A method for preparation of hydrogel microcapsules for stem cell bioprocessing and stem cell therapy.

A method for the preparation of suspension culture microcapsules used in the bioprocessing of human mesenchymal stem cells (hMSCs) is reported. The microcapsules are prepared from a semi-synthetic hydrogel comprising Pluronic®F127 conjugated to denatured fibrinogen. The Pluronic-fibrinogen adducts display a lower critical solubility temperature (LCST) at ∼30 °C, thus enabling mild, cell-compatible physical crosslinking of the microcapsules in a warm gelation bath. Cell-laden microgels were prepared from a solution of Pluronic-fibrinogen hydrogel precursor and hMSCs; these were cultivated for up to 15 days in laboratory-scale suspension bioreactors and harvested by reducing the temperature of the microcapsules to disassemble the physical polymer network. The viability, proliferation and cell recovery yields of the hMSCs were shown to be better than photo-chemically crosslinked microcapsules made from a similar material. The cell culture yields, which exceeded 300% after 15 days in suspension culture, were comparable to other microcarrier systems used for the mass production of hMSCs. The simplicity of this methodology, both in terms of the cell inoculation and mild recovery conditions, represent distinct advantages for stem cell bioprocessing with suspension culture bioreactors.

Stimulus-responsive hydrogels made from biosynthetic fibrinogen conjugates for tissue engineering: structural characterization.

Nanostructured hydrogels based on "smart" polymer conjugates of poloxamers and protein molecules were developed in order to form stimulus-responsive materials with bioactive properties for 3-D cell culture. Functionalized Pluronic F127 was covalently attached to a fibrinopeptide backbone and cross-linked into a structurally versatile and mechanically stable polymer network endowed with bioactivity and temperature-responsive structural features. Small angle X-ray scattering and transmission electron microscopy combined with rheology were used to characterize the structural and mechanical features of this biosynthetic conjugate, both in solution and in hydrogel form. The temperature at which the chemical cross-linking of F127-fibrinopeptide conjugates was initiated had a profound influence on the mechanical properties of the thermo-responsive hydrogel. The analysis of the scattering data revealed modification in the structure of the protein backbone resulting from increases in ambient temperature, whereas the structure of the polymer was not affected by ambient temperature. The hydrogel cross-linking temperature also had a major influence on the modulus of the hydrogel, which was rationally correlated to the molecular structure of the polymer network. The hydrogel structure exhibited a small mesh size when cross-linked at low temperatures and a larger mesh size when cross-linked at higher temperatures. The mesh size was nicely correlated to the mechanical properties of the hydrogels at the respective cross-linking temperatures. The schematic charts that model this material's behavior help to illustrate the relationship that exists between the molecular structure, the cross-linking temperature, and the temperature-responsive features for this class of protein-polymer conjugates. The precise control over structural and mechanical properties that can be achieved with this bioactive hydrogel material is essential in designing a tissue-engineering scaffold for clinical applications.

Defining a turnover index for the correlation of biomaterial degradation and cell based extracellular matrix synthesis using fluorescent tagging techniques.

Non-destructive protocols which can define a biomaterial's degradation and its associated ability to support proliferation and/or promote extracellular matrix deposition will be an essential in vitro tool. In this study we investigate fluorescently tagged biomaterials, with varying rates of degradation and their ability to support cell proliferation and osteogenic differentiation. Changes in fluorescence of the biomaterials and the release of fluorescent soluble by-products were confirmed as accurate methods to quantify degradation. It was demonstrated that increasing rates of the selected biomaterials' degradation led to a decrease in cell proliferation and concurrently an increase in osteogenic matrix production. A novel turnover index (TI), which directly describes the effect of degradation of a biomaterial on cell behaviour, was calculated. Lower TIs for proliferation and high TIs for osteogenic marker production were observed on faster degrading biomaterials, indicating that these biomaterials supported an upregulation of osteogenic markers. This TI was further validated using an ex vivo chick femur model, where the faster degrading biomaterial, fibrin, led to an increased TI for mineralisation within an epiphyseal defect. This in vitro tool, TI, for monitoring the effect of biomaterial degradation on extracellular matrix production may well act as predictor of the selected biomaterials' performance during in vivo studies. STATEMENT OF SIGNIFICANCE: This paper outlines a novel metric, Turnover Index (TI), which can be utilised in tissue-engineering for the comparison of a range of biomaterials. The metric sets out to define the relationship between the rate of degradation of biomaterials with the rate of cell proliferation and ECM synthesis, ultimately allowing us to tailor material for set clinical requirements. We have discovered some novel comparative findings that cells cultured on biomaterials with increased rates of degradation have lower rates of proliferation but alternatively have a greater production of osteogenic markers compared to materials which degrade slower. By making comparisons in a rigorous manner, we can begin to define a useful matrix for materials which ultimately may aid for clinical selection.

Protein composition alters in vivo resorption of PEG-based hydrogels as monitored by contrast-enhanced MRI.

We report on the use of magnetic resonance imaging (MRI)-based non-invasive monitoring to document the role of protein adjuvants in hydrogel implant integration in vivo. Polyethylene glycol (PEG) hydrogels were formed with different protein constituents, including albumin, fibrinogen and gelatin. The hydrogels were designed to exhibit similar material properties, including modulus, swelling and hydrolytic degradation kinetics. The in vivo resorption properties of these PEG-based hydrogels, which contained a tethered gadolinium contrast agent, were characterized by MRI and histology, and compared to their in vitro characteristics. MRI data revealed that PEG-Albumin implants remained completely intact throughout the experiments, PEG-Fibrinogen implants lost about 10% of their volume and PEG-Gelatin implants underwent prominent swelling and returned to their initial volume by day 25. Fully synthetic PEG-diacrylate (PEG-DA) control hydrogels lost about half of their volume after 25 days in vivo. Transverse MRI cross-sections of the implants revealed distinct mechanisms of the hydrogel's biodegradation: PEG-Fibrinogen and PEG-Albumin underwent surface erosion, whereas PEG-Gelatin and PEG-DA hydrogels mainly underwent bulk degradation. Histological findings substantiated the MRI data and demonstrated significant cellular response towards PEG-DA and PEG-Gelatin scaffolds with relatively low reaction towards PEG-Fibrinogen and PEG-Albumin hydrogels. These findings demonstrate that PEG-protein hydrogels can degrade via a different mechanism than PEG hydrogels, and that this difference can be linked to a reduced foreign body response.

Steric Interference of Adhesion Supports In-Vitro Chondrogenesis of Mesenchymal Stem Cells on Hydrogels for Cartilage Repair.

Recent studies suggest the presence of cell adhesion motifs found in structural proteins can inhibit chondrogenesis. In this context, the current study aims to determine if a polyethylene glycol (PEG)-modified fibrinogen matrix could support better chondrogenesis of human bone marrow mesenchymal stem cells (BM-MSC) based on steric interference of adhesion, when compared to a natural fibrin matrix. Hydrogels used as substrates for two-dimensional (2D) BM-MSC cultures under chondrogenic conditions were made from cross-linked PEG-fibrinogen (PF) and compared to thrombin-activated fibrin. Cell morphology, protein expression, DNA and sulfated proteoglycan (GAG) content were correlated to substrate properties such as stiffness and adhesiveness. Cell aggregation and chondrogenic markers, including collagen II and aggrecan, were observed on all PF substrates but not on fibrin. Shielding fibrinogen's adhesion domains and increasing stiffness of the material are likely contributing factors that cause the BM-MSCs to display a more chondrogenic phenotype. One composition of PF corresponding to GelrinC™--a product cleared in the EU for cartilage repair--was found to be optimal for supporting chondrogenic differentiation of BM-MSC while minimizing hypertrophy (collagen X). These findings suggest that semi-synthetic biomaterials based on ECM proteins can be designed to favourably affect BM-MSC towards repair processes involving chondrogenesis.

Matrix stiffness determines the fate of nucleus pulposus-derived stem cells.

Intervertebral disc (IVD) degeneration and consequent low-back pain present a major medical challenge. Nucleus pulposus-derived stem cells (NP-SCs) may lead to a novel therapy for this severe disease. It was recently shown that survival and function of mature NP cells are regulated in part by tissue stiffness. We hypothesized that modification of matrix stiffness will influence the ability of cultured NP-SCs to proliferate, survive, and differentiate into mature NP cells. NP-SCs were subcultured in three-dimensional matrices of varying degrees of stiffness as measured by the material's shear storage modulus. Cell survival, activity, and rate of differentiation toward the chondrogenic or osteogenic lineage were analyzed. NP-SCs were found to proliferate and differentiate in all matrices, irrespective of matrix stiffness. However, matrices with a low shear storage modulus (G' = 1 kPa) promoted significantly more proliferation and chondrogenic differentiation, whereas matrices with a high modulus (G' = 2 kPa) promoted osteogenic differentiation. Imaging performed via confocal and scanning electron microscopes validated cell survival and highlighted stiffness-dependent cell-matrix interactions. These results underscore the effect of the matrix modulus on the fate of NP-SCs. This research may facilitate elucidation of the complex cross-talk between NP-SCs and their surrounding matrix in healthy as well as pathological conditions.

The effect of matrix stiffness of injectable hydrogels on the preservation of cardiac function after a heart attack.

This study compares the effect of four injectable hydrogels with different mechanical properties on the post-myocardial infarction left ventricle (LV) remodeling process. The bioactive hydrogels were synthesized from Tetronic-fibrinogen (TF) and PEG-fibrinogen (PF) conjugates; each hydrogel was supplemented with two levels of additional cross-linker to increase the matrix stiffness as measured by the shear storage modulus (G'). Infarcts created by ligating the left anterior descending coronary artery in a rodent model were treated with the hydrogels, and all four treatment groups showed an increase in wall thickness, arterial density, and viable cardiac tissue in the peri-infarct areas of the LV. Echocardiography and hemodynamics data of the PF/TF treated groups showed significant improvement of heart function associated with the attenuated effects of the remodeling process. Multi-factorial regression analysis indicated that the group with the highest modulus exhibited the best rescue of heart function and highest neovascularization. The results of this study demonstrate that multiple properties of an injectable bioactive biomaterial, and notably the matrix stiffness, provide the multifaceted stimulation necessary to preserve cardiac function and prevent adverse remodeling following a heart attack.

The biocompatibility of PluronicF127 fibrinogen-based hydrogels.

Our research is focused on the design of hydrogel biomaterials that can be used for 3-D cell encapsulation and tissue engineering. In this study, our goal was to engineer a temperature-responsive biomaterial to possess bioactive properties using polymer and protein chemistry, and at the same time provide the biomaterial with susceptibility to cell-mediated remodeling. Toward this goal, we developed a biomimetic material that can harness the bioactive properties of fibrinogen and the unique structural properties of PluronicF127. PluronicF127 is a synthetic block copolymer that exhibits reverse thermal gelation (RTG) in response to small changes in ambient temperature. We conjugated fibrinogen to Pluronic)F127 to create a biosynthetic precursor with tunable physicochemical properties based on the relationship between the two constituents. A hydrogel matrix was formed from the fibrinogen-F127 adducts by free-radical polymerization using light activation (photo-polymerization). These materials displayed a reversible temperature-induced physical sol-gel transition and an irreversible light-activated chemical cross-linking. The susceptibility of this hydrogel biomaterial to protease degradation and consequent cell-mediated remodeling was controlled by the PluronicF127 constituent. The protein-based material also conveyed inductive signals to cells through bioactive sites on the fibrinogen backbone, as well as through structural properties such as the matrix modulus. We apply these materials as a tissue engineering hydrogel scaffold for 3-D in vitro culture of dermal fibroblasts in order to gain a better understanding of how the material bioactivity and matrix properties can independently affect cell morphology and remodeling.