Laura A. Smith Callahan

Strain-Promoted Crosslinking of PEG-based Hydrogels via Copper-Free Cycloaddition

The synthesis of a 4-dibenzocyclooctynol (DIBO) functionalized polyethylene glycol (PEG) and fabrication of hydrogels via strain-promoted, metal-free, azide-alkyne cycloaddition is reported. The resulting hydrogel materials provide a versatile alternative in which to encapsulate cells that are sensitive to photochemical or chemical crosslinking mechanisms.

Resorbable, amino acid-based poly(ester urea)s crosslinked with osteogenic growth peptide with enhanced mechanical properties and bioactivity.

Materials currently used for the treatment of bone defects include ceramics, polymeric scaffolds and composites, which are often impregnated with recombinant growth factors and other bioactive substances. While these materials have seen instances of success, each has inherent shortcomings including prohibitive expense, poor protein stability, poorly defined growth factor release and less than desirable mechanical properties. We have developed a novel class of amino acid-based poly(ester urea)s (PEU) materials which are biodegradable in vivo and possess mechanical properties superior to conventionally used polyesters (<3.5 GPa) available currently to clinicians and medical providers. We report the use of a short peptide derived from osteogenic growth peptide (OGP) as a covalent crosslinker for the PEU materials. In addition to imparting specific bioactive signaling, our crosslinking studies show that the mechanical properties increase proportionally when 0.5% and 1.0% concentrations of the OGP crosslinker are added. Our results in vitro and in an in vivo subcutaneous rat model show the OGP-based crosslinkers, which are small fragments of growth factors that are normally soluble, exhibit enhanced proliferative activity, accelerated degradation properties and concentration dependent bioactivity when immobilized.

Primary human chondrocyte extracellular matrix formation and phenotype maintenance using RGD-derivatized PEGDM hydrogels possessing a continuous Young’s modulus gradient

Efficient ex vivo methods for expanding primary human chondrocytes while maintaining the phenotype is critical to advancing the sourcing of autologous cells for tissue engineering applications. While there has been significant research reported in the literature, systematic approaches are necessary to determine and optimize the chemical and mechanical scaffold properties for hyaline cartilage generation using limited cell numbers. Functionalized hydrogels possessing continuous variations in physico-chemical properties are, therefore, an efficient three-dimensional platform for studying several properties simultaneously. Herein we describe a polyethylene glycol dimethacrylate (PEGDM) hydrogel system with a modulus gradient (~27,000-3800 Pa) containing a uniform concentration of arginine-glycine-aspartic acid (RGD) peptide to enhance cell adhesion in order to correlate primary human osteoarthritic chondrocyte proliferation, phenotype maintenance, and extracellular matrix (ECM) production with hydrogel properties. Cell number and chondrogenic phenotype (CD14:CD90 ratios) were found to decline in regions with a higher storage modulus (>13,100 Pa), while regions with a lower storage modulus maintained their cell number and phenotype. Over 3 weeks culture hydrogel regions possessing a lower Youngs modulus experienced an increase in ECM content (~200%) compared with regions with a higher storage modulus. Variations in the amount and organization of the cytoskeletal markers actin and vinculin were observed within the modulus gradient, which are indicative of differences in chondrogenic phenotype maintenance and ECM expression. Thus scaffold mechanical properties have a significant impact in modulating human osteoarthritic chondrocyte behavior and tissue formation.

OGP functionalized phenylalanine-based poly(ester urea) for enhancing osteoinductive potential of human mesenchymal stem cells.

Amino acid-based poly(ester urea)s (PEU) are high modulus, resorbable polymers with many potential uses, including the surgical repair of bone defects. In vitro and in vivo studies have previously shown that phenylalanine-based PEUs have nontoxic hydrolytic byproducts and tunable degradation times. Phenylalanine PEUs (poly(1-PHE-6)) have been further modified by tethering osteogenic growth peptide (OGP) to tyrosine-based monomer subunits. These OGP-tethered PEUs have been fabricated into porous scaffolds and cultured in vitro to examine their effect on differentiation of human mesenchymal stem cells (hMSCs) toward the osteogenic lineage. The influence of tethered OGP on the hMSC proliferation and differentiation profile was measured using immunohistochemistry, biochemistry, and quantitative real time polymerase chain reaction (qRT-PCR). In vitro data indicated an enhanced expression of BSP by 130-160% for hMSCs on OGP-tethered scaffolds compared to controls. By 4 weeks, there was a significant drop (60-85% decrease) in BSP expression on OGP-functionalized scaffolds, which is characteristic of osteogenic differentiation. ALP and OSC expression was significantly enhanced for OGP-functionalized scaffolds by week 4, with values reaching 145% and 300% greater, respectively, compared to nonfunctionalized controls. In vivo subcutaneous implantation of poly(1-PHE-6) scaffolds revealed significant tissue-scaffold integration, as well as the promotion of both osteogenesis and angiogenesis.

ECM production of primary human and bovine chondrocytes in hybrid PEG hydrogels containing type i collagen and hyaluronic acid

The development of advanced materials that facilitate hyaline cartilage formation and regeneration in aging populations is imperative. Critical to the success of this endeavor is the optimization of ECM production from clinically relevant cells. However, much of the current literature focuses on the investigation of primary bovine chondrocytes from young calves, which differ significantly than osteoarthritic cells from human sources. This study examines the levels of extracellular matrix (ECM) production using various levels of type I collagen and hyaluronic acid in poly(ethylene glycol) dimethacrylate (PEGDM) hydrogels in total knee arthroplasties, compared with the results from bovine chondrocytes. The addition of type 1 collagen in both the presence and absence of low levels of hyaluronic acid increased ECM production and/or retention in scaffolds containing either bovine or human chondrocytes. These findings are supported consistently with colorimetric quantification, whole mount extracellular matrix staining for both cell types, and histological staining for glycoaminoglycans and collagen of human chondrocyte containing samples. While exhibiting similar trends, the relative ECM productions levels for the primary human chondrocytes are significantly less than the bovine chondrocytes which reinforces the need for additional optimization.

Optimization of adhesive conditions for neural differentiation of murine embryonic stem cells using hydrogels functionalized with continuous Ile-Lys-Val-Ala-Val concentration gradients

Stem cell therapies, which aim to restore neurological function after central nervous system injury, have shown increased efficacy when a tissue engineering matrix is implanted with cells compared to implantation of the cells alone. However, much work still needs to be done to characterize materials that can be used to facilitate and direct the differentiation of implanted cells. In the current study, polyethylene glycol hydrogels functionalized with continuous Ile-Lys-Val-Ala-Val (IKVAV) concentration gradients were fabricated and utilized to systematically study and optimize the adhesive conditions for neural differentiation of mouse embryonic stem cells in two- and three-dimensional environments. The results suggest that 570 μM and 60 μM are the optimal IKVAV concentrations for 2D and 3D neural differentiation, respectively, to maximize mRNA expression of neuron-specific markers and neurite extension while minimizing apoptotic activities in cultured cells compared to those exposed to higher IKVAV concentrations. The combinatorial approach presented in this work demonstrates that hydrogels functionalized with bioactive peptides provide a defined and tunable platform that can be employed to characterize and improve culture conditions for superior survival, maturation and integration of implanted cells, leading to enhanced restoration of neurological function for those receiving stem cell therapies after traumatic brain and spinal cord injuries.

Maximizing phenotype constraint and extracellular matrix production in primary human chondrocytes using arginine–glycine–aspartate concentration gradient hydrogels

New systematic approaches are necessary to determine and optimize the chemical and mechanical scaffold properties for hyaline cartilage generation using the limited cell numbers obtained from primary human sources. Peptide functionalized hydrogels possessing continuous variations in physico-chemical properties are an efficient three-dimensional platform for studying several properties simultaneously. Herein, we describe a polyethylene glycol dimethacrylate (PEGDM) hydrogel system possessing a gradient of arginine-glycine-aspartic acid peptide (RGD) concentrations from 0mM to 10mM. The system is used to correlate primary human osteoarthritic chondrocyte proliferation, phenotype maintenance and extracellular matrix (ECM) production to the gradient hydrogel properties. Cell number and chondrogenic phenotype (CD14:CD90 ratios) were found to decline in regions with higher RGD concentrations, while regions with lower RGD concentrations maintained cell number and phenotype. Over three weeks of culture, hydrogel regions containing lower RGD concentrations experience an increase in ECM content compared to regions with higher RGD concentrations. Variations in actin amounts and vinculin organization were observed within the RGD concentration gradients that contribute to the differences in chondrogenic phenotype maintenance and ECM expression.

Influence of discrete and continuous culture conditions on human mesenchymal stem cell lineage choice in RGD concentration gradient hydrogels.

Stem cells have shown lineage-specific differentiation when cultured on substrates possessing signaling groups derived from the native tissue. A distinct determinant in this process is the concentration of the signaling motif. While several groups have been working actively to determine the specific factors, concentrations, and mechanisms governing the differentiation process, many have been turning to combinatorial and gradient approaches in attempts to optimize the multiple chemical and physical parameters needed for the next advance. However, there has not been a direct comparison between the cellular behavior and differentiation of human mesenchymal stem cells cultured in gradient and discrete substrates, which quantitates the effect of differences caused by cell-produced, soluble factors due to design differences between the culture systems. In this study, the differentiation of human mesenchymal stem cells in continuous and discrete polyethylene glycol dimethacrylate (PEGDM) hydrogels containing an RGD concentration gradient from 0 to 14 mM were examined to study the effects of the different culture conditions on stem-cell behavior. Culture condition was found to affect every osteogenic (alkaline phosphatase, Runx 2, type 1 collagen, bone sailoprotein, and calcium content) and adipogenic marker (oil red and peroxisome proliferator-activated receptor gamma) examined regardless of RGD concentration. Only in the continuous gradient culture did RGD concentration affect human mesenchymal stem-cell lineage commitment with low RGD concentrations expressing higher osteogenic differentiation than high RGD concentrations. Conversely, high RGD concentrations expressed higher adipogenic differentiation than low RGD concentrations. Cytoskeletal actin organization was only affected by culture condition at low RGD concentrations, indicating that it played a limited role in the differences in lineage commitment observed. Therefore, the role of discrete versus gradient strategies in high-throughput experimentation needs to be considered when designing experiments as we show that the respective strategies alter cellular outcomes even though base scaffolds have similar material and chemical properties.

Neurite extension and neuronal differentiation of human induced pluripotent stem cell derived neural stem cells on polyethylene glycol hydrogels containing a continuous Young's Modulus gradient

Mechanotransduction in neural cells involves multiple signaling pathways that are not fully understood. Differences in lineage and maturation state are suggested causes for conflicting reports on neural cell mechanosensitivity. To optimize matrices for use in stem cell therapy treatments transplanting human induced pluripotent stem cell derived neural stem cells (hNSC) into lesions after spinal cord injury, the effects of Youngs Modulus changes on hNSC behavior must be understood. The present study utilizes polyethylene glycol hydrogels containing a continuous gradient in Youngs modulus to examine changes in the Youngs Modulus of the culture substrate on hNSC neurite extension and neural differentiation. Changes in the Youngs Modulus of the polyethylene glycol hydrogels was found to affect neurite extension and cellular organization on the matrices. hNSC cultured on 907 Pa hydrogels were found to extend longer neurites than hNSC cultured on other tested Youngs Moduli hydrogels. The gene expression of β tubulin III and microtubule-associated protein 2 in hNSC was affected by changes in the Youngs Modulus of the hydrogel. The combinatory method approach used in the present study demonstrates that hNSC are mechanosensitive and the matrix Youngs Modulus should be a design consideration for hNSC transplant applications.

Concentration dependent neural differentiation and neurite extension of mouse ESC on primary amine-derivatized surfaces

Cell sourcing continues to be a significant limitation to regenerative medicine especially in neural lineages where population heterogeneity during in vitro culture prevents definitive phenotype assessment. For nearly 40 years, the biological community has worked with amine-derivated surfaces and hydrogels, especially alginate, with little quantitative assessment of how local amine concentration influences the extent of neural differentiation and neurite extension. In this manuscript we show that the local concentration of amines distinctly influences mouse embryonic stem cell (ESC) lineage commitment and the length of neurite extensions both of which are early indicators of differentiation. The well-defined amine gradients are a highly relevant tool for identifying these critical concentrations and thresholds. We feel these results will be of critical importance to researchers developing new ex vivo culture materials for neural applications as well as the community exploring nerve regeneration in vivo.