Wednesday, November 7, 2007

In the US alone, roughly 1.4 million patients undergo operations requiring arterial prostheses annually (1). Tissue engineering (TE) represents a potential means to construct functional small diameter vascular grafts in situations where autologous tissue is unavailable and conventional synthetic materials fail. While initial results with many of the TE vascular grafts (TEVGs) constructed to date are very encouraging, potential for thrombosis, hyperplasia, and mechanical failure have limited their success (1). In the present study, we characterize the effects of novel inorganic-organic hydrogel scaffolds generated by the photo-cure of hydrophobic star polydimethylsiloxane (PDMSstar) (inorganic polymer) and hydrophilic linear poly(ethylene oxide) (PEO) on smooth muscle (SM) progenitor cell differentiation and extracellular matrix (ECM) production. Initial studies have shown these hybrid scaffolds to be biocompatible, nonthrombogenic, and highly elastic. In addition, they display unique microstructural, biochemical, and biomechanical properties that can be precisely tuned over a broad range, enabling systematic exploration of the effects of scaffold material properties on cell behavior towards TEVG optimization. Materials and Methods: Synthesis of PDMSstar-MA, Diacrylate-Terminated PEO (PEO-DA), and Monoacrylate Derivatized RGDS. Briefly, PDMSstar-SiH (I) was prepared by combining octamethylcyclotetrasiloxane and tetrakis(dimethylsiloxane)silane (2) in an N2 purged flask. Triflic acid was then added and the reaction stirred for 1 h at 90C. The silane terminal groups of I were converted into photo-sensitive methacrylate (MA) moieties by the subsequent hydrosilylation reaction of I and allyl MA (3). Photo-sensitive acrylate groups were introduced to the terminal ends of linear 6000 Da PEO with known methods (1). Cell adhesion peptide RGDS was reacted with excess ACRL-PEG-NHS at pH 8.5 (1). The products were characterized by H NMR and/or GPC. TEVG Construct Preparation and Maintenance. Hydrogels were prepared by the photopolymerization of aqueous mixtures of PDMSstar-MA and PEO-DA. PDMS:PEO were dispersed in PBS at a total concentration of 10 mg/mL at the following weight ratios: (1) 0:100, (2) 1:99, (3) 5:95, (4) 10:90. 10 mL of DMAP photoinitiator solution and 1 mM ACRL-PEG-RGDS was added per mL of aqueous mixture. The mixtures were sterile-filtered and 10T1⁄2 SM progenitor cells were suspended in each gel formulation at 2x10 cells/mL. The mixtures were pipetted between two glass plates separated by 1.1 mm spacers and polymerized by exposure to 10 mW/cm 365 nm UV light for 2 min. After 1 wk and 3 wk of culture, constructs were collected for biochemical, histological, and biomechanical analyses (1). Biochemical Analyses. Construct cellularity (Picogreen assay), collagen (hydroxyproline assay), elastin (ninhydrin assay), and GAG (Blyscan assay) production were analyzed following overnight digestion of the constructs with 0.1 N NaOH (1). Histological, Microstructural, Biochemical, and Biomechanical assays. Immunohistochemical staining for ECM proteins collagen type I and elastin, and cell differentiation indicators SM-a-actin, calponin h1,and SM-g-actin were conducted. To determine hydrogel modulus, rectangular samples were subjected to a uniaxial strain in tension configuration until failure. Statistical Analyses. Data sets were compared using ANOVA followed by pairwise Tukey post-hoc tests. P values less than 0.05 were considered significant. Results/Discussion: By varying the ratio of PDMSstar to PEO, the modulus of the hydrogels remained constant. However, gradual alterations in scaffold hydrophobicity and microscale morphology were introduced. These alterations in morphology impacted cellular differentiation (Figure 1) and ECM production (Figure 2). Specifically, progenitor cells demonstrated increasing levels of differentiation as indicated by relative expression of calponin h1 and SM-g-actin as PDMSstar levels increased from 0 to 5%. Similarly, the levels of collagen and elastin production varied significantly with hydrogel PDMSstar concentration. Conclusions: By modifying the composition of novel inorganic-organic PDMSstar:PEO hydrogels, we are able to modulate the ECM production, and differentiation of encapsulated SM progenitor cells. Future investigation of the impact of systematic alterations in the microstructural, biochemical, and biomechanical properties of these hybrid scaffolds on SM cell behavior should therefore yield profound insight into the dependence of cell behavior on material properties.