regulatory effect on macrophage M2 polarization, reducing the inflammatory factor release and higher secretion of VEGF (vascular endothelial growth factor), which can accelerate the endothelialization of the implant.

Heparin-functionalized hydrogels as growth factor-signaling substrates.

Tuneable, bioactive hydrogels present an attractive option as cell-instructive substrates for tissue regeneration. Properties mimicking the extracellular matrix at the site of injury are sought after, in particular the ability to regulate growth factors that are key to the regeneration process. This study demonstrates the successful formation of hydrogels with heparin functionalities and fibroblast growth factor-2 (FGF-2). Poly(2-hydroxyethyl methacrylate)-heparin hydrogels were capable of retaining FGF-2 by specific binding to heparin and subsequently showed sustained presentation of the growth factor to mesenchymal stromal cells (MSC). Heparin acted as stable anchoring molecules for FGF-2 on the substrate and the synergistic effect of the ensuing heparin-FGF-2 complex was evident in supporting long term cell growth. The presence of heparin during 3D scaffold formation was also found to introduce surface roughness and microporosity to the resulting hydrogels. While FGF-2 has been known to encourage MSC growth and maintain their multilineage potential, other heparin-binding ligands such as bone morphogenetic proteins are potent differentiation stimuli for MSC. Therefore preserving MSC multipotency or a push toward a differentiation pathway may be pursued by the choice of ligand applied to and bound by the heparin functionalities on the current substrate.

Thermosensitive bFGF-Modified Hydrogel with Dental Pulp Stem Cells on Neuroinflammation of Spinal Cord Injury.

Acute spinal cord injury (SCI) induces severe neuroinflammation, which increases intermediary filaments and neurodegeneration. Previous studies have shown that a basic fibroblast growth factor (bFGF) and dental pulp stem cells (DPSCs) contribute to a protective effect on injured neuronal cells, but the mechanism of SCI repair is still unclear. In this study, in situ heparin (HeP) hydrogel injection containing bFGF and DPSCs (HeP-bFGF-DPSCs), as well as in vitro studies of bFGF and DPSCs, proved an effective control over inflammation. The in vivo application of HeP-bFGF-DPSCs regulated inflammatory reactions and accelerated the nerve regeneration through microtubule stabilization and tissue vasculature. Our mechanistic investigation also showed that bFGF-DPSCs treatment inhibited microglia/macrophage proliferation and activation. Furthermore, HeP-bFGF-DPSCs prevented microglia/macrophage activation and reduced proinflammatory cytokine release. In this paper, we discovered that bFGF and DPSCs worked together to attenuate tissue inflammation of the injured spinal cord, resulting in a superior nerve repair. Our results indicated that a thermosensitive hydrogel delivering bFGF and DPSCs could serve as a promising treatment option for spinal cord injuries.

Investigation of Sustained BMP Delivery in the Prevention of Medication-Related Osteonecrosis of the Jaw (MRONJ) in a Rat Model.

Medication-related osteonecrosis of the jaw (MRONJ) poses an ongoing challenge for clinicians and researchers. Currently, there is a lack of preventative measures available for at-risk patients undergoing tooth extractions, especially those with prior bisphosphonate treatment due to osteoporosis or bone metastasis diagnoses. Here, these issues are addressed using a preventative tissue engineering strategy against MRONJ development. This study evaluates the efficacy of a poly(ethylene glycol)-heparin hydrogel as a tool for the delivery of arginylglycylaspartic acid (RGD) and recombinant human bone morphogenic protein-2 (rhBMP-2). Three groups of skeletally mature rats each receive two doses of intravenous zoledronic acid prior to surgery and undergo extraction of the right first mandibular molar with gingival closure. Experimental groups either have the sockets left empty, filled with hydrogel minus rhBMP-2, or filled with hydrogel plus rhBMP-2. Eight weeks postoperatively specimens are analyzed using radiological, histological, and scanning electron microscopy (SEM) techniques. µCT analysis shows increased bone formation with hydrogel/rhBMP-2 delivery compared to the empty socket. Hydrogel-treated groups display increased presence of osteocytes and increased osteoclastic action compared to the empty sockets. These results represent the first step toward improved delivery of rhBMP-2 and a potential MRONJ preventative for patients undergoing bisphosphonate treatment.

Correction to: The effect of heparin hydrogel embedding on glutaraldehyde fixed bovine pericardial tissues: Mechanical behavior and anticalcification potential.

The original version of this article unfortunately contained a few errors. The captions of Figs. 4, 5, 6, 7, and 8 were mixed up and they were misreferred in the text. The correct captions and their references in text are given below.

The effect of heparin hydrogel embedding on glutaraldehyde fixed bovine pericardial tissues: Mechanical behavior and anticalcification potential.

Heart valve diseases remain common in industrialized countries. Bioprosthetic heart valves, introduced as free of anticoagulation therapy alternatives to mechanical substitutes. Still they suffer from long term failure due to calcification. Different treatment methods introduced to inhibit calcification, have so far been limited in success. Glycosaminoglycans (GAGs) possess properties including high negative charge, anticoagulation and anti-inflammatory activity that make them a potential solution for calcification problem. In this study, heparin hydrogel was prepared and characterized both chemically and mechanically. After that, heparin hydrogel embedded bovine pericardial tissues, fixed with glutaraldehyde, were produced and tested for their mechanical behavior and anticalcifcation potential in vitro using the constant composition model. In the calcification experiments, tissues were divided into three groups: a) Controls without treatment, b) Hydrogel treated tissues and c) Tissues with raw heparin dissolved in the calcification solution. The results showed that embedding of tissue with hydrogel had no stiffening effect on its mechanical behavior. Calcification assessment showed a significant efficacy on inhibition of calcium phosphate deposition of hydrogel treated (second group) in comparison to untreated tissues (control, first group). Calcification inhibition potential was very similar in both the second and raw heparin (third group). Histological data confirmed the obtained results, suggesting that heparin treatment is a promising anticalcification agent.

Analyzing the antiseptic capacity of silver-functionalized poly(ethylene glycol)-heparin hydrogels after human whole blood exposure.

Advanced blood contacting biomaterials are designed to combine antiseptic and anticoagulant functionalities. Here, we present a new in vitro methodology for the analysis of bacterial adhesion and growth after the preceding human whole blood incubation of the tested materials. Poly(styrene) surfaces as well as thrombin-responsive and non-responsive poly(ethylene glycol)-heparin hydrogel coatings, with and without silver functionalization, were analyzed with this approach using freshly drawn human whole blood and various human pathogens (Staphylococcus epidermidis, Staphylococcus aureus, Pseudomonas aeruginosa, and Escherichia coli). Adsorbed blood proteins and adherent immune cells were observed to suppress bacterial colonization on poly(styrene) surfaces. Silver functionalization of responsive and non-responsive poly(ethylene glycol)-heparin hydrogels had no influence on microbial attachment but decreased bacterial proliferation and viability. Whole blood pre-incubation did not affect the antimicrobial properties of the tested silver-modified hydrogels. In sum, our introduced multistage incubation test revealed the antibacterial effects as well as antiseptic-permissive characteristics of blood-borne interfacial layers on polymeric biomaterials.