High-throughput screening of a small molecule library for promoters and inhibitors of mesenchymal stem cell osteogenic differentiation.

The use of high-throughput screening (HTS) techniques has long been employed by the pharmaceutical industry to increase discovery rates for new drugs that could be useful for disease treatment, yet this technology has only been minimally applied in other applications such as in tissue regeneration. In this work, an assay for the osteogenic differentiation of human mesenchymal stem cells (hMSCs) was developed and used to screen a library of small molecules for their potential as either promoters or inhibitors of osteogenesis, based on levels of alkaline phosphatase activity and cellular viability. From a library of 1,040 molecules, 36 promoters, and 20 inhibitors were identified as hits based on statistical criteria. Osteopromoters from this library were further investigated using standard culture techniques and a wider range of outcomes to verify that these compounds drive cellular differentiation. Several hits led to some improvement in the expression of alkaline phosphatase, osteogenic gene expression, and matrix mineralization by hMSCs when compared to the standard dexamethasone supplemented media and one molecule was investigated in combination with a recently identified biodegradable and osteoconductive polymer. This work illustrates the ability of HTS to more rapidly identify potential molecules to control stem cell differentiation.

Identification of osteoconductive and biodegradable polymers from a combinatorial polymer library.

Combinatorial polymer syntheses are now being utilized to create libraries of materials with potential utility for a wide variety of biomedical applications. We recently developed a library of photopolymerizable and biodegradable poly(beta-amino ester)s (PBAEs) that possess a range of tunable properties. In this study, the PBAE library was assessed for candidate materials that met design criteria (e.g., physical properties such as degradation and mechanical strength and in vitro cell viability and osteoconductive behavior) for scaffolding in mineralized tissue repair. The most promising candidate, A6, was then processed into three-dimensional porous scaffolds and implanted subcutaneously and only presented a mild inflammatory response. The scaffolds were then implanted intramuscularly and into a critical-sized cranial defect either alone or loaded with bone morphogenetic protein-2 (BMP-2). The samples in both locations displayed mineralized tissue formation in the presence of BMP-2, as evident through radiographs, micro-computed tomography, and histology, whereas samples without BMP-2 showed minimal or no mineralized tissue. These results illustrate a process to identify a candidate scaffolding material from a combinatorial polymer library, and specifically for the identification of an osteoconductive scaffold with osteoinductive properties via the inclusion of a growth factor.

High-throughput and combinatorial technologies for tissue engineering applications.

As the field of tissue engineering progresses, new technology is essential to accelerate the identification of potentially translatable approaches for the repair of tissues damaged due to disease or trauma. The development of high-throughput and combinatorial technologies is helping to speed up research that is applicable to all aspects of the tissue engineering paradigm. This diverse technology can be used for both the rapid synthesis of polymers and their characterization with respect to local and bulk properties in a high-throughput fashion. The interactions of cells with many diverse materials in both two- and three-dimensions can be assessed rapidly through the use of microarrays and rapid outcome measures and with microfluidic devices for investigation of soluble factor and material combinations. Finally, small molecule screening of large libraries is being used to identify molecules that exhibit control over cell behavior, including stem cell differentiation. All of these approaches are aimed to move beyond traditional iterative methods to identify unique materials and molecules that would not have been identified otherwise. Although much of this work is only applicable for in vitro studies, future methods may translate into rapid screening of these approaches in vivo.

Electrospinning of photocrosslinked and degradable fibrous scaffolds.

Electrospun fibrous scaffolds are being developed for the engineering of numerous tissues. Advantages of electrospun scaffolds include the similarity in fiber diameter to elements of the native extracellular matrix and the ability to align fibers within the scaffold to control and direct cellular interactions and matrix deposition. To further expand the range of properties available in fibrous scaffolds, we developed a process to electrospin photocrosslinkable macromers from a library of multifunctional poly(beta-amino ester)s. In this study, we utilized one macromer (A6) from this library for initial examination of fibrous scaffold formation. A carrier polymer [poly(ethylene oxide) (PEO)] was used for fiber formation because of limitations in electrospinning A6 alone. Various ratios of A6 and PEO were successfully electrospun and influenced the scaffold fiber diameter and appearance. When electrospun with a photoinitiator and exposed to light, the macromers crosslinked rapidly to high double bond conversions and fibrous scaffolds displayed higher elastic moduli compared to uncrosslinked scaffolds. When these fibers were deposited onto a rotating mandrel and crosslinked, organized fibrous scaffolds were obtained, which possessed higher moduli (approximately 4-fold) in the fiber direction than perpendicular to the fiber direction, as well as higher moduli (approximately 12-fold) than that of nonaligned crosslinked scaffolds. With exposure to water, a significant mass loss and a decrease in mechanical properties were observed, correlating to a rapid initial loss of PEO which reached an equilibrium after 7 days. Overall, these results present a process that allows for formation of fibrous scaffolds from a wide variety of possible photocrosslinkable macromers, increasing the diversity and range of properties achievable in fibrous scaffolds for tissue regeneration.

Controlling poly(beta-amino ester) network properties through macromer branching.

Photopolymerizable and degradable biomaterials are becoming important in the development of advanced materials in the fields of tissue engineering, drug delivery, and microdevices. We have recently developed a library of poly(beta-amino ester)s (PBAEs) that form networks with a wide range of mechanical properties and degradation rates that are controlled by simple alterations in the macromer molecular weight or chemical structure. In this study, the influence of macromer branching on network properties was assessed by adding the trifunctional monomer pentaerythritol triacrylate (PETA) during synthesis. This led to a dose-dependent increase in the network compressive modulus, tensile modulus, and glass transition temperature, and a decrease in the network soluble fraction, yet led to only minor variations in degradation profiles and reaction behavior. For instance, the tensile modulus increased from 1.98+/-0.09MPa to 3.88+/-0.20MPa when the macromer went from a linear structure to a more branched structure with the addition of PETA. When osteoblast-like cells were grown on thin films, there was an increase in cell adhesion and spreading as the amount of PETA incorporated during synthesis increased. Towards tissue engineering applications, porous scaffolds were fabricated by photopolymerizing around a poragen and then subsequently leaching the poragen. Interconnected pores were observed in the scaffolds and observed trends translated to the porous scaffold (i.e., increasing mechanics with increasing branching). These findings demonstrate a simple variation during macromer synthesis that can be used to further tune the physical properties of scaffolds for given applications, particularly for candidates from the PBAE library.

Influence of macromer molecular weight and chemistry on poly(beta-amino ester) network properties and initial cell interactions.

A library of photocrosslinkable poly(beta-amino ester)s (PBAEs) was recently synthesized to expand the number of degradable polymers that can be screened and developed for a variety of biological applications. In this work, the influence of variations in macromer chemistry and macromer molecular weight (MMW) on network reaction behavior, overall bulk properties, and cell interactions were investigated. The MMW was controlled through alterations in the initial diacrylate to amine ratio (> or =1) during synthesis and decreased with an increase in this ratio. Lower MMWs reacted more quickly and to higher double bond conversions than higher MMWs, potentially due to the higher concentration of reactive groups. Additionally, the lower MMWs led to networks with higher compressive and tensile moduli that degraded slower than networks formed from higher MMWs because of an increase in the crosslinking density and decrease in the number of degradable units per crosslink. The adhesion and spreading of osteoblast-like cells on polymer films was found to be dependent on both the macromer chemistry and the MMW. In general, the number of cells was similar on networks formed from a range of MMWs, but the spreading was dramatically influenced by MMW (higher spreading with lower MMWs). These results illustrate further diversity in photocrosslinkable PBAE properties and that the chemistry and macromer structure must be carefully selected for the desired application.

Resveratrol and estradiol rapidly activate MAPK signaling through estrogen receptors alpha and beta in endothelial cells.

Vascular endothelial cells (EC) are an important target of estrogen action through both the classical genomic (i.e. nuclear-initiated) activities of estrogen receptors alpha and beta (ERalpha and ERbeta) and the rapid "non-genomic" (i.e. membrane-initiated) activation of ER that stimulates intracellular phosphorylation pathways. We tested the hypothesis that the red wine polyphenol trans-resveratrol activates MAPK signaling via rapid ER activation in bovine aortic EC, human umbilical vein EC, and human microvascular EC. We report that bovine aortic EC, human umbilical vein EC, and human microvascular EC express ERalpha and ERbeta. We demonstrate that resveratrol and estradiol (E(2)) rapidly activated MAPK in a MEK-1, Src, matrix metalloproteinase, and epidermal growth factor receptor-dependent manner. Importantly, resveratrol activated MAPK and endothelial nitric-oxide synthase (eNOS) at nm concentrations (i.e. an order of magnitude less than that required for ER genomic activity) and concentrations possibly achieved transiently in serum following oral red wine consumption. Co-treatment with ER antagonists ICI 182,780 or 4-hydroxytamoxifen blocked resveratrol- or E(2)-induced MAPK and eNOS activation, indicating ER dependence. We demonstrate for the first time that ERalpha-and ERbeta-selective agonists propylpyrazole triol and diarylpropionitrile, respectively, stimulate MAPK and eNOS activity. A red but not a white wine extract also activated MAPK, and activity was directly correlated with the resveratrol concentration. These data suggest that ER may play a role in the rapid effects of resveratrol in EC and that some of the atheroprotective effects of resveratrol may be mediated through rapid activation of ER signaling in EC.