Integrin and syndecan binding peptide-conjugated alginate hydrogel for modulation of nucleus pulposus cell phenotype.

Biomaterial based strategies have been widely explored to preserve and restore the juvenile phenotype of cells of the nucleus pulposus (NP) in degenerated intervertebral discs (IVD). With aging and maturation, NP cells lose their ability to produce necessary extracellular matrix and proteoglycans, accelerating disc degeneration. Previous studies have shown that integrin or syndecan binding peptide motifs from laminin can induce NP cells from degenerative human discs to re-express juvenile NP-specific cell phenotype and biosynthetic activity. Here, we engineered alginate hydrogels to present integrin- and syndecan-binding peptides alone or in combination (cyclic RGD and AG73, respectively) to introduce bioactive features into the alginate gels. We demonstrated human NP cells cultured upon and within alginate hydrogels presented with cRGD and AG73 peptides exhibited higher cell viability, biosynthetic activity, and NP-specific protein expression over alginate alone. Moreover, the combination of the two peptide motifs elicited markers of the NP-specific cell phenotype, including N-Cadherin, despite differences in cell morphology and multicellular cluster formation between 2D and 3D cultures. These results represent a promising step toward understanding how distinct adhesive peptides can be combined to guide NP cell fate. In the future, these insights may be useful to rationally design hydrogels for NP cell-transplantation based therapies for IVD degeneration.

Copper-Free Azide-Alkyne Cycloaddition for Peptide Modification of Alginate Hydrogels.

Alginate, a biocompatible polymer naturally derived from algae, is widely used as a synthetic analogue of the extracellular matrix in tissue engineering. Integrin-binding peptide motifs, including RGD, a derivative of fibronectin, are typically grafted to the alginate polymer through carbodiimide reactions between peptide amines and alginate uronic acids. However, lack of chemo-selectivity of carbodiimide reactions can lead to side reactions that lower peptide bioactivity. To overcome these limitations, we developed an approach for copper-free, strain-promoted azide-alkyne cycloaddition (SPAAC)-mediated conjugation of azide-modified adhesive peptides (azido-cyclo-RGD, Az-cRGD) onto alginate. Successful conjugation of azide-reactive cyclooctynes onto alginates using a heterobifunctional crosslinker was confirmed by azido-coumarin fluorescent assay, NMR, and through click reactions with azide-modified fluorescent probes. Compared to cyclo-RGD peptides directly conjugated to alginate polymers with standard carbodiimide chemistry, Az-cyclo-RGD peptides exhibited higher bioactivity, as demonstrated by cell adhesion and proliferation assays. Finally, Az-cRGD peptides enhanced the effects of recombinant bone morphogenetic proteins on inducing osteogenesis of osteoblasts and bone marrow stromal stem cells in 3D alginate gels. SPAAC-mediated click approaches for peptide-alginate bioconjugation overcome the limitations of previous alginate bioconjugation approaches and potentially expand the range of ligands that can be grafted to alginate polymers for tissue engineering applications.

In-situ tissue regeneration through SDF-1α driven cell recruitment and stiffness-mediated bone regeneration in a critical-sized segmental femoral defect.

In-situ tissue regeneration aims to utilize the body's endogenous healing capacity through the recruitment of host stem or progenitor cells to an injury site. Stromal cell-derived factor-1α (SDF-1α) is widely discussed as a potent chemoattractant. Here we use a cell-free biomaterial-based approach to (i) deliver SDF-1α for the recruitment of endogenous bone marrow-derived stromal cells (BMSC) into a critical-sized segmental femoral defect in rats and to (ii) induce hydrogel stiffness-mediated osteogenic differentiation in-vivo. Ionically crosslinked alginate hydrogels with a stiffness optimized for osteogenic differentiation were used. Fast-degrading porogens were incorporated to impart a macroporous architecture that facilitates host cell invasion. Endogenous cell recruitment to the defect site was successfully triggered through the controlled release of SDF-1α. A trend for increased bone volume fraction (BV/TV) and a significantly higher bone mineral density (BMD) were observed for gels loaded with SDF-1α, compared to empty gels at two weeks. A trend was also observed, albeit not statistically significant, towards matrix stiffness influencing BV/TV and BMD at two weeks. However, over a six week time-frame, these effects were insufficient for bone bridging of a segmental femoral defect. While mechanical cues combined with ex-vivo cell encapsulation have been shown to have an effect in the regeneration of less demanding in-vivo models, such as cranial defects of nude rats, they are not sufficient for a SDF-1α mediated in-situ regeneration approach in segmental femoral defects of immunocompetent rats, suggesting that additional osteogenic cues may also be required. STATEMENT OF SIGNIFICANCE: Stromal cell-derived factor-1α (SDF-1α) is a chemoattractant used to recruit host cells for tissue regeneration. The concept that matrix stiffness can direct mesenchymal stromal cell (MSC) differentiation into various lineages was described a decade ago using in-vitro experiments. Recently, alginate hydrogels with an optimized stiffness and ex-vivo encapsulated MSCs were shown to have an effect in the regeneration of skull defects of nude rats. Here, we apply this material system, loaded with SDF-1α and without encapsulated MSCs, to (i) recruit endogenous cells and (ii) induce stiffness-mediated osteogenic differentiation in-vivo, using as model system a load-bearing femoral defect in immunocompetent rats. While a cell-free approach is of great interest from a translational perspective, the current limitations are described.

Hydrogels with tunable stress relaxation regulate stem cell fate and activity.

Natural extracellular matrices (ECMs) are viscoelastic and exhibit stress relaxation. However, hydrogels used as synthetic ECMs for three-dimensional (3D) culture are typically elastic. Here, we report a materials approach to tune the rate of stress relaxation of hydrogels for 3D culture, independently of the hydrogel's initial elastic modulus, degradation, and cell-adhesion-ligand density. We find that cell spreading, proliferation, and osteogenic differentiation of mesenchymal stem cells (MSCs) are all enhanced in cells cultured in gels with faster relaxation. Strikingly, MSCs form a mineralized, collagen-1-rich matrix similar to bone in rapidly relaxing hydrogels with an initial elastic modulus of 17 kPa. We also show that the effects of stress relaxation are mediated by adhesion-ligand binding, actomyosin contractility and mechanical clustering of adhesion ligands. Our findings highlight stress relaxation as a key characteristic of cell-ECM interactions and as an important design parameter of biomaterials for cell culture.

Matrix elasticity of void-forming hydrogels controls transplanted-stem-cell-mediated bone formation.

The effectiveness of stem cell therapies has been hampered by cell death and limited control over fate. These problems can be partially circumvented by using macroporous biomaterials that improve the survival of transplanted stem cells and provide molecular cues to direct cell phenotype. Stem cell behaviour can also be controlled in vitro by manipulating the elasticity of both porous and non-porous materials, yet translation to therapeutic processes in vivo remains elusive. Here, by developing injectable, void-forming hydrogels that decouple pore formation from elasticity, we show that mesenchymal stem cell (MSC) osteogenesis in vitro, and cell deployment in vitro and in vivo, can be controlled by modifying, respectively, the hydrogel's elastic modulus or its chemistry. When the hydrogels were used to transplant MSCs, the hydrogel's elasticity regulated bone regeneration, with optimal bone formation at 60 kPa. Our findings show that biophysical cues can be harnessed to direct therapeutic stem cell behaviours in situ.

Substrate stress relaxation regulates cell spreading.

Studies of cellular mechanotransduction have converged upon the idea that cells sense extracellular matrix (ECM) elasticity by gauging resistance to the traction forces they exert on the ECM. However, these studies typically utilize purely elastic materials as substrates, whereas physiological ECMs are viscoelastic, and exhibit stress relaxation, so that cellular traction forces exerted by cells remodel the ECM. Here we investigate the influence of ECM stress relaxation on cell behaviour through computational modelling and cellular experiments. Surprisingly, both our computational model and experiments find that spreading for cells cultured on soft substrates that exhibit stress relaxation is greater than cells spreading on elastic substrates of the same modulus, but similar to that of cells spreading on stiffer elastic substrates. These findings challenge the current view of how cells sense and respond to the ECM.

Ultrasound-triggered disruption and self-healing of reversibly cross-linked hydrogels for drug delivery and enhanced chemotherapy.

Biological systems are exquisitely sensitive to the location and timing of physiologic cues and drugs. This spatiotemporal sensitivity presents opportunities for developing new therapeutic approaches. Polymer-based delivery systems are used extensively for attaining localized, sustained release of bioactive molecules. However, these devices typically are designed to achieve a constant rate of release. We hypothesized that it would be possible to create digital drug release, which could be accelerated and then switched back off, on demand, by applying ultrasound to disrupt ionically cross-linked hydrogels. We demonstrated that ultrasound does not permanently damage these materials but enables nearly digital release of small molecules, proteins, and condensed oligonucleotides. Parallel in vitro studies demonstrated that the concept of applying temporally short, high-dose "bursts" of drug exposure could be applied to enhance the toxicity of mitoxantrone toward breast cancer cells. We thus used the hydrogel system in vivo to treat xenograft tumors with mitoxantrone, and found that daily ultrasound-stimulated drug release substantially reduced tumor growth compared with sustained drug release alone. This approach of digital drug release likely will be applicable to a broad variety of polymers and bioactive molecules, and is a potentially useful tool for studying how the timing of factor delivery controls cell fate in vivo.

Harnessing traction-mediated manipulation of the cell/matrix interface to control stem-cell fate.

Stem cells sense and respond to the mechanical properties of the extracellular matrix. However, both the extent to which extracellular-matrix mechanics affect stem-cell fate in three-dimensional microenvironments and the underlying biophysical mechanisms are unclear. We demonstrate that the commitment of mesenchymal stem-cell populations changes in response to the rigidity of three-dimensional microenvironments, with osteogenesis occurring predominantly at 11-30 kPa. In contrast to previous two-dimensional work, however, cell fate was not correlated with morphology. Instead, matrix stiffness regulated integrin binding as well as reorganization of adhesion ligands on the nanoscale, both of which were traction dependent and correlated with osteogenic commitment of mesenchymal stem-cell populations. These findings suggest that cells interpret changes in the physical properties of adhesion substrates as changes in adhesion-ligand presentation, and that cells themselves can be harnessed as tools to mechanically process materials into structures that feed back to manipulate their fate.

Infection-mimicking materials to program dendritic cells in situ.

Cancer vaccines typically depend on cumbersome and expensive manipulation of cells in the laboratory, and subsequent cell transplantation leads to poor lymph-node homing and limited efficacy. We propose that materials mimicking key aspects of bacterial infection may instead be used to directly control immune-cell trafficking and activation in the body. It is demonstrated that polymers can be designed to first release a cytokine to recruit and house host dendritic cells, and subsequently present cancer antigens and danger signals to activate the resident dendritic cells and markedly enhance their homing to lymph nodes. Specific and protective anti-tumour immunity was generated with these materials, as 90% survival was achieved in animals that otherwise die from cancer within 25 days. These materials show promise as cancer vaccines, and more broadly suggest that polymers may be designed to program and control the trafficking of a variety of cell types in the body.

Cyclic arginine-glycine-aspartate peptides enhance three-dimensional stem cell osteogenic differentiation.

The role of morphogens in bone regeneration has been widely studied, whereas the effect of matrix cues, particularly on stem cell differentiation, are less well understood. In this work, we investigated the effects of arginine-glycine-aspartate (RGD) ligand conformation (linear vs cyclic RGD) on primary human bone marrow stromal cell (hBMSC) and D1 stem cell osteogenic differentiation in three-dimensional (3D) culture and compared their response with that of committed MC3T3-E1 preosteoblasts to determine whether the stage of cell differentiation altered the response to the adhesion ligands. Linear RGD densities that promoted osteogenic differentiation of committed cells (MC3T3-E1 preosteoblasts) did not induce differentiation of hBMSCs or D1 stem cells, although matrices presenting the cyclic form of this adhesion ligand enhanced osteoprogenitor differentiation in 3D culture. This may be due to enhanced integrin-ligand binding. These studies indicate that biomaterial design parameters optimized for differentiated cell types may not directly translate to stem cell populations, because less-committed cells may require more instruction than differentiated cells. It is likely that design of synthetic extracellular matrices tailored to promote stem cell differentiation may enhance bone regeneration by transplanted cells.

Analysis of sterilization protocols for peptide-modified hydrogels.

Concerns about the efficacy of ethanol disinfection for implanted biomaterials prompted investigation of an alternative sterilization process, ultraviolet irradiation, for terminal sterilization of N-isopropylacrylamide-based hydrogels containing biomimetic peptides. Ultraviolet irradiation is more easily applied on a laboratory scale than gamma irradiation or electron beam, two commercially utilized methods; thus, UVC irradiation was investigated as a low-cost sterilization procedure that might be performed in laboratories prior to in vivo studies. UVC irradiation at 400 muW/cm(2) for up to 15 h did not prevent growth of Escherichia coli within the hydrogels, while ethanol disinfection did prevent growth for the duration of the experiment (120 h). Furthermore, UVC irradiation caused progressive degradation of peptides containing the Arg-Gly-Arg (RGD) domain. UVC irradiation cannot be used as a terminal sterilization process for peptide-modified materials. The system used in this study is not intended to be adequate for evaluating the sterility of medical devices in accordance with current Good Manufacturing Practice (cGMP); however, it remains a useful, low-cost system for the preliminary evaluation of sterilization procedures in terms of their ability to eliminate pathogenic organisms while preserving the structure of biologically active molecules within in a laboratory setting. Ethanol treatment is still the preferred method for disinfection of bioactive materials containing peptides or UV-degradable groups.