David Dean

Photocrosslinking characteristics and mechanical properties of diethyl fumarate/poly(propylene fumarate) biomaterials

The development of tissue engineered materials for the treatment of large bone defects would provide attractive alternatives to the autografts, allografts, non-degradable polymers, ceramics, and metals that are currently used in clinical settings. To this end, poly(propylene fumarate) (PPF), a viscous polyester synthesized from diethyl fumarate (DEF), has been studied for use as an engineered bone graft. We have investigated the photocrosslinking of PPF dissolved in its precursor, DEF, using the photoinitiator bis(2,4,6-trimethylbenzoyl) phenylphosphine oxide (BAPO) and low levels of ultraviolet light exposure. A three factor, 2 x 2 x 4 factorial design was developed, studying the effects of PPF number average molecular weight, BAPO initiator content, and DEF content upon photocrosslinking characteristics and mechanical properties. Uncured DEF/PPF solution viscosity fell over three orders of magnitude as DEF content was increased from 0% to 75%. The exothermic photocrosslinking reaction released low levels of heat, with no more than 160J/g released from any formulation tested. As a result, the maximum photocrosslinking temperature remained below 47 degrees C for all samples. Both sol fraction and swelling degree generally increased with increasing DEF content. Compressive mechanical properties were within the range of trabecular bone, with the strongest samples possessing an elastic modulus of 195.3 +/- 17.5 MPa and a fracture strength of 68.8 +/- 9.4MPa. Finally, the results indicate that PPF crosslinking was facilitated at low DEF precursor concentrations, but hindered at higher precursor concentrations. These novel DEF/PPF solutions may be preferred over pure PPF as the basis for an engineered bone graft because they (1) exhibit reduced viscosity and thus are easily handled, (2) form polymer networks with compressive strength at fracture suitable for consideration for trabecular bone replacement, and (3) may be readily fabricated into solids with a wide range of structures.

Synthesis and properties of photocross-linked poly(propylene fumarate) scaffolds

The photocross-linking of poly(propylene fumarate) (PPF) to form porous scaffolds for bone tissue engineering applications was investigated. PPF was cross-linked using the photoinitiator bis(2,4,6-trimethylbenzoyl) phenylphosphine oxide (BAPO) and exposure to 30 min of long wavelength ultraviolet (UV) light. The porous photocross-linked PPF scaffolds (6.5 mm diameter cylinders) were synthesized by including a NaCl porogen (70, 80, and 90 wt% at cross-linking) prior to photocross-linking. After UV exposure, the samples were placed in water to remove the soluble porogen, revealing the porous PPF scaffold. As porogen leaching has not been used often with cross-linked polymers, and even more rarely with photoinitiated cross-linking, a study of the efficacy of this strategy and the properties of the resulting material was required. Results show that the inclusion of a porogen does not significantly alter the photoinitiation process and the resulting scaffolds are homogeneously cross-linked throughout their diameter. It was also shown that porosity can be generally controlled by porogen content and that scaffolds synthesized with at least 80 wt% porogen possess an interconnected pore structure. Compressive mechanical testing showed scaffold strength to decrease with increasing porogen content. The strongest scaffolds with interconnected pores had an elastic modulus of 2.3 ± 0.5 MPa and compressive strength at 1% yield of 0.11 ± 0.02 MPa. This work has shown that a photocross-linking/porogen leaching technique is a viable method to form porous scaffolds from photoinitiated materials.

Effect of Initial Cell Seeding Density on Early Osteogenic Signal Expression of Rat Bone Marrow Stromal Cells Cultured on Cross-Linked Poly(propylene fumarate) Disks

The intercellular signaling mechanisms among a transplanted cell population are largely determined by the cell population itself as well as the surrounding environment. Changes in cell-to-cell paracrine signaling distance can be obtained by altering cell density, and signal expression of growth factors can be enhanced by auto/paracrine signal transduction. To examine these relationships, we investigated the effect of cell seeding density on viability, proliferation, differentiation, and the endogenous osteogenic signal expression among rat bone marrow stromal cells (BMSCs) cultured on a 2D disk. Rat BMSCs were isolated from rats and then cultured for 8 days on biodegradable poly(propylene fumarate) disks with three different seeding densities (0.06, 0.15, and 0.30 million cells/disk). At days 1, 4, and 8, viability by live/dead fluorescent staining, DNA amount, osteogenic differentiation by alkaline phosphatase and osteocalcin mRNA expression, calcium deposition, and osteogenic growth factor mRNA expression were assayed. Osteogenic signal expression was evaluated using quantitative reverse transcriptase-polymerase chain reaction, and signals of interest include bone morphogenetic protein-2, transforming growth factor-β(1), fibroblast growth factor-2, and platelet-derived growth factor-A. The results from this study demonstrate that rat BMSCs were viable over 8 days without being affected by cell density and that both cell proliferation rate and early osteogenic differentiation were stimulated by lower cell seeding density. Most importantly, this study has demonstrated for the first time that the temporal gene expression profiles of endogenous growth factors can be controlled by altering the initial cell seeding density on poly(propylene fumarate) disks. Therefore, our results suggest that changes in the paracrine signal distance by altering cell seeding density may be a useful strategy to optimize the cell-biomaterial construct microenvironments to enhance the osteogenic signal expression.

Computer aided design of large-format prefabricated cranial plates.

The authors objective in this project was to replace current state-of-the-art manual methods for preoperative production (i.e., prefabrication) of large-format (>100 cm2) cranioplasties with a system for computer-aided design and direct computer-aided manufacture of the implants shape. This system uses standard 3D CT data, requires no specialized training, and produces an accurately fitting cranioplasty that can be recast in the physicians material of choice (e.g., polymethylmethacrylate [PMMA] or pre-bent titanium plating). The authors begin by locating the cranial defect margin on a skull surface image generated from a 3D head CT-scan. A right-to-left mirrored or average 3D skull surface template image is then fit to the patients skull surface image. The area around the defect is cut out and stitched to the previously isolated defect margin. This defect-filling surface is then tapered and 3D printed. The 3D printed implant model is then recast in a biocompatible material. Manually generated cranial implants produced for five patients were compared with implants resulting from this new computer-based method. All five computer-generated implants were better fitting and more cosmetically suitable than the manually generated skull plates received by these patients. These well-fitting implants are more likely to protect the brain from trauma and infection. Therefore, the authors conclude that their new production method provides a better result with less expense than current methods for preoperative or intraoperative fabrication of large-format cranioplasties.

The influence of stereolithographic scaffold architecture and composition on osteogenic signal expression with rat bone marrow stromal cells

Scaffold design parameters, especially physical construction factors such as mechanical stiffness of substrate materials, pore size of 3D porous scaffolds, and channel geometry, are known to influence the osteogenic signal expression and subsequent differentiation of a transplanted cell population. In this study of photocrosslinked poly(propylene fumarate) (PPF) and diethyl fumarate (DEF) scaffolds, the effect of DEF incorporation ratio and pore size on the osteogenic signal expression of rat bone marrow stromal cells (BMSCs) was investigated. Results demonstrated that DEF concentrations and pore sizes that led to increased scaffold mechanical stiffness also upregulated osteogenic signal expression, including bone morphogenic protein-2 (BMP-2), fibroblast growth factors-2 (FGF-2), transforming growth factor-β1 (TGF-β1), vascular endothelial growth factor (VEGF), and Runx2 transcriptional factor. Similar scaffold fabrication parameters supported rapid BMSC osteoblastic differentiation, as demonstrated by increased alkaline phosphatase (ALP) and osteocalcin expression. When scaffolds with random architecture, fabricated by porogen leaching, were compared to those with controlled architecture, fabricated by stereolithography (SLA), results showed that SLA scaffolds with the highly permeable and porous channels also have significantly higher expression of FGF-2, TGF-β1, and VEGF. Subsequent ALP expression and osteopontin secretion were also significantly increased in SLA scaffolds. Based upon these results, we conclude that scaffold properties provided by additive manufacturing techniques such as SLA fabrication, particularly increased mechanical stiffness and high permeability, may stimulate dramatic BMSC responses that promote rapid bone tissue regeneration.

Early osteogenic signal expression of rat bone marrow stromal cells is influenced by both hydroxyapatite nanoparticle content and initial cell seeding density in biodegradable nanocomposite scaffolds.

Incorporation of hydroxyapatite (HA) within a degradable polymeric scaffold may provide a favorable synthetic microenvironment that more closely mimics natural bone tissue physiology. Both incorporation of HA nanoparticles and alterations of the paracrine cell-cell signaling distance may affect the intercellular signaling mechanism and facilitate enhanced osteogenic signal expression among the implanted cell population. In this study we investigate the effect of the incorporation of HA nanoparticles into poly(propylene fumarate) (PPF) scaffolds on the surface properties of composite scaffolds and early osteogenic growth factor gene expression in relation to initial cell seeding density. The results of surface characterization indicated that HA addition improved the surface properties of PPF/HA composite scaffolds by increasing the roughness, hydrophilicity, protein adsorption, and initial cell attachment. Rat bone marrow stromal cells (BMSCs), which were CD34-, CD45-, CD29+, and CD90+, were cultured on three-dimensional (3-D) macroporous PPF/HA scaffolds at two different initial cell seeding densities (0.33 and 1.00 million cells per scaffold) for 8 days. The results demonstrated that endogenous osteogenic signal expression profiles, including bone morphogenetic protein-2, fibroblast growth factor-2, and transforming growth factor-β1, as well as the transcriptional factor Runx2, were affected by both HA amount and initial cell seeding density. Up-regulated expression of osteogenic growth factor genes was related to subsequent osteoblastic differentiation of rat BMSCs on 3-D scaffolds, as characterized by alkaline phosphatase activity, osteocalcin mRNA expression, and calcium deposition. Thus, the PPF/HA composite scaffold construction parameters, including amount of HA incorporated and initial cell seeding density, may be utilized to induce the osteoblastic differentiation of transplanted rat BMSCs.

A three-dimensional fractal analysis method for quantifying white matter structure in human brain

Fractal dimension (FD) is increasingly used to quantify complexity of brain structures. Previous research that analyzed FD of human brain mainly focused on two-dimensional measurements. In this study, we developed a three-dimensional (3D) box-counting method to measure FD of human brain white matter (WM) interior structure, WM surface and WM general structure simultaneously. This method, which firstly incorporates a shape descriptor (3D skeleton) representing interior structure and combines the three features, provides a more comprehensive characterization of WM structure. WM FD of different brain segments was computed to test robustness of the method. FDs of fractal phantoms were computed to test the accuracy of the method. The consistency of the computed and theoretical FD values suggests that our method is accurate in measuring FDs of fractals. Statistical analysis was performed to examine sensitivity of the method in detecting WM structure differences in a number of young and old subjects. FD values of the WM skeleton and surface were significantly greater in young than old individuals, indicating more complex WM structures in young people. These results suggest that our method is accurate in quantifying three-dimensional brain WM structures and sensitive in detecting age-related degeneration of the structures.

Quantifying degeneration of white matter in normal aging using fractal dimension

Although degeneration of brain white matter (WM) in aging is a well-recognized problem, its quantification has mainly relied on volumetric measurements, which lack detail in describing the degenerative adaptation. In this study, WM structural complexity was evaluated in healthy old and young adults by analyzing the three-dimensional fractal dimension (FD) of WM segmented from magnetic resonance images of brain. FDs detected in the old were significantly smaller than in the young subjects. Specifically, WM interior structure complexity degenerated in the left hemisphere in old men but in the right hemisphere in old women. Men showed more complex WM patterns than women. An asymmetrical (right-greater-than-left-hemisphere) complexity pattern was observed in the interior and general structures of WM, yet the surface complexity was symmetrical across WM structures of the two hemispheres. WM volumes were also measured, but no significant decline was found with aging. These results suggest that the deterioration of WM complexity is not uniformly distributed between the genders and across brain hemispheres.

Evaluation of the in vitro cytotoxicity of cross-linked biomaterials.

This study evaluated the in vitro cytotoxicity of poly(propylene fumarate) (PPF). PPF is an aliphatic biodegradable polymer that has been well characterized for use in bone tissue engineering scaffolds. Four different cell types, human mesenchymal stem cells (hMSC), fibroblasts (L929), preosteoblasts (MC3T3), and canine mesenchymal stem cells (cMSC), were used to evaluate the cytotoxicity of PPF. These cell types represent the tissues that PPF would interact with in vivo as a bone tissue scaffold. The sol fraction of the PPF films was measured and then utilized to estimate cross-linking density. Cytotoxicity was evaluated using XTT assay and fluorescence imaging. Results showed that PPF supported similar cell metabolic activities of hMSC, L929, MC3T3, and cMSC compared to the noncytotoxic control, high-density polyethylene (HDPE) and were statistically different than those cultured with the cytotoxic control, a polyurethane film containing 0.1% zinc diethyldithiocarbamate (ZCF). Results showed differing cellular responses to ZCF, the cytotoxic control. The L929 cells had the lowest cell metabolic activity levels after exposure to ZCF compared to the cell metabolic activity levels of the MC3T3, hMSC, or cMSC cells. Qualitative verification of the results using fluorescence imaging demonstrated no change in cell morphology, vacuolization, or detachment when cultured with PPF compared to HDPE or blank media cultures. Overall, the cytotoxicity response of the cells to PPF was demonstrated to be similar to the cytotoxic response of cells to known noncytotoxic materials (HDPE).

Continuous Digital Light Processing (cDLP): Highly Accurate Additive Manufacturing of Tissue Engineered Bone Scaffolds

Highly accurate rendering of the external and internal geometry of bone tissue engineering scaffolds affects fit at the defect site, loading of internal pore spaces with cells, bioreactor-delivered nutrient and growth factor circulation, and scaffold resorption. It may be necessary to render resorbable polymer scaffolds with 50 µm or better accuracy to achieve these goals. This level of accuracy is available using Continuous Digital Light Processing (cDLP) which utilizes a DLP® (Texas Instruments, Dallas, TX) chip. One such additive manufacturing device is the envisionTEC (Ferndale, MI) Perfactory®. To use cDLP we integrate a photo-crosslinkable polymer, a photo-initiator, and a biocompatible dye. The dye attenuates light, thereby limiting the depth of polymerization. In this study we fabricated scaffolds using the well-studied resorbable polymer, poly(propylene fumarate) (PPF), titanium dioxide (TiO2) as a dye, Irgacure® 819 (BASF [Ciba], Florham Park, NJ) as an initiator, and diethyl fumarate as a solvent to control viscosity.