Cryogel Scaffolds Research Articles

Evaluation of PVA/Chitosan Cryogels as Potential Tissue Engineering Scaffolds; Synthesis, cytotoxicity and genotoxicity investigations

Cryogelation has become an advantageous method to obtain macro-porous materials with well-defined, interconnected pores for tissue engineering applications. Herein, polyvinyl alcohol and chitosan polymers (PVA-CHI) were used to produce cryogel scaffolds via cryogelation. Glutaraldehyde was used as a crosslinking agent and the effect of crosslinking amount on the properties of scaffolds investigated. Glutaraldehyde amount was divided into 5, 10, and 15% total amount of polymer concentration. The optimized pore morphology was obtained as a scaffold containing 5% glutaraldehyde amount. In addition to the FTIR, SEM, swelling, and degradation analyses, mechanical tests were performed to present the characterization properties of the cryogels. Direct and indirect cytotoxicity test and genotoxicity experiments were performed with Mouse Embryonic Fibroblasts (MEF). In addition, cell morphologies on scaffolds were analyzed with SEM. The results showed that PVA-CHI based cryogels had no genotoxic and cytotoxic effects on MEF cells and have a potential for tissue engineering applications.

A Biodegradable Chitosan-Polyurethane Cryogel with Switchable Shape Memory.

Cryogels are matrices that are formed in moderately frozen solutions of monomeric or polymeric precursors. They have the advantages of interconnected macropores, structural stability, and compressibility. Meanwhile, thermally induced shape memory is an attractive feature of certain functional materials. Although there have been several studies concerning shape-memory cryogels, little work has been conducted on shape-memory cryogels with biodegradability. In this study, a water-based biodegradable difunctional polyurethane with a shape-memory property was synthesized and used as the nanoparticulate crosslinker to react with chitosan to form a shape-memory cryogel. The thermally induced shape-memory mechanism was clarified using in situ wide-angle X-ray scattering (WAXS) and small-angle X-ray scattering (SAXS) during the shape-memory process. The in situ WAXS showed the changes of crystallinity in the crosslinker and the cryogel during the shape fixation and recovery processes. The in situ SAXS revealed the orientation of crystallinity of the crosslinker and the cryogel as the mechanism for shape memory. The strip-shape cryogel was deformed at 50 °C to U-shape and fixed at - 20 °C, which was squeezable at 25 °C and returned to the strip-shape at 50 °C in air. The shape recovery was further tested in water at two different temperatures. The injected cryogel recovered the U-shape in 4 °C water, representing elastic recovery, and transformed to a long strip in 37 °C water, representing the switchable shape memory. Moreover, the shape-memory cryogel sheet with a large dimension (10 mm × 10 mm × 1.1 mm cryogel sheet) or with complex structures (N, T, and U shapes) could be fixed as a rod, injected through a 16 G needle, and return to its original shape in 37 °C water, all of which could not be achieved by the conventional cryogel. Human mesenchymal stem cells grown in the shape-memory cryogel scaffolds displayed long-term proliferation and chondrogenic potential. Their unique injectability and cytocompatibility suggested potential applications of shape-memory cryogels as injectable and expandable templates for tissue engineering and minimally invasive surgery.

Sulfonated cryogel scaffolds for focal delivery in ex-vivo brain tissue cultures

The human brain has unique features that are difficult to study in animal models, including the mechanisms underlying neurodevelopmental and psychiatric disorders. Despite recent advances in human primary brain tissue culture systems, the use of these models to elucidate cellular disease mechanisms remains limited. A major reason for this is the lack of tools available to precisely manipulate a specific area of the tissue in a reproducible manner. Here we report an easy-to-use tool for site-specific manipulation of human brain tissue in culture. We show that line-shaped cryogel scaffolds synthesized with precise microscale dimensions allow the targeted delivery of a reagent to a specific region of human brain tissue in culture. 3-sulfopropyl acrylate (SPA) was incorporated into the cryogel network to yield a negative surface charge for the reversible binding of molecular cargo. The fluorescent dyes BODIPY and DiI were used as model cargos to show that placement of dye loaded scaffolds onto brain tissue in culture resulted in controlled delivery without a burst release, and labelling of specific regions without tissue damage. We further show that cryogels can deliver tetrodotoxin to tissue, inhibiting neuronal function in a reversible manner. The robust nature and precise dimensions of the cryogel resulted in a user-friendly and reproducible tool to manipulate primary human tissue cultures. These easy-to-use cryogels offer an innovate approach for more complex manipulations of ex-vivo tissue.

Hyaluronic Acid-Based Shape-Memory Cryogel Scaffolds for Focal Cartilage Defect Repair

Traumatic joint injuries can result in significant cartilage defects, which can greatly increase the risk of osteoarthritis development. Due to the limited self-healing capacity of avascular cartilage, tissue engineering approaches are required for filling defects and promoting cartilage regeneration. Current approaches utilize invasive surgical procedures for extraction and implantation of autologous chondrocytes; therefore, injectable biomaterials have gained interest to minimize the risk of infection as well as patient pain and discomfort. In this study, we engineered biomimetic, hyaluronic acid (HA)-based cryogel scaffolds that possess shape-memory properties as they contract and regain their shape after syringe injection to noninvasively fill cartilage defects. The cryogels, fabricated with HA and glycidyl methacrylate at -20°C, resulted in an elastic, macroporous, and highly interconnected network that provided a conducive microenvironment for chondrocytes to remain viable and metabolically active after injection through a syringe needle. Chondrocytes seeded within cryogels and cultured for 15 days exhibited enhanced cell proliferation, metabolism, and production of cartilage extracellular matrix glycosaminoglycans compared with HA-based hydrogels. Furthermore, immunohistochemical staining revealed production of collagen type II from chondrocyte-seeded cryogels, indicating the maintenance of cell phenotype. These results demonstrate the potential of chondrocyte-seeded, HA-based, injectable cryogel scaffolds to promote regeneration of cartilage tissue for nonsurgically invasive defect repair. Impact statement Hyaluronic acid-based shape-memory cryogels provide a conducive microenvironment for chondrocyte adhesion, proliferation, and matrix biosynthesis for use in repair of cartilage defects. Due to their sponge-like elastic properties, cryogels can fully recover their original shape back after injection while not impacting metabolism or viability of encapsulated cells. Clinically, they provide an opportunity for filling focal cartilage defects by using a single, minimally invasive injection of a cell encapsulating biocompatible three-dimensional scaffold that can return to its original structure to fit the defect geometry and enable matrix regeneration.

Differential anti-inflammatory properties of chitosan-based cryogel scaffolds depending on chitosan/gelatin ratio

Chitosan/gelatine-based materials have been widely used as biocompatible scaffolds in the tissue engineering field. Chitosan suppresses the inflammatory activities of macrophages whereas gelatine induces inflammatory cytokine production by these cells. Cryogel form of the scaffolds created an effect that was mostly dominated by chitosan activity. Since independent of chitosan to gelatine ratio, the cryogels eliminated the inflammatory cytokine production by the activated macrophages. This will enable suppression of inflammatory reactions by macrophages during implant procedure while enabling a nest of the matrix for the macrophages to reside. Determining the immunomodulatory effect of these materials during the decay is crucial to assess their biocompatibility and safety. Our results suggest that when the chitosan ratio was higher than that of gelatine the materials had anti-inflammatory activity in their powder forms based on TNFα production levels by LPS activated macrophages, whereas higher gelatine to chitosan ratio eliminated this effect. To our knowledge, this is the first study to assess the powder vs. gel forms of the chitosan/gelatine-based materials for their immunomodulatory potentials as well as how the ratio of chitosan to gelatine might affect these materials immunomodulatory effects on the activated macrophages. HIGHLIGHTS Chitosan/gelatin composite cryogels have anti-inflammatory activities. Different ratios of chitosan to gelatin content altered the immunomodulatory activities. They can be safely and effectively used as implant materials for tissue engineering applications. They will also reduce the use of anti-inflammatory drugs during implantation.

Photothermally Active Cryogel Devices for Effective Release of Antimicrobial Peptides: On-Demand Treatment of Infections.

There has been significant interest in the use of peptides as antimicrobial agents, and peptide containing hydrogels have been proposed as biological scaffolds for various applications. Limited stability and rapid clearance of small molecular weight peptides pose challenges to their widespread implementation. As a common approach, antibacterial peptides are physically loaded into hydrogel scaffolds, which leads to continuous release through the passive mode with spatial control but provides limited control over drug dosage. Although utilization of peptide covalent linkage onto hydrogels addresses partially this problem, the peptide release is commonly too slow. To alleviate these challenges, in this work, maleimide-modified antimicrobial peptides are covalently conjugated onto furan-based cryogel (CG) scaffolds via the Diels-Alder cycloaddition at room temperature. The furan group offers a handle for specific loading of the peptides, thus minimizing passive and burst drug release. The porous nature of the CG matrix provides rapid loading and release of therapeutic peptides, apart from high water uptake. Interfacing the peptide adduct containing a CG matrix with a reduced graphene oxide-modified Kapton substrate allows "on-demand" photothermal heating upon near-infrared (NIR) irradiation. A fabricated photothermal device enables tunable and efficient peptide release through NIR exposure to kill bacteria. Apart from spatial confinement offered by this CG-based bandage, the selective ablation of planktonic Staphylococcus aureus is demonstrated. It can be envisioned that this modular "on-demand" peptide-releasing device can be also employed for other topical applications by appropriate choice of therapeutic peptides.

Needle-injectable microcomposite cryogel scaffolds with antimicrobial properties

Porous three-dimensional hydrogel scaffolds have an exquisite ability to promote tissue repair. However, because of their high water content and invasive nature during surgical implantation, hydrogels are at an increased risk of bacterial infection. Recently, we have developed elastic biomimetic cryogels, an advanced type of polymeric hydrogel, that are syringe-deliverable through hypodermic needles. These needle-injectable cryogels have unique properties, including large and interconnected pores, mechanical robustness, and shape-memory. Like hydrogels, cryogels are also susceptible to colonization by microbial pathogens. To that end, our minimally invasive cryogels have been engineered to address this challenge. Specifically, we hybridized the cryogels with calcium peroxide microparticles to controllably produce bactericidal hydrogen peroxide. Our novel microcomposite cryogels exhibit antimicrobial properties and inhibit antibiotic-resistant bacteria (MRSA and Pseudomonas aeruginosa), the most common cause of biomaterial implant failure in modern medicine. Moreover, the cryogels showed negligible cytotoxicity toward murine fibroblasts and prevented activation of primary bone marrow-derived dendritic cells ex vivo. Finally, in vivo data suggested tissue integration, biodegradation, and minimal host inflammatory responses when the antimicrobial cryogels, even when purposely contaminated with bacteria, were subcutaneously injected in mice. Collectively, these needle-injectable microcomposite cryogels show great promise for biomedical applications, especially in tissue engineering and regenerative medicine.

A powerful combination in designing polymeric scaffolds: 3D bioprinting and cryogelation

Three-dimensional (3D) bioprinting technologies have great attention in different researching areas such as tissue engineering, medicine, etc. due to its maximum mimetic property of natural biomaterials by providing cell combination, growth factors, and other biomaterials. Bioprinting of tissues, organs, or drug delivery systems emerged layer-by-layer deposition of bioinks. 3D bioprinting technique has some complexity such as choice of bioink combination, cell type, growth, and differentiation. In this study, a composite material in 3D bioprinting studies has been developed for biofabrication of the cell carrying scaffolds namely cryogenic scaffolds. Cryogenic scaffolds are highly elastic and have a continuous interconnected macroporous structure in 3D biomaterials that enable the cell attachment, viability, and proliferation. Freeze-drying cryogelation process for the formation of cryogel scaffolds has been achieved firstly among 3D bioprinting studies. Cryogenic gelatin–hyaluronic acid (Gel–HA)-based 3D-bioprinted scaffolds have been fabricated and characterized by scanning electron microscope (SEM), optical microscope images, tensile tests, determination of swelling degree, and porosity. Then, L929 cells from mouse C3H/An have been attached to cryogenic Gel–HA scaffolds. Cell attachment, viability, and proliferation on cryogenic scaffolds have been investigated for 7 days. The results showed that a combination of 3D bioprinting technologies and cryogenic process provided a new direction on biomedical scaffolds.

Development of Hypericum perforatum oil incorporated antimicrobial and antioxidant chitosan cryogel as a wound dressing material

In this study, a herbal infused oil (Hypericum perforatum, HP) incorporated chitosan (CS) cryogel as a wound dressing material was produced in order to be used in wound healing process. The main strategy is to combine the traditional perspective of using medicinal oils with polymeric scaffolds manufactured by an engineering approach to fabricate a potential tissue engineering product that provides both new tissue formation and wound healing. The scaffolds manufactured by cryogelation were soft, spongy, highly porous, physically stable, elastic and could be easily cut in any desired shape. Physicochemical, mechanical and morphological analyzes were used to characterize the produced cryogels. Young modulus of the plain chitosan cryogel was about 21 kPa whereas it increased with increasing HP oil content and became 61 kPa for 20% HP oil ratio. Further, the antimicrobial studies, antioxidant and DNA cleavage effects were investigated. Samples including the highest ratio of oil (CS4) showed the highest DPPH scavenging activity as 69.9%. In addition, 20% HP oil loaded chitosan cryogel demonstrated single strain DNA cleavage activitiy at 500 μg/mL concentration. Antimicrobial studies were applied against seven strains. The lowest activities were obtained against E. hirae and B. cereus, the highest against E. coli and L. pneumophila. This study concluded that the newly developed HP oil loaded chitosan cryogel scaffolds with unique antimicrobial and antioxidant properties are promising candidates to be used in tissue engineering applications as wound dressing for exudative and long-term healing wounds.

Biofabrication of gold nanoparticles with bone remodeling potential: an in vitro and in vivo assessment

Recent thrust in the biomaterials research has shifted towards developing bioactive and resorbable scaffolds with nanotechnological interventions. Next-generation bone scaffolds need to be multifunctional with desired osteo-integration ability, conformed resorption rate and in vivo imaging potential. In this study, we synthesized biogenic multifunctional gold nanoparticles (AuNPs) using immobilized crude enzyme (ImCE) of Bacillus licheniformis. The multifunctional characteristics of AuNPs and reusability of ImCE for AuNP synthesis can make the process and application cost-effective. ImCE catalyzed the bioreduction of gold ions to AuNPs of 18.4 ± 3.3 nm. AuNPs showed good hemocompatibility and antioxidant property without any toxic effects. Further, AuNPs demonstrated the increase in alkaline phosphatase activity and calcium deposition through MC3T3-E1 murine preosteoblast cell line and rat bone marrow stromal cells (BMSCs). The osteoinductive effect of AuNPs was also investigated in vivo wherein the composite cryogel scaffold functionalized with bone morphogenic protein-2 (rhBMP-2), zoledronic acid (ZA), and AuNPs was implanted in a muscle pouch of Wistar rats. AuNPs had synergistic effect on bone formation along with rhBMP-2 and ZA as seen from micro-computed tomography and histological analysis. The AuNPs were found to exhibit autofluorescence property and enhanced the degradation of scaffold at the implanted site in vivo. The developed process of using ImCE presents a novel approach for biogenic fabrication of AuNPs with the potential orthopedic application.

Hyaluronic acid-based shape memory cryogel scaffolds for focal cartilage defect repair

Purpose: Traumatic joint injuries can result in focal cartilage defects that regenerate poorly due to a lack of blood supply and low chondrocyte density. Cell-laden hydrogel scaffolds are under study but provide little flexibility in modulating their pore size, interconnectivity, swelling and stiffness, properties required for a conducive biomimetic environment. In this study, we engineered an injectable and biomimetic shape memory hyaluronic acid (HA)-based cryogel scaffolds with a macroporous and highly interconnected network in three-dimensions seeded with chondrocytes. Due to their unique ability to withstand up to 90% strains and rapidly recover their shape back, they can be arthroscopically delivered to fill up focal chondral defects for cartilage repair. Here we studied whether chondrocytes encapsulated within cryogels remain viable and metabolically active following injection and provide a superior micro-environment than conventional (nanoporous) hydrogels for cartilage regeneration. We also investigated how RGD-functionalized cryogel affects chondrocyte adhesion, migration and proliferation. Methods: Synthesis of HA-based Cryogels and Hydrogels: HA cryogels were prepared using HA modified with glycidyl methacrylate (HAGM) and either Acryl-PEG-G4RGDS to synthesis gels functionalized with RGD (Cryo RGD+) or Acryl-PEG-methoxy to synthesize gels without RGD (Cryo RGD-) in deionized (DI) water. The mixture was cast in 6 mm diam x 1.5 mm cylindrical discs using APS/TEMED as our initiator system. The samples were frozen at -20°C and allowed to cryopolymerize overnight. Next, cryogels were brought to RT and washed/sanitized prior to cell seeding. HA hydrogels (Hydro RGD+) were fabricated via photopolymerization by mixing HAGM, RGD, and lithium phenyl-2,4,6- trimethylbenzoylphosphinate (LAP) photo-initiator in DI water. Chondrocyte seeding and in-vitro culture:Chondrocytes harvested from calf knee femoral condyle cartilage were seeded onto each scaffold and cultured for 15 days. Injectability test: Cell-laden cryogels were incubated overnight to enable complete cell adhesion and subsequently syringe injected through a 16-gauge needle before incubation for another 24h following which cell viability and metabolism were quantified. Biochemical analysis: At the end of 15-day culture, gel samples were digested to quantify the cell metabolism (Alamar Blue), DNA (PicoGreen) and GAG (DMMB) content. Cell viability and cytoskeleton staining: Gel samples were fixed and stained for nucleus, actin, dead cells and confocal imaged at 40x. Results: Shape memory HA-based cryogels fully recovered their shape back following syringe injection through a 16-gauge needle without impacting cell metabolism or viability (Figs 1A-C). Scanning electron microscopy (SEM) images showed an average pore size of 49.2 ± 15 μm (Fig 1D) rendering a macroporous structure with high interconnectivity, which was 7x higher than hydrogels (Fig 1E). This unique macrostructural architecture enables efficient transport of nutrients and metabolites thereby offering a superior microenvironment for chondrocytes; both cell metabolism and GAG content was 2x higher in cryogels than hydrogels after 15-day culture (Figs 1F-G). Additionally, a significant amount of GAG (∼70 μg) was released from both cryogels into media owing to their macroporous structure; no released GAG was measured in media from Hydro RGD+ (Fig 1G). The presence of RGD (cell-adhesion peptide) did not improve cell adhesion or biosynthesis rates. Chondrocytes formed larger organoid-like structures in Cryo RGD- while remaining homogenously dispersed in the presence of RGD in Cryo RGD+ (Fig 1H). Conclusions: Syringe injectable HA-based shape memory cryogels provide a conducive microenvironment for chondrocyte adhesion, proliferation and matrix biosynthesis for use in repair of cartilage defects.

Abstract B106: Engineering cryogels to modulate oxygen tension in reconstructed tumor microenvironments

Abstract Introduction: Hypoxia, defined as low oxygen tension, is a characteristic feature of solid tumors and a hallmark of aggressive cancers. The rapid growth of tumors often results in the development of a hypoxic microenvironment, leading to tumor cell growth and invasion, resistance to apoptosis, and multidrug resistance. Multicellular tumor spheroids (MCTS) have been used to model solid tumors and emulate key aspects of tumor biology such as hypoxia. However, challenges with MCTS formation and reproducibility, inadequate biomechanical cues provided to cells, and uncontrolled oxygen depletion among other limitations led to nonphysiologic tumor cell responses. As a result, predicting clinical responses to anticancer drugs with MCTS remains a major challenge in cancer treatment. To model solid tumors more accurately, our team has recently developed an innovative approach using a cryogel scaffold inducing rapid oxygen depletion while enabling cell rearrangement into 3D spherical cellular aggregates. Our main objectives were: (1) to engineer hypoxia-inducing cryogels (HIC) to generate cellular hypoxia; (2) to provide a biophysical support enabling tumor cell attachment, proliferation, and remodeling; and (3) to evaluate acquired resistance of hypoxic B16-F10 melanoma cells to a common anticancer drug. Methods: HIC were fabricated via a cryogelation process using methacrylated hyaluronic acid (HAGM). RGD peptide, catalase, and glucose oxidase (GO) were incorporated into the gel to promote cell attachment and viability while depleting oxygen. Oxygen depletion was monitored using needle-type oxygen microprobes in the presence or absence of glucose. B16-F10 cell viability was evaluated by confocal imaging. Cellular hypoxia was monitored using a fluorescent hypoxyprobe. Finally, alamarblue assay was performed to evaluate drug resistance of B16-F10 cells to various concentrations of doxorubicin (DOX). Results: We demonstrated that HICs induce a controlled and sustained depletion of oxygen leading to hypoxia (~1% O2) up to 48h. Hypoxia induction is dependent on both GO and glucose contents. Unlike regular cryogels (control), HIC promoted cell remodeling into MCTS-like structure with high cell viability (95 ± 2%), while inducing a substantial level of cellular hypoxia (94 ± 3%) after 24h. Finally, HIC significantly induced melanoma resistance to DOX. With our highest DOX concentration (2μM), 100% and 77% of drug-resistant B16-F10 cells in HIC remained alive after 24h and 48h treatment, respectively. In comparison, all B16-F10 cells died within 48h in our control condition (blank cryogels). Conclusions: We have engineered advanced cryogels, namely HIC, capable of inducing hypoxia while promoting tumor cell remodeling and anticancer drug resistance. This suggests that tumor-laden HIC may mimic key aspects of aggressive tumor microenvironments, making them a promising platform for drug screening and potentially improving preclinical drug discovery and testing. Citation Format: Thibault Colombani, James Sinoimeri, Sidi A. Bencherif. Engineering cryogels to modulate oxygen tension in reconstructed tumor microenvironments [abstract]. In: Proceedings of the AACR Special Conference on Tumor Immunology and Immunotherapy; 2019 Nov 17-20; Boston, MA. Philadelphia (PA): AACR; Cancer Immunol Res 2020;8(3 Suppl):Abstract nr B106.

Combination of a biomolecule-aided biphasic cryogel scaffold with a barrier membrane adhering PDGF-encapsulated nanofibers to promote periodontal regeneration.

To achieve periodontal regeneration, numerous investigations have concentrated on biomolecule supplement and optimization of bone substitute or barrier membrane. This study evaluated the benefit of combining these strategies for periodontal regeneration. Biphasic cryogel scaffold (BCS) composed of gelatin (ligament phase) and gelatin with beta-tricalcium phosphate/hydroxyapatite (BH) (bone phase) was designed as tested bone substitute, and both enamel matrix derivatives (EMD) and bone morphogenetic protein-2 (BMP-2) were applied to formulate a biomolecule-aided BCS (BBS). Functionally graded membrane (FGM) was designed as tested barrier membrane by adhering PDGF-encapsulated poly(L-lactide-co-D/L-lactide) nanofibers on the conventional membrane (CM). BBS and FGM were characterized and tested for biocompatibility in vitro. Thirty 4×4×5mm3 periodontal intrabony defects were created on 6 Beagle dogs. Each defect was evenly assigned to one of the following treatments including BH-CM, BCS-CM, BBS-CM, BH-FGM, BCS-FGM, and BBS-FGM, for 12weeks. The therapeutic efficiency was assessed by micro-CT and histology. BCS and FGM sustained the release of biomolecules. The viability of MSCs was maintained in both phases of BCS and was promoted while seeding on the PDGF-encapsulated nanofibers. In CM-covered sites, BBS showed significantly greater osteogenesis (P<.01) and early defect fill (P<.05) relative to BH. FGM significantly promoted osteogenesis (P<.05) in BH-treated sites but showed limited benefit in BBS-treated sites. On denuded roots, cementum deposition was evident in BBS-treated sites. PDGF-loaded FGM promoted periodontal osteogenesis, and BBS with EMD-BMP-2 had potential for reconstructing alveolar ridge, periodontal ligament, and cementum. FGM and BBS combination provided limited additional benefit.

Static and dynamic 3D culture of neural precursor cells on macroporous cryogel microcarriers

Neural precursor cells have been much studied to further our understanding of the far-reaching and controversial question of adult neurogenesis. Currently, differentiation of primary neural precursor cells from the mouse dentate gyrus via 2-dimentional in vitro culture yields low numbers of neurons, a major hindrance to the field of study. 3-dimentional “neurosphere” culture allows better 3D cell-cell contact, but control over cell differentiation is poor because nutrition and oxygen restrictions at the core of the sphere causes spontaneous differentiation, predominantly to glial cells, not neurons. Our group has developed macroporous scaffolds, which overcome the above-mentioned problems, allowing long-term culture of neural stem cells, which can be differentiated into a much higher yield of neurons. Herein we describe a method for culturing neural precursor cells on RGD peptide functionalized-heparin containing cryogel scaffolds, either in standard non-adherent well-plates (static culture) or in spinner flasks (dynamic culture). This method includes:•The synthesis and characterization of heparin based microcarriers.•A “static” 3D culture method for that does not require spinner flask equipment.•“Dynamic” culture in which cell loaded microcarriers are transferred to a spinner flask.

A Biomimicking Polymeric Cryogel Scaffold for Repair of Critical-Sized Cranial Defect in a Rat Model.

Mineralized polymeric cryogels with interconnective macroporous structure have demonstrated their potential as promising scaffolding material in bone tissue engineering. However, their capability in inducing osteogenic differentiation of mesenchymal stem cells (MSCs) in vitro and osteogenesis in vivo has not been explored yet. In this work, the roles of the mineralized cryogel on osteogenesis are systematically studied. Mineralized macroporous poly(ethylene glycol)-co-2-hydroxyethyl methacrylate cryogel promotes osteogenic differentiation of rat MSCs, particularly in upregulating the activity of alkaline phosphatase (ALP, ∼5.7-folds) and expression of related osteogenic gene markers (ALP ∼16-folds, osteocalcin ∼133-folds) at 14 days. In vivo implantation reveals that mineralized cryogels could promote fast osteogenesis and angiogenesis in critical-sized cranial bone defect of a Sprague-Dawley rat model in 4 weeks. The adsorption, entrapment, and concentration of osteogenic growth factors (bone morphogenetic protein 2) and angiogenesis growth factor (vascular endothelial growth factor [VEGF]) in the matrices in vivo may possibly participate in the process of osteogenesis and angiogenesis. Notably, the adsorption of larger amount of VEGF in nonmineralized cryogels facilitates obvious angiogenesis and comparable osteogenesis in bone defect in 8 weeks. Graphical abstract [Figure: see text] Impact Statement The current work reported the fabrication and characterization of a biomimicking mineralized polymeric cryogel as scaffolding material in bone regeneration. In addition to its three dimensional porous structure and the osteogenic potential, this biomimicking scaffold was also found to enhance the adsorption of biochemical cues, which in turn greatly promoted the angiogenesis as well as the tissue regeneration.

Osteogenic potential of induced pluripotent stem cells from human adipose-derived stem cells

BackgroundBone regeneration is a crucial and challenging issue in clinical practice. Bone tissue engineering (BTE) with an optimal cell source may provide an ideal strategy for the reconstruction of bone defects. This study examined whether induced pluripotent stem cells (iPSCs) derived from adipose-derived stem cells (ASCs) could act as an osteogenic substitute and whether these ASC-iPSCs yield more new bone formation than ASCs in hydrogel scaffolds.MethodsASC-iPSCs were reprogrammed from ASCs through a retroviral system. ASCs were harvested and isolated from adipose tissue of humans. An aliquot of cell suspension (1 × 106 cells/mL) was seeded directly onto the nHAP-gelatin cryogel scaffolds. Nude mice back implantation of cell-seeded scaffolds was designed for in vivo comparison of osteogenic potentials between ASCs and ASC-iPSCs. Samples were harvested 4 and 8 weeks after implantation for further analysis based on histology and RT-PCR.ResultsASC-iPSCs were successfully obtained from human adipose-derived stem cells. PCR results also showed that specific genes of iPSCs with the ability to cause the differentiation of cells into the three germ layers were expressed. In our in vivo experiments, iPSCs were subcutaneously injected into nude mice to induce teratoma formation. The morphology of the three germ layers was confirmed by histological staining. ASC is an essential cell source for BTE with benefits of high volume and less-invasive acquisition. With additional transforming Yamanaka factors, ASC-iPSCs showed higher osteogenic differentiation and elevated expression of collagen type I (Col I), osteocalcin (OCN), alkaline phosphate (ALP), and runt-related transcription factor 2 (RunX-2).ConclusionsThis report suggests that ASC-iPSCs could be a superior cell source in BTE with better osteogenic differentiation efficacy for future clinical applications.

Preparation of Gelatin and Gelatin/Hyaluronic Acid Cryogel Scaffolds for the 3D Culture of Mesothelial Cells and Mesothelium Tissue Regeneration.

Mesothelial cells are specific epithelial cells that are lined in the serosal cavity and internal organs. Nonetheless, few studies have explored the possibility to culture mesothelial cells in a three-dimensional (3D) scaffold for tissue engineering applications. Towards this end, we fabricated macroporous scaffolds from gelatin and gelatin/hyaluronic acid (HA) by cryogelation, and elucidated the influence of HA on cryogel properties and the cellular phenotype of mesothelial cells cultured within the 3D scaffolds. The incorporation of HA was found not to significantly change the pore size, porosity, water uptake kinetics, and swelling ratios of the cryogel scaffolds, but led to a faster scaffold degradation in the collagenase solution. Adding 5% HA in the composite cryogels also decreased the ultimate compressive stress (strain) and toughness of the scaffold, but enhanced the elastic modulus. From the in vitro cell culture, rat mesothelial cells showed quantitative cell viability in gelatin (G) and gelatin/HA (GH) cryogels. Nonetheless, mesothelial cells cultured in GH cryogels showed a change in the cell morphology and cytoskeleton arrangement, reduced cell proliferation rate, and downregulation of the mesothelium specific maker gene expression. The production of key mesothelium proteins E-cadherin and calretinin were also reduced in the GH cryogels. Choosing the best G cryogels for in vivo studies, the cell/cryogel construct was used for the transplantation of allograft mesothelial cells for mesothelium reconstruction in rats. A mesothelium layer similar to the native mesothelium tissue could be obtained 21 days post-implantation, based on hematoxylin and eosin (H&E) and immunohistochemical staining.

Cryogel scaffolds for regionally constrained delivery of lysophosphatidylcholine to central nervous system slice cultures: A model of focal demyelination for multiple sclerosis research

The pathology of multiple sclerosis (MS) is typified by focal demyelinated areas of the brain and spinal cord, which results in axonal degeneration and atrophy. Although the field has made much progress in developing immunomodulatory therapies to reduce the occurrence of these focal lesions, there is a conspicuous lack of licensed effective therapies to reduce axonal degeneration or promote repair. Remyelination, carried out by oligodendrocytes, does occur in MS, and is protective against axonal degeneration. Unfortunately, remyelination is not very efficient, and ultimately fails and so there is a research focus to generate new therapeutics to enhance remyelination leading to neuroprotection.To develop these therapies, we need preclinical models that well reflect remyelination in MS. We have previously characterized an ex vivo model that uses lysophosphatidylcholine (LPC) to cause acute and global demyelination of tissue slices, followed by spontaneous remyelination, which has been widely used as a surrogate for in vivo rodent models of demyelination. However, this ex vivo model lacks the focal demyelinated lesions seen in MS, surrounded by normal tissue from which the repairing oligodendrocytes are derived. Therefore, to improve the model, we have developed and characterized small macroporous cryogel scaffolds for controlled/regional delivery of LPC with diameters of either 0.5, 1 or 2 mm. Placement of LPC loaded scaffolds adjacent to ex vivo cultured mouse brain and spinal cord slices induced focal areas of demyelination in proximity to the scaffold. To the best of our knowledge, this is the first such report of spatial mimicry of the in vivo condition in ex vivo tissue culture. This will allow not only the investigation into focal lesions, but also provides a better platform technology with which to test remyelination-promoting therapeutics. Statement of SignificanceThis manuscript is the first report of using macroporous hydrogels (cryogels) as a research tool for lysophosphatidylcholine (LPC) delivery, in order to create an ex vivo model of focal demyelination in the brain and spinal cord, which is of great relevance to multiple sclerosis research.Here, we transform an existing ex vivo model of demyelination by delivering LPC to focal regions of brain and spinal cord slice cultures. We have developed an easy-to-handle cylindrical and macroporous PEG-based sponge-like scaffold material (cryogel) that can deliver LPC only to a small area of the slice. Such cryogels are ideal as a delivery system in this culture model as they exhibit a soft but robust nature, with high mechanical deformability in their dry and swollen state, with no need to stay permanently hydrated. In addition, the synthesis of these cryogels is simple and easy to reproduce via photochemical cryopolymerisation using a PEG-diacrylate monomer and a photoinitiator, which are both commercially available.This more accurate model of demyelination will not only allow researchers to gain a better understanding of the CNS remyelination process in diseases such as MS, but also provides a platform technology, which could be utilized to screen and test pro-remyelination compounds which may help to find new therapeutics for progressive MS.

Rational design of gelatin/nanohydroxyapatite cryogel scaffolds for bone regeneration by introducing chemical and physical cues to enhance osteogenesis of bone marrow mesenchymal stem cells.

Identification of key components in the chemical and physical milieu for directing osteogenesis is a requirement in the investigation of tissue engineering scaffolds for advancement of bone regeneration. In this study, we engineered different gelatin-based cryogels and studied the effect of nanohydroxyapatite (nHAP) and crosslinking agents on scaffold properties and its osteogenic response towards bone marrow stem cells (BMSCs). The cryogels examined are 5% gelatin and 5% gelatin/2.5% nHAP, crosslinked either with 1-ethyl-3-(3-dimethylaminopropyl)-carbodiimide (EDC) or glutaraldehyde (GA). We confirmed that nHAP or the crosslinking agent has no effects on scaffold pore size and porosity. Nonetheless, incorporation of nHAP increased mechanical strength, swelling ratio and degree of crosslinking, but decreased degradation rate. Cryogels crosslinked with EDC showed faster degradation and promoted osteogenic differentiation of BMSCs while those prepared from GA crosslinking promoted proliferation of BMSCs. Furthermore, osteogenic differentiation was always enhanced in the presence of nHAP irrespective of the culture medium (normal or osteogenic) used but osteogenic medium always provide a higher extent of osteogenic differentiation. Employing gelatin/nHAP cryogel crosslinked by EDC in a bioreactor for dynamic culture of BMSCs, cyclic compressive mechanical simulation was found to be beneficial for both cell proliferation and osteogenic differentiation. However, the optimum conditions for osteogenic differentiation and cell proliferation were found at 30% and 60% strain, respectively. We thus demonstrated that osteogenic differentiation of BMSCs could be tuned by taking advantages of chemical cues generated from scaffold chemistry or physical cues generated from dynamic cell culture in vitro. Furthermore, by combining the best cryogel preparation and in vitro cell culture condition for osteogenesis, we successfully employed in vitro cultured cryogel/BMSCs constructs for repair of rabbit critical-sized cranial bone defects.

Investigating Manuka Honey Antibacterial Properties When Incorporated into Cryogel, Hydrogel, and Electrospun Tissue Engineering Scaffolds.

Honey is well-known for its wound healing capability and Manuka honey (MH) contains a unique Manuka factor, providing an additional antibacterial agent. Previously, there has not been a practical way to apply MH to a wound site, which renders treatment for an extended period extremely difficult. Tissue-engineered scaffolds offer an alternative treatment method to standard dressings by providing varying geometries to best treat the specific tissue. MH was incorporated into cryogels, hydrogels, and electrospun scaffolds to assess the effect of scaffold geometry on bacterial clearance and adhesion, as well as cellular adhesion. Electrospun scaffolds exhibited a faster release due to the nanoporous fibrous geometry which led to a larger partial bacterial clearance as compared to the more three-dimensional cryogels (CG) and hydrogels (HG). Similarly, the fast release of MH from the electrospun scaffolds resulted in reduced bacterial adhesion. Overall, the fast MH release of the electrospun scaffolds versus the extended release of the HG and CG scaffolds provides differences in cellular/bacterial adhesion and advantages for both short and long-term applications, respectively. This manuscript provides a comparison of the scaffold pore structures as well as bacterial and cellular properties, providing information regarding the relationship between varying scaffold geometry and MH efficacy.