Aleksander Skardal

Bioprinting vessel-like constructs using hyaluronan hydrogels crosslinked with tetrahedral polyethylene glycol tetracrylates.

Bioprinting enables deposition of cells and biomaterials into spatial orientations and complexities that mirror physiologically relevant geometries. To facilitate the development of bioartificial vessel-like grafts, two four-armed polyethylene glycol (PEG) derivatives with different PEG chain lengths, TetraPEG8 and TetraPEG13, were synthesized from tetrahedral pentaerythritol derivatives. The TetraPEGs are unique multi-armed PEGs with a compact and symmetrical core. The TetraPEGs were converted to tetra-acrylate derivatives (TetraPAcs) which were used in turn to co-crosslink thiolated hyaluronic acid and gelatin derivatives into extrudable hydrogels for printing tissue constructs. First, the hydrogels produced by TetraPAc crosslinking showed significantly higher shear storage moduli when compared to PEG diacrylate (PEGDA)-crosslinked synthetic extracellular matrices (sECMs) of similar composition. These stiffer hydrogels have rheological properties more suited to bioprinting high-density cell suspensions. Second, TetraPAc-crosslinked sECMs were equivalent or superior to PEGDA-crosslinked gels in supporting cell growth and proliferation. Third, the TetraPac sECMs were employed in a proof-of-concept experiment by encapsulation of NIH 3T3 cells in sausage-like hydrogel macrofilaments. These macrofilaments were then printed into tubular tissue constructs by layer-by-layer deposition using the Fab@Home printing system. LIVE/DEAD viability/cytotoxicity-stained cross-sectional images showed the bioprinted cell structures to be viable in culture for up to 4 weeks with little evidence of cell death. Thus, biofabrication of cell suspensions in TetraPAc sECMs demonstrates the feasibility of building bioartificial blood vessel-like constructs for research and potentially clinical uses.

Dynamically Crosslinked Gold Nanoparticle – Hyaluronan Hydrogels

Bioprinting employs three-dimensional (3D) deposition of cells and biomaterials to create organized structures with organappropriate architecture. Such engineered organs could offer alternatives to inadequate donor organ supplies, [ 1 , 2 ] and bioprinted human tissues could improve predictability during preclinical evaluation of therapeutic agents. [ 3 ] However, scalability of bioprinting is limited by lack of extrudable, biocompatible materials that can retain form, be remodeled by cells, be removed to create lumens, and offer layer-to-layer connectivity following assembly. To address these needs, we developed dynamically crosslinkable materials using gold nanoparticles (AuNPs) as multivalent crosslinkers. Specifi cally, 24 nm AuNPs and thiolmodifi ed biomacromonomers derived from hyaluronic acid (HA) and gelatin were used to form printable semi-synthetic extracellular matrix (sECM) hydrogels. AuNP-sECMs are unique in having dynamic crosslinks; that is, both intra-gel and inter-gel covalent interactions can form and reform during and after printing. Moreover, AuNP-thiol crosslinking is reversible in the presence of benign thiols such as cysteine. In a proof-ofconcept experiment, AuNP-sECMs were used to print tubular tissue constructs using an automated bioprinting system. In bioprinting, cells (the “bio-ink”) and hydrogels (the “biopaper”) are deposited into precise 3D geometries by a 3-axis printer in a fashion enabling maturation into functional tissues. [ 4,5 ] Recently, cell aggregates and cell rods were printed into tubular assemblies that fused into seamless structures. [ 6,7 ]

Engineered extracellular matrices with cleavable crosslinkers for cell expansion and easy cell recovery.

An unmet need for expansion of primary cells and progenitor cells in three dimensions (3-D) is a synthetic mimic of the extracellular matrix (ECM) with user-controllable composition that would permit rapid recovery of viable cells under mild, non-enzymatic conditions. Three block copolymers based on disulfide-containing polyethylene glycol diacrylate crosslinkers were synthesized, and were used to crosslink thiol-modified hyaluronan and gelatin macromonomers in the presence of cells. The triblock PEGSSDA contained a single disulfide-containing block, the pentablock PEG(SS)(2)DA contained two disulfide blocks, and the heptablock PEG(SS)(3)DA contained three disulfide blocks. For each hydrogel composition, four cell types were encapsulated in 3-D, and growth and proliferation were evaluated. Murine NIH 3T3 fibroblasts, human HepG2 C3A hepatocytes, human bone marrow-derived mesenchymal stem cells (MSCs), and human umbilical vein endothelial cells (HUVECs) all showed excellent viability and growth during expansion in 3-D in the three disulfide block copolymer crosslinkers. After cell expansion, the hydrogels were dissociated using the thiol-disulfide exchange reaction in the presence of N-acetyl-cysteine or glutathione, which dissolved the hydrogel network. After dissolution, cells were recovered in high yield and with high viability by gentle centrifugation.

The generation of 3-D tissue models based on hyaluronan hydrogel-coated microcarriers within a rotating wall vessel bioreactor

With the increasing necessity for functional tissue- and organ equivalents in the clinic, the optimization of techniques for the in vitro generation of organotypic structures that closely resemble the native tissue is of paramount importance. The engineering of a variety of highly differentiated tissues has been achieved using the rotating wall vessel (RWV) bioreactor technology, which is an optimized suspension culture allowing cells to grow in three-dimensions (3-D). However, certain cell types require the use of scaffolds, such as collagen-coated microcarrier beads, for optimal growth and differentiation in the RWV. Removal of the 3-D structures from the microcarriers involves enzymatic treatment, which disrupts the delicate 3-D architecture and makes it inapplicable for potential implantation. Therefore, we designed a microcarrier bead coated with a synthetic extracellular matrix (ECM) composed of a disulfide-crosslinked hyaluronan and gelatin hydrogel for 3-D tissue engineering, that allows for enzyme-free cell detachment under mild reductive conditions (i.e. by a thiol-disulfide exchange reaction). The ECM-coated beads (ECB) served as scaffold to culture human intestinal epithelial cells (Int-407) in the RWV, which formed viable multi-layered cell aggregates and expressed epithelial differentiation markers. The cell aggregates remained viable following dissociation from the microcarriers, and could be returned to the RWV bioreactor for further culturing into bead-free tissue assemblies. The developed ECBs thus offer the potential to generate scaffold-free 3-D tissue assemblies, which could further be explored for tissue replacement and remodeling.

A Murine Model of Inflammatory Bladder Disease: Cathelicidin Peptide Induced Bladder Inflammation and Treatment With Sulfated Polysaccharides

PURPOSE Studies show that LL-37 is a naturally occurring urinary defensin peptide that is up-regulated during urinary tract infections. Although normal urinary LL-37 levels are antimicrobial, we propose that increased LL-37 may trigger bladder inflammation. We further suggest that anti-inflammatory sulfated polysaccharides known as semi-synthetic glycosaminoglycan ether compounds can treat/prevent LL-37 mediated bladder inflammation. MATERIALS AND METHODS C57BL/6 mice were catheterized/instilled with LL-37 (320 μM, 150 μl) for 45 minutes. Animals were sacrificed at 12 and 24 hours, and tissues were examined using hematoxylin and eosin. Separate experiments were performed for myeloperoxidase to quantify inflammation. GM-1111 semi-synthetic glycosaminoglycan ether treatments involved instillation of 10 mg/ml for 45 minutes directly before or after LL-37. Tissues were harvested at 24 hours. To compare semi-synthetic glycosaminoglycan ether efficacy, experiments were performed using 10 mg/ml heparin. Finally, tissue localization of semi-synthetic glycosaminoglycan ether was examined using a fluorescent GM-1111-Alexa Fluor® 633 conjugate. RESULTS Profound bladder inflammation developed after LL-37. Greater tissue inflammation occurred after 24 hours compared to that at 12 hours. Myeloperoxidase assays revealed a 21 and 61-fold increase at 12 and 24 hours, respectively. Semi-synthetic glycosaminoglycan ether treatment after LL-37 showed mild attenuation of inflammation with myeloperoxidase 2.5-fold below that of untreated bladders. Semi-synthetic glycosaminoglycan ether treatment before LL-37 demonstrated almost complete attenuation of inflammation. Myeloperoxidase results mirrored those in controls. In heparin treated bladders minimal attenuation of inflammation occurred. Finally, instillation of GM-1111-Alexa Fluor 633 revealed urothelial coating, significant tissue penetration and binding to endovasculature. CONCLUSIONS We developed what is to our knowledge a new model of inflammatory bladder disease by challenge with the naturally occurring urinary peptide LL-37. We also noted that a new class of anti-inflammatory sulfated polysaccharides prevents and mitigates bladder inflammation.

Cross-linked hydrogel and polyester resorbable ventilation tubes in a Chinchilla model

To determine the resorption rate and biocompatibility characteristics of novel cross‐linked hydrogel ventilation tubes and varied formulations of polyester ventilation tubes in a Chinchilla model.