Macroporous biohybrid cryogels for co-housing pancreatic islets with mesenchymal stromal cells.

UNLABELLED: Intrahepatic transplantation of allogeneic pancreatic islets offers a promising therapy for type 1 diabetes. However, long-term insulin independency is often not achieved due to severe islet loss shortly after transplantation. To improve islet survival and function, extrahepatic biomaterial-assisted transplantation of pancreatic islets to alternative sites has been suggested. Herein, we present macroporous, star-shaped poly(ethylene glycol) (starPEG)-heparin cryogel scaffolds, covalently modified with adhesion peptides, for the housing of pancreatic islets in three-dimensional (3D) co-culture with adherent mesenchymal stromal cells (MSC) as accessory cells. The implantable biohybrid scaffolds provide efficient transport properties, mechanical protection, and a supportive extracellular environment as a desirable niche for the islets. MSC colonized the cryogel scaffolds and produced extracellular matrix proteins that are important components of the natural islet microenvironment known to facilitate matrix-cell interactions and to prevent cellular stress. Islets survived the seeding procedure into the cryogel scaffolds and secreted insulin after glucose stimulation in vitro. In a rodent model, intact islets and MSC could be visualized within the scaffolds seven days after subcutaneous transplantation. Overall, this demonstrates the potential of customized macroporous starPEG-heparin cryogel scaffolds in combination with MSC to serve as a multifunctional islet supportive carrier for transplantation applications. STATEMENT OF SIGNIFICANCE: Diabetes results in the insufficient production of insulin by the pancreatic ß-cells in the islets of Langerhans. Transplantation of pancreatic islets offers valuable options for treating the disease; however, many transplanted islets often do not survive the transplantation or die shortly thereafter. Co-transplanted, supporting cells and biomaterials can be instrumental for improving islet survival, function and protection from the immune system. In the present study, islet supportive hydrogel sponges were explored for the co-transplantation of islets and mesenchymal stromal cells. Survival and continued function of the supported islets were demonstrated in vitro. The in vivo feasibility of the approach was shown by transplantation in a mouse model.

Neurotropic growth factors and glycosaminoglycan based matrices to induce dopaminergic tissue formation.

Current cell replacement therapies in Parkinson's disease (PD) are limited by low survival of transplanted cell and lacking regeneration of neuronal circuitries. Therefore, bioartificial cell carriers and growth/differentiation factors are applied to improve the integration of transplants and maximize newly generated and/or residual dopaminergic function. In this work, biohybrid poly(ethylene glycol) (starPEG)-heparin hydrogels releasing fibroblast growth factor 2 (FGF-2) and glial-derived neurotrophic factor (GDNF) were used to trigger dopaminergic tissue formation by primary murine midbrain cells in vitro. Matrix-delivered FGF-2 enhanced cell viability while release of GDNF had a pro-neuronal/dopaminergic effect. Combined delivery of both factors from the glycosaminoglycan-based matrices resulted in a tremendous improvement in survival and maturation capacity of dopaminergic neurons as obvious from tyrosine hydroxylase expression and neurite outgrowth. The reported data demonstrate that glycosaminoglycan-based hydrogels can facilitate the administration of neurotrophic factors and are therefore instrumental in potential future treatments of PD.

Cryogel micromechanics unraveled by atomic force microscopy-based nanoindentation.

Cell-instructive physical characteristics of macroporous scaffolds, developed for tissue engineering applications, often remain difficult to assess. Here, an atomic force microscopy-based nanoindentation approach is adapted to quantify the local mechanical properties of biohybrid glycosaminoglycan-poly(ethylene glycol) cryogels. Resulting from cryoconcentration effects upon gel formation, cryogel struts are observed to feature a higher stiffness compared to the corresponding bulk hydrogel materials. Local Young's moduli, porosity, and integral moduli of the cryogel scaffolds are compared in dependence on gel formation parameters. The results provide valuable insights into the cryogelation process and a base for adjusting physical characteristics of the obtained cryogel scaffolds, which can critically influence the cellular response.

Macroporous starPEG-heparin cryogels.

Macroporous scaffolds with adaptable mechanical and biomolecular properties can be instrumental in enabling cell-based therapies. To meet these requirements, a cryostructuration method was adapted to prepare spongy hydrogels based on chemically cross-linked star-shaped poly(ethylene glycol) (starPEG) and heparin. Subzero temperature treatment of the gel forming reaction mixtures and subsequent lyophilization of the incompletely frozen gels resulted in macroporous biohybrid cryogels showing rapid swelling, porosity of up to 92% with interconnected large pores (30-180 µm), low bulk stiffness, and high mechanical stability upon compression. The applicability of the cryogel scaffolds was investigated using human umbilical vein endothelial cells. Cell attachment and three-dimensional spreading resulted in evenly distributed viable cells within the macroporous starPEG-heparin materials, demonstrating the significant translational potential of the developed three-dimensional cell carriers.

FGF-2 and VEGF functionalization of starPEG-heparin hydrogels to modulate biomolecular and physical cues of angiogenesis.

Tissue engineering therapies require biomaterials capable of encouraging an angiogenic response. To dissect the influence of different pro-angiogenic stimuli a set of starPEG-heparin hydrogels with varied physicochemical properties was used as a highly efficient reservoir and tunable delivery system for basic fibroblast growth factor (FGF-2) and vascular endothelial growth factor (VEGF). The engineered gel materials could be precisely tailored by decoupling the biomolecular functionalization from the variation of the viscoelastic matrix characteristics. Culture experiments with human umbilical vein endothelial cells (HUVECs) revealed the interplay of growth factor presentation, adhesive characteristics and elasticity of the gel matrices in triggering differential cellular behavior which allowed identifying effective pro-angiogenic conditions.

A star-PEG-heparin hydrogel platform to aid cell replacement therapies for neurodegenerative diseases.

Biofunctional matrices for in vivo tissue engineering strategies must be modifiable in both biomolecular composition and mechanical characteristics. To address this challenge, we present a modular system of biohybrid hydrogels based on covalently cross-linked heparin and star-shaped poly(ethylene glycols) (star-PEG) in which network characteristics can be gradually varied while heparin contents remain constant. Mesh size, swelling and elastic moduli were shown to correlate well with the degree of gel component cross-linking. Additionally, secondary conversion of heparin within the biohybrid gels allowed the covalent attachment of cell adhesion mediating RGD peptides and the non-covalent binding of soluble mitogens such as FGF-2. We applied the biohybrid gels to demonstrate the impact of mechanical and biomolecular cues on primary nerve cells and neural stem cells. The results demonstrate the cell type-specific interplay of synergistic signaling events and the potential of biohybrid materials to selectively stimulate cell fate decisions. These findings suggest important future uses for this material in cell replacement based-therapies for neurodegenerative diseases.

Engineered extracellular matrices modulate the expression profile and feeder properties of bone marrow-derived human multipotent mesenchymal stromal cells.

The bone marrow harbors multipotent mesenchymal stromal cells (MSCs) that nurture hematopoietic stem cells (HSCs). The extracellular matrix (ECM) is an integral part of the bone marrow, and the aim of this study was therefore to examine the effect of engineered ECM substrates on MSC gene expression over time and to determine quantitatively the functional ability of ECM-cultured MSCs to support HSCs. ECMs were surface immobilized using thin films of maleic anhydride to covalently immobilize tropocollagen or fibrillar collagen type I to the substrate. Where indicated, collagen type I fibrils were supplemented with heparin or hyaluronic acid. All surfaces maintained MSC viability and supported cell expansion. Microarray analysis of MSCs cultured on engineered ECM substrates revealed that culture time, as well as substrate composition, significantly affected expression levels. Based on these studies, it was possible to predict the effect of these substrates on in vitro HSC clonogenicity and self-renewal. The ability to regulate the expression of stromal factors using engineered ECM is exciting and warrants further studies to identify the ECM components and combinations that maximize the expansion of clonogenic HSCs.

Heparin intercalation into reconstituted collagen I fibrils: Impact on growth kinetics and morphology.

Collagen type I fibrils, reconstituted in vitro in the presence of heparin, exhibit an unusually thick and straight shape. A detailed structural analysis by scanning force and scanning electron microscopy revealed a non-linear dependence in size distribution, width-to-length ratio, and morphology over a wide range of glycosaminoglycan (GAG) concentrations. By varying molecular weight, degree of sulphation, charge, and concentration of different GAGs we are able to correlate the morphological data with kinetic turbidimetric measurements, and quantitation of fibril-bound GAG. The experiments imply a pronounced impact of the prenucleation phase on the cofibril morphology as a result of the strong electrostatic interaction of heparin with tropocollagen. Heparin is assumed to stabilize the collagen microfibrils and to enhance their parallel accretion during cofibrillogenesis with preservation of the typical asymmetric collagen banding pattern. The heparin quantitation data show heparin to be intercalated as a linker molecule with one specific binding site inside the cofibrils. The reconstituted cofibrils with their unusual morphology and GAG intercalation-a phenomenon not reported in vivo-can be expected to exhibit interesting mechanical and biochemical behaviours as a biomaterial for extracellular matrix scaffolds.

Electrostatic interactions modulate the conformation of collagen I.

The pH- and electrolyte-dependent charging of collagen I fibrils was analyzed by streaming potential/streaming current experiments using the Microslit Electrokinetic Setup. Differential scanning calorimetry and circular dichroism spectroscopy were applied in similar electrolyte solutions to characterize the influence of electrostatic interactions on the conformational stability of the protein. The acid base behavior of collagen I was found to be strongly influenced by the ionic strength in KCl as well as in CaCl(2) solutions. An increase of the ionic strength with KCl from 10(-4) M to 10(-2) M shifts the isoelectric point (IEP) of the protein from pH 7.5 to 5.3. However, a similar increase of the ionic strength in CaCl(2) solutions shifts the IEP from 7.5 to above pH 9. Enhanced thermal stability with increasing ionic strength was observed by differential scanning calorimetry in both electrolyte systems. In line with this, circular dichroism spectroscopy results show an increase of the helicity with increasing ionic strength. Better screening of charged residues and the formation of salt bridges are assumed to cause the stabilization of collagen I with increasing ionic strength in both electrolyte systems. Preferential adsorption of hydroxide ions onto intrinsically uncharged sites in KCl solutions and calcium binding to negatively charged carboxylic acid moieties in CaCl(2) solutions are concluded to shift the IEP and influence the conformational stability of the protein.

Functional films of maleic anhydride copolymers under physiological conditions.

Reactivity and swelling of nanometer films of alternating maleic anhydride copolymers were investigated in dependence on the kind of comonomer and molar mass of copolymer in aqueous solution at pH 7.4 and pH 3.0 in order to reveal their characteristics under physiological conditions. Fully hydrolyzed (maleic acid) chains of the copolymers with styrene, propene, and ethylene comonomers covalently bound to SiO2 substrates showed a "mushroom" swelling behavior at pH 7.4 with a layer thickness scaling of N3/5. Decreasing the environmental pH was found to induce a comonomer-dependent shrinking or collapse of the immobilized polymers due to the change in ionization. From the swelling kinetics of non-hydrolyzed chains, the time constants and characteristics of swelling and anhydride hydrolysis were determined and found to depend on the type of comonomer. The short- and long-term swelling kinetics [l approximately t and approximately ln(t)1/2] were found to be in agreement with theoretical models of polymer swelling, while at intermediate time scales enhanced swelling was observed due to hydrolysis reaction of maleic anhydride groups. The findings elucidate the variety of properties of maleic anhydride copolymer films under physiological conditions, which can advantageously be applied for biofunctionalization of different templates.

In vitro reconstitution of fibrillar collagen type I assemblies at reactive polymer surfaces.

The reconstitution of fibrillar collagen and its assemblies with heparin and hyaluronic acid was studied in vitro. Fibril formation kinetics were analyzed by turbidity and depletion measurements in solutions containing varied concentrations of collagen and glycosaminoglycans. Fibril-forming collagen solutions were further applied for the coating of planar substrates which had been modified with alternating maleic anhydride copolymer films before. The immobilized collagen assemblies were characterized with respect to the deposited amount of protein using ellipsometry and acidic hydrolysis/HPLC-based amino acid analysis, respectively. AFM, SEM, and cLSM were utilized to gain information on structural features and patterns formed by surface-attached fibrils depending on the initial solution concentrations of collagen. The results revealed that the addition of heparin and hyaluronic acid affected both the fibril dimensions and the meshwork characteristics of the surface-bound fibrils.