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1.  Poly(Propylene Fumarate) Reinforced Dicalcium Phosphate Dihydrate Cement Composites for Bone Tissue Engineering 
Calcium phosphate cements have many desirable properties for bone tissue engineering, including osteoconductivity, resorbability, and amenability to rapid prototyping based methods for scaffold fabrication. In this study, we show that dicalcium phosphate dihydrate (DCPD) cements, which are highly resorbable but also inherently weak and brittle, can be reinforced with poly(propylene fumarate) (PPF) to produce strong composites with mechanical properties suitable for bone tissue engineering. Characterization of DCPD-PPF composites revealed significant improvements in mechanical properties for cements with a 1.0 powder to liquid ratio. Compared to non-reinforced controls, flexural strength improved from 1.80 ± 0.19 MPa to 16.14 ± 1.70 MPa, flexural modulus increased from 1073.01 ± 158.40 MPa to 1303.91 ± 110.41 MPa, maximum displacement during testing increased from 0.11 ± 0.04 mm to 0.51 ± 0.09 mm, and work of fracture improved from 2.74 ± 0.78 J/m2 to 249.21 ± 81.64 J/m2. To demonstrate the utility of our approach for scaffold fabrication, 3D macroporous scaffolds were prepared with rapid prototyping technology. Compressive testing revealed that PPF reinforcement increased scaffold strength from 0.31 ± 0.06 MPa to 7.48 ± 0.77 MPa. Finally, 3D PPF-DCPD scaffolds were implanted into calvarial defects in rabbits for 6 weeks. Although the addition of mesenchymal stem cells to the scaffolds did not significantly improve the extent of regeneration, numerous bone nodules with active osteoblasts were observed within the scaffold pores, especially in the peripheral regions. Overall, the results of this study suggest that PPF-DCPD composites may be promising scaffold materials for bone tissue engineering.
PMCID: PMC3360828  PMID: 22489012
2.  Two-Dimensional Nanostructure- Reinforced Biodegradable Polymeric Nanocomposites for Bone Tissue Engineering 
Biomacromolecules  2013;14(3):900-909.
This study investigates the efficacy of two dimensional (2D) carbon and inorganic nanostructures as reinforcing agents of crosslinked composites of the biodegradable and biocompatible polymer polypropylene fumarate (PPF) as a function of nanostructure concentration. PPF composites were reinforced using various 2D nanostructures: single- and multi-walled graphene oxide nanoribbons (SWGONRs, MWGONRs), graphene oxide nanoplatelets (GONPs), and molybdenum di-sulfite nanoplatelets (MSNPs) at 0.01–0.2 weight% concentrations. Cross-linked PPF was used as the baseline control, and PPF composites reinforced with single- or multi-walled carbon nanotubes (SWCNT, MWCNT) were used as positive controls. Compression and flexural testing show a significant enhancement (i.e., compressive modulus = 35–108%, compressive yield strength = 26–93%, flexural modulus = 15–53%, and flexural yield strength = 101–262% greater than the baseline control) in the mechanical properties of the 2D-reinforced PPF nanocomposites. MSNPs nanocomposites consistently showed the highest values among the experimental or control groups in all the mechanical measurements. In general, the inorganic nanoparticle MSNPs showed a better or equivalent mechanical reinforcement compared to carbon nanomaterials, and 2-D nanostructures (GONP, MSNP) are better reinforcing agents compared to 1-D nanostructures (e.g. SWCNTs). The results also indicate that the extent of mechanical reinforcement is closely dependent on the nanostructure morphology and follows the trend nanoplatelets > nanoribbons > nanotubes. Transmission electron microscopy of the cross-linked nanocomposites indicates good dispersion of nanomaterials in the polymer matrix without the use of a surfactant. The sol-fraction analysis showed significant changes in the polymer cross-linking in the presence of MSNP (0.01–0.2 wt %) and higher loading concentrations of GONP and MWGONR (0.1–0.2 wt%). The analysis of surface area and aspect ratio of the nanostructures taken together with the above results indicates differences in nanostructure architecture (2D vs. 1D nanostructures), as well as the chemical compositions (inorganic vs. carbon nanostructures), number of functional groups, and structural defects for the 2D nanostructures maybe key properties that affect the mechanical properties of 2D nanostructure-reinforced PPF nanocomposites, and the reason for the enhanced mechanical properties compared to the controls.
PMCID: PMC3601907  PMID: 23405887
3.  In vivo bone biocompatibility and degradation of porous fumarate-based polymer/alumoxane nanocomposites for bone tissue engineering 
The objective of this study was to determine how the incorporation of surface-modified alumoxane nanoparticles into a biodegradable fumarate-based polymer affects in vivo bone biocompatibility (characterized by direct bone contact and bone ingrowth) and in vivo degradability. Porous scaffolds were fabricated from four materials: poly(propylene fumarate)/propylene fumarate-diacrylate (PPF/PF-DA) polymer alone; a macrocomposite consisting of PPF/PF-DA polymer with boehmite microparticles; a nanocomposite composed of PPF/PF-DA polymer and mechanically-reinforcing surface-modified alumoxane nanoparticles; and a low molecular weight PPF polymer alone (tested as a degradation control). Scaffolds were implanted in the lateral femoral condyle of adult goats for 12 weeks and evaluated by micro-computed tomography and histological analysis. For all material groups, small amounts of bone, some soft tissue, and a few inflammatory elements were observed within the pores of scaffolds, though many pores remained empty or filled with fluid only. Direct contact between scaffolds and surrounding bone tissue was also observed in all scaffold types, though less commonly. Minimal in vivo degradation occurred during the 12 weeks of implantation in all materials. These results demonstrate that the incorporation of alumoxane nanoparticles into porous PPF/PF-DA scaffolds does not significantly alter in vivo bone biocompatibility or degradation.
PMCID: PMC2797574  PMID: 19191316
Bone tissue engineering; Nanocomposite; Biocompatibility; Nanobiomaterials; Micro-computed tomography
4.  Effect of Autologous Bone Marrow Stromal Cell Seeding and Bone Morphogenetic Protein-2 Delivery on Ectopic Bone Formation in a Microsphere/Poly(Propylene Fumarate) Composite 
Tissue Engineering. Part A  2008;15(3):587-594.
A biodegradable microsphere/scaffold composite based on the synthetic polymer poly(propylene fumarate) (PPF) holds promise as a scaffold for cell growth and sustained delivery vehicle for growth factors for bone regeneration. The objective of the current work was to investigate the in vitro release and in vivo bone forming capacity of this microsphere/scaffold composite containing bone morphogenetic protein-2 (BMP-2) in combination with autologous bone marrow stromal cells (BMSCs) in a goat ectopic implantation model. Three composites consisting of 0, 0.08, or 8 μg BMP-2 per mg of poly(lactic-co-glycolic acid) microspheres, embedded in a porous PPF scaffold, were combined with either plasma (no cells) or culture-expanded BMSCs. PPF scaffolds impregnated with a BMP-2 solution and combined with BMSCs as well as empty PPF scaffolds were also tested. The eight different composites were implanted subcutaneously in the dorsal thoracolumbar area of goats. Incorporation of BMP-2–loaded microspheres in the PPF scaffold resulted in a more sustained in vitro release with a lower burst phase, as compared to BMP-2–impregnated scaffolds. Histological analysis after 9 weeks of implantation showed bone formation in the pores of 11/16 composites containing 8 μg/mg BMP-2–loaded microspheres with no significant difference between composites with or without BMSCs (6/8 and 5/8, respectively). Bone formation was also observed in 1/8 of the BMP-2–impregnated scaffolds. No bone formation was observed in the other conditions. Overall, this study shows the feasibility of bone induction by BMP-2 release from microspheres/scaffold composites.
PMCID: PMC2810278  PMID: 18925831
5.  Segmental Bone Regeneration Using a Load Bearing Biodegradable Carrier of Bone Morphogenetic Protein-2 
Biomaterials  2006;28(3):459-467.
Segmental defect regeneration has been a clinical challenge. Current tissue engineering approach using porous biodegradable scaffolds to delivery osteogenic cells and growth factors demonstrated success in facilitating bone regeneration in these cases. However, due to the lack of mechanical property, the porous scaffolds were evaluated in non-load bearing area or were stabilized with stress-shielding devices (bone plate or external fixation). In this paper, we tested a scaffold that does not require a bone plate because it has sufficient biomechanical strength. The tube-shaped scaffolds were manufactured from poly(propylene) fumarate/tricalcium phosphate (PPF/TCP) composites. Dicalcium phosphate dehydrate (DCPD) were used as bone morphogenetic protein -2 (BMP-2) carrier. Twenty two scaffolds were implanted in 5 mm segmental defects in rat femurs stabilized with k-wire for 6 and 15 weeks with and without 10 μg of rhBMP-2. Bridging of the segmental defect was evaluated first radiographically and was confirmed by histology and micro- computer tomography (μ-CT) imaging. The scaffolds in the BMP group maintained the bone length throughout the duration of the study and allow for bridging. The scaffolds in the control group failed to induce bridging and collapsed at 15 weeks. Peripheral computed tomography (pQCT) showed that BMP-2 does not increase the bone mineral density in the callus. Finally, the scaffold in BMP group was found to restore the mechanical property of the rat femur after 15 weeks. Our results demonstrated that the load-bearing BMP-2 scaffold can maintain bone length and allow successfully regeneration in segmental defects.
PMCID: PMC1986795  PMID: 16996588
6.  Fabrication of Porous Ultra-Short Single-Walled Carbon Nanotube Nanocomposite Scaffolds for Bone Tissue Engineering 
Biomaterials  2007;28(28):4078-4090.
We investigated the fabrication of highly porous scaffolds made of three different materials [poly(propylene fumarate (PPF) polymer, an ultra-short single-walled carbon nanotube (US-tube) nanocomposite, and a dodecylated US-tube (F-US-tube) nanocomposite] in order to evaluate the effects of material composition and porosity on scaffold pore structure, mechanical properties, and marrow stromal cell culture. All scaffolds were produced by a thermal-crosslinking particulate-leaching technique at specific porogen contents of 75, 80, 85, and 90 vol%. Scanning electron microcopy, microcomputed tomography, and mercury intrusion porosimetry were used to analyze the pore structures of scaffolds. The porogen content was found to dictate the porosity of scaffolds. There was no significant difference in porosity, pore size, and interconnectivity among the different materials for the same porogen fraction. Nearly 100% of the pore volume was interconnected through 20 μm or larger connections for all scaffolds. While interconnectivity through larger connections improved with higher porosity, compressive mechanical properties of scaffolds declined at the same time. However, the compressive modulus, offset yield strength, and compressive strength of F-US-tube nanocomposites were higher than or similar to the corresponding properties for the PPF polymer and US-tube nanocomposites for all the porosities examined. As for in vitro osteoconductivity, marrow stromal cells demonstrated equally good cell attachment and proliferation on all scaffolds made of different materials at each porosity. These results indicate that functionalized ultra-short single-walled carbon nanotube nanocomposite scaffolds with tunable porosity and mechanical properties hold great promise for bone tissue engineering applications.
PMCID: PMC3163100  PMID: 17576009
7.  Bone Tissue-Engineering Material Poly(propylene fumarate): Correlation between Molecular Weight, Chain Dimensions, and Physical Properties 
Biomacromolecules  2006;7(6):1976-1982.
Poly(propylene fumarate) (PPF) is an important biodegradable and crosslinkable polymer designed for bone tissue-engineering applications. For the first time we report the extensive characterization of this biomaterial including molecular weight dependences of physical properties such as glass transition temperature Tg, thermal degradation temperature Td, density ρ melt viscosity η0, hydrodynamic radius RH, and intrinsic viscosity [η]. The temperature dependence of η0 changes progressively with molecular weight, while it can be unified when the temperature is normalized to Tg. The plateau modulus GN0 and entanglement molecular weight Me have been obtained from the rheological master curves. A variety of chain microstructure parameters such as the Mark-Houwink-Sakurada constants K and α, characteristic ratio C∞, unperturbed chain dimension r02/M, packing length p, Kuhn length b, and tube diameter a have been deduced. Further correlation between the microstructure and macroscopic physical properties has been discussed in light of recent progress in polymer dynamics to supply a better understanding about this unsaturated polyester to advance its biomedical uses. The molecular weight dependence of Tg for six polymer species including PPF has been summarized to support that Me is irrelevant for the finite length effect on glass transition, while surprisingly these polymers can be divided into two groups when their normalized Tg is plotted simply against Mw to indicate the deciding roles of inherent chain properties such as chain fragility, intermolecular cooperativity, and chain end mobility.
PMCID: PMC2530912  PMID: 16768422
8.  Mechanical and Cell Viability Properties of Crosslinked Low and High Molecular Weight Poly(ethylene glycol) Diacrylate Blends 
There is a strong need for tissue engineering scaffolds that are mechanically robust, exhibit good biocompatibility, and can be made from readily available materials. To this end, blends of commercially available poly(ethylene glycol) diacrylate (PEGDA) with molecular weights of 400 and 3400 were UV-crosslinked at total polymer concentrations that varied systematically from 20 to 40 wt %. The compressive strength and cell viability were determined for each PEGDA mixture. The compressive modulus of the blends was maximized when the wt % ratio PEGDA3400/400 was about 40/60, with the compressive strength reaching 1.7 MPa. Cell viability results with a LIVE/DEAD fluorescence assay show an average viability of ~ 80% at a total PEGDA concentration of 20 wt %, for all blends. Increasing the total polymer concentration increased the compressive modulus of a polymer, but adversely affected cell viability for all the PEGDA blend compositions. The blend composition affected the mechanical behavior of the discs, where a higher degree of crosslinking was achieved by increasing the concentration of shorter chained PEGDA400, whereas elasticity was gained by incorporating longer chained PEGDA3400 into the blends. These results can be exploited for use in tissue engineering applications, where a mechanically robust scaffold is advantageous.
PMCID: PMC2845736  PMID: 19585581
Cell encapsulation; Hydrogel; Mechanical properties; Photopolymerization; Tissue Engineering
9.  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 
Acta biomaterialia  2010;7(3):1249-1264.
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 alteration of paracrine cell-cell signaling distances may affect the intercellular signaling mechanism and facilitate the enhanced osteogenic signal expressions 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 result of surface characterization indicated that HA addition improved surface properties of PPF/HA composite scaffolds by showing increased roughness, hydrophilicity, protein adsorption, and initial cell attachment. Rat bone marrow stromal cells (BMSCs), which were CD34(−), CD45(−), CD29(+), and CD90(+), were cultured on 3D macroporous PPF/HA scaffolds with two different initial cell seeding densities (0.33 and 1.00 million cells per scaffold) for 8 days. 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. Upregulated expression of osteogenic growth factor genes was related to subsequent osteoblastic differentiation of rat BMSCs on 3D scaffolds, as characterized by alkaline phosphatase activity, osteocalcin mRNA expression, and calcium deposition. Thus PPF/HA composite scaffold construction parameters, including incorporated HA amount and initial cell seeding density, may be utilized to induce the osteoblastic differentiation of transplanted rat BMSCs.
PMCID: PMC3031657  PMID: 21074640
osteogenic signaling; hydroxyapatite; BMP-2; RT-PCR; bone marrow stromal cells; poly(propylene fumarate)
10.  Evaluation of Osteoconductive Scaffolds in the Canine Femoral Multi-Defect Model 
Tissue Engineering. Part A  2013;19(5-6):634-648.
Treatment of large segmental bone defects remains an unsolved clinical challenge, despite a wide array of existing bone graft materials. This project was designed to rapidly assess and compare promising biodegradable osteoconductive scaffolds for use in the systematic development of new bone regeneration methodologies that combine scaffolds, sources of osteogenic cells, and bioactive scaffold modifications. Promising biomaterials and scaffold fabrication methods were identified in laboratories at Rutgers, MIT, Integra Life Sciences, and Mayo Clinic. Scaffolds were fabricated from various materials, including poly(L-lactide-co-glycolide) (PLGA), poly(L-lactide-co-ɛ-caprolactone) (PLCL), tyrosine-derived polycarbonate (TyrPC), and poly(propylene fumarate) (PPF). Highly porous three-dimensional (3D) scaffolds were fabricated by 3D printing, laser stereolithography, or solvent casting followed by porogen leaching. The canine femoral multi-defect model was used to systematically compare scaffold performance and enable selection of the most promising substrate(s) on which to add cell sourcing options and bioactive surface modifications. Mineralized cancellous allograft (MCA) was used to provide a comparative reference to the current clinical standard for osteoconductive scaffolds. Percent bone volume within the defect was assessed 4 weeks after implantation using both MicroCT and limited histomorphometry. Bone formed at the periphery of all scaffolds with varying levels of radial ingrowth. MCA produced a rapid and advanced stage of bone formation and remodeling throughout the defect in 4 weeks, greatly exceeding the performance of all polymer scaffolds. Two scaffold constructs, TyrPCPL/TCP and PPF4SLA/HAPLGA Dip, proved to be significantly better than alternative PLGA and PLCL scaffolds, justifying further development. MCA remains the current standard for osteoconductive scaffolds.
PMCID: PMC3568967  PMID: 23215980
11.  Towards excimer-laser-based stereolithography: a rapid process to fabricate rigid biodegradable photopolymer scaffolds 
We demonstrate high-resolution photocross-linking of biodegradable poly(propylene fumarate) (PPF) and diethyl fumarate (DEF) using UV excimer laser photocuring at 308 nm. The curing depth can be tuned in a micrometre range by adjusting the total energy dose (total fluence). Young's moduli of the scaffolds are found to be a few gigapascal, high enough to support bone formation. The results presented here demonstrate that the proposed technique is an excellent tool for the fabrication of stiff and biocompatible structures on a micrometre scale with defined patterns of high resolution in all three spatial dimensions. Using UV laser photocuring at 308 nm will significantly improve the speed of rapid prototyping of biocompatible and biodegradable polymer scaffolds and enables its production in a few seconds, providing high lateral and horizontal resolution. This short timescale is indeed a tremendous asset that will enable a more efficient translation of technology to clinical applications. Preliminary cell tests proved that PPF : DEF scaffolds produced by excimer laser photocuring are biocompatible and, therefore, are promising candidates to be applied in tissue engineering and regenerative medicine.
PMCID: PMC3479907  PMID: 22696484
laser photocuring; scaffolds; tissue engineering; biocompatibility; poly(propylene fumarate)
12.  Biodegradable Composite Scaffolds Incorporating an Intramedullary Rod and Delivering Bone Morphogenetic Protein-2 for Stabilization and Bone Regeneration in Segmental Long Bone Defects 
Acta biomaterialia  2011;7(10):3627-3637.
In this study, a two part bone tissue engineering scaffold was investigated consisting of a solid poly(propylene fumarate) (PPF) intramedullary rod for mechanical support surrounded by a porous PPF sleeve for osseointegration and delivery of poly(DL-lactic-co-glycolic acid) (PLGA) microspheres with adsorbed recombinant human bone morphogenetic protein-2 (rhBMP-2). Scaffolds were implanted into critical size rat segmental femoral defects with internal fixation for 12 weeks. Bone formation was assessed throughout the study via radiography, and following euthanasia via micro-CT and histology. Mechanical stabilization was evaluated further via torsional testing. Experimental implant groups included the PPF rod alone and the rod with a porous PPF sleeve containing PLGA microspheres with 0, 2, or 8 μg of rhBMP-2 adsorbed onto their surface. Results showed that presence of the scaffold increased mechanical stabilization of the defect, as evidenced by the increased torsional stiffness of the femurs by the presence of a rod compared to the empty defect. Although the presence of a rod decreased bone formation, the presence of a sleeve combined with a low or high dose of rhBMP-2 increased the torsional stiffness to 2.06 ± 0.63 N·mm and 1.68 ± 0.56 N·mm, respectively, from 0.56 ± 0.24 N·mm for the rod alone. The results indicate that, while scaffolds may provide structural support to regenerating tissues and increase their mechanical properties, the presence of scaffolds within defects may hinder overall bone formation if they interfere with cellular processes.
PMCID: PMC3163689  PMID: 21757034
Bone Tissue Engineering; Bone Morphogenetic Protein; Bone Regeneration; Intramedullary Rod; Composite Scaffold
13.  Biodegradable Fibrous Scaffolds with Tunable Properties Formed from Photocrosslinkable Poly(glycerol sebacate) 
ACS applied materials & interfaces  2009;1(9):1878-1892.
It is becoming increasingly apparent that the architecture and mechanical properties of scaffolds, particularly with respect to mimicking features of natural tissues, are important for tissue engineering applications. Acrylated poly(glycerol sebacate) (Acr-PGS) is a material that can be crosslinked upon exposure to ultraviolet light, leading to networks with tunable mechanical and degradation properties through simple changes during Acr-PGS synthesis. For example, the number of acrylate functional groups on the macromer dictates the concentration of crosslinks formed in the resulting network. Three macromers were synthesized that form networks that vary dramatically with respect to their tensile modulus (~30 kPa to 6.6 MPa) and degradation behavior (~20 to 100% mass loss at 12 weeks) based on the extent of acrylation (~1 to 24%). These macromers were processed into biodegradable fibrous scaffolds using electrospinning, with gelatin as a carrier polymer to facilitate fiber formation and cell adhesion. The resulting scaffolds were also diverse with respect to their mechanics (tensile modulus ranging from ~60 kPa to 1 MPa) and degradation (~45 to 70% mass loss by 12 weeks). Mesenchymal stem cell adhesion and proliferation on all fibrous scaffolds was indistinguishable from controls. The scaffolds showed similar diversity when implanted on the surface of hearts in a rat model of acute myocardial infarction and demonstrated a dependence on scaffold thickness and chemistry in the host response. In summary, these diverse scaffolds with tailorable chemical, structural, mechanical and degradation properties are potentially useful for the engineering of a wide range of soft tissues.
PMCID: PMC2765054  PMID: 20160937
electrospinning; elastomer; tissue engineering; scaffold; stem cells
14.  Synthesis and Characterization of Degradable Bioconjugated Hydrogels with Hyperbranched Multifunctional Crosslinkers 
Acta biomaterialia  2010;6(11):4189-4198.
Hyperbranched poly(ester amide) polymer (Hybrane™ S1200; Mn 1200 g/mol) was functionalized with maleic anhydride (MA) and propylene sulfide, to obtain multifunctional crosslinkers with fumaric and thiol-end groups, S1200MA and S1200SH, respectively. The degree of substitution of maleic acid groups (DS) was controlled by varying the molar ratio of MA to S1200 in the reaction mixture. Hydrogels were obtained by UV crosslinking of functionalized S1200 and poly(ethyleneglycol) diacrylate (PEGDA) in aqueous solutions. Compressive modulus increased with decreasing the S1200/PEG ratio and also depended on the DS of the multifunctional crosslinker (S1200). Also, heparin-based macromonomers together with functionalized hyperbranched polymers were used to construct novel functional hydrogels. The multivalent hyperbranched polymers allowed high crosslinking densities in heparin modified gels while introducing biodegradation sites. Both heparin presence and acrylate/thiol ratio have an impact on degradation profiles and morphologies. Hyperbranched crosslinked hydrogels showed no evidence of cell toxicity. Overall, the multifunctional crosslinkers afford hydrogels with promising properties that suggest that these may be suitable for tissue engineering applications.
PMCID: PMC3023059  PMID: 20561601
bioconjugated hydrogels; hyperbranched polymers; multifunctional crosslinkers; biodegradable; heparin
15.  A poly(propylene glycol-co-fumaric acid) based bone graft extender for lumbar spinal fusion: in vivo assessment in a rabbit model 
European Spine Journal  2005;15(6):936-943.
Study design: An animal model of posterolateral intertransverse process lumbar spinal fusion compared fusion rates amongst autologous bone (group 1), a porous, bioabsorbable, scaffold based on the biopolymer, poly(propylene glycol-co-fumaric acid) (PPF) (group 2), and a combination of autograft and the bioabsorbable scaffold (group 3). Objectives: To evaluate the feasibility of augmenting spinal fusion with an osteoconductive and bioabsorbable scaffold as an alternative or as an adjunct, i.e., an extender, to autograft. Summary of background data: There is little preclinical data on applications of bioabsorable bone graft extenders in spinal fusion. Methods: New Zealand White rabbits underwent single-level lumbar posterolateral intertransverse process fusion. Animals were treated with one of three materials: autologous bone (group 1), a bioabsorable material based on PPF (group 2), and the PPF biopolymer scaffold with autologous bone graft (group 3). Animals were evaluated at 6 weeks, and fusion was evaluated by manual palpation, and radiographic, histologic, and histomorphometric analyses. Results: Radiographic and manual palpation showed evidence of fusion in all three groups. Histomorphometric measurement of bone ingrowth showed the highest quantity of new bone in group 3 (91%), followed by group 1 (72%) and group 2 (53%). Conclusions: Results of this study suggested that osteoconductive bioabsorbable scaffolds prepared from PPF might be used as an autograft extender when applied as an adjunct to spinal fusion.
PMCID: PMC3489455  PMID: 16133085
Spinal fusion; Bone graft extender; Bioabsorbable scaffold
16.  Synthesis of Poly(Propylene Fumarate) 
Nature protocols  2009;4(4):518-525.
This protocol describes the synthesis of 500 – 4000 Da poly(propylene fumarate) by a two-step reaction of diethyl fumarate and propylene glycol through a bis(hydroxypropyl) fumarate diester intermediate. Purified PPF can be covalently crosslinked to form degradable polymer networks, which have been widely explored for biomedical applications. The properties of crosslinked PPF networks depend upon the molecular properties of the constituent polymer, such as the molecular weight. The purity of the reactants and the exclusion of water from the reaction system are of utmost importance in the generation of high-molecular-weight PPF products. Additionally, the reaction time and temperature influence the molecular weight of the PPF product. The expected time required to complete this protocol is 3 d.
PMCID: PMC3076598  PMID: 19325548
biodegradable; biomaterial; injectable material; tissue engineering scaffold
17.  Development of Biodegradable and Injectable Macromers Based on Poly(Ethylene Glycol) and Diacid Monomers 
Novel biodegradable injectable poly(ethylene glycol) (PEG) based macromers were synthesized by reacting low molecular weight PEG (MW: 200) and dicarboxylic acids such as sebacic acid or terephthalic acid. Chemical structures of the resulting polymers were confirmed by Fourier transform infrared (FTIR) and nuclear magnetic resonance (NMR) spectroscopy characterizations. Differential scanning calorimetry (DSC) showed that these polymers were completely amorphous above room temperature. After photopolymerization, dynamic elastic shear modulus of the crosslinked polymers was up to 1.5 MPa and compressive modulus was up to 2.2 MPa depending on the polymer composition. The in vitro degradation study showed that mass losses of these polymers were gradually decreased over 23 weeks of period in simulated body fluid. By incorporating up to 30 wt% of 2-hydroxyethyl methylmethacrylate (HEMA) into the crosslinking network, the dynamic elastic modulus and compressive modulus was significantly increased up to 7.2 MPa and 3.2 MPa, respectively. HEMA incorporation also accelerated degradation as indicated by significantly higher mass loss of up to 27% after 20 weeks of incubation. Cytocompatability studies using osteoblasts and neural cells revealed that cell metabolic activity on these polymers with or without HEMA was close to the control tissue culture polystyrene. The PEG based macromers developed in this study may be useful as scaffolds or cell carriers for tissue engineering applications.
PMCID: PMC2857720  PMID: 18655146
Polyethylene glycol; dicarboxylic acid; HEMA; tissue engineering; biodegradation
18.  Synthesis, Material Properties and Biocompatibility of a Novel Self-Crosslinkable Poly(caprolactone fumarate) as an Injectable Tissue Engineering Scaffold 
Biomacromolecules  2005;6(5):2503-2511.
A novel self-crosslinkable and biodegradable macromer poly(caprolactone fumarate) (PCLF) has been developed for guided bone regeneration. This macromer is a copolymer of fumaryl chloride, which contains double bonds for in-situ crosslinking, and poly(ε-caprolactone) that has a flexible chain to facilitate self-crosslinkability. PCLF was characterized with Fourier transform infrared (FTIR) spectroscopy, 1H and 13C nuclear magnetic resonance (NMR) spectroscopy, and gel permeation chromatography (GPC). Porous scaffolds were fabricated with sodium chloride particles as the porogen and a chemical initiation system. The PCLF scaffolds were characterized with scanning electron microscopy (SEM) and micro-computed tomography (micro-CT). The cytotoxicity and in vivo biocompatibility of PCLF were also assessed. Our results suggest that this novel copolymer, PCLF, is an injectable, self-crosslinkable, and biocompatible macromer that may be potentially used as a scaffold for tissue engineering applications.
PMCID: PMC2530909  PMID: 16153086
19.  Physical Properties and Cellular Responses to Crosslinkable Poly(Propylene Fumarate)/Hydroxyapatite Nanocomposites 
Biomaterials  2008;29(19):2839-2848.
A series of crosslinkable nanocomposites has been developed using hydroxyapatite (HA) nanoparticles and poly(propylene fumarate) (PPF). PPF/HA nanocomposites with four different weight fractions of HA nanoparticles have been characterized in terms of thermal and mechanical properties. To assess surface chemistry of crosslinked PPF/HA nanocomposites, their hydrophilicity and capability of adsorbing proteins have been determined using static contact angle measurement and MicroBCA protein assay kit after incubation with 10% fetal bovine serum (FBS), respectively. In vitro cell studies have been performed using MC3T3-E1 mouse pre-osteoblast cells to investigate the ability of PPF/HA nanocomposites to support cell attachment, spreading, and proliferation after 1, 4, and 7 days. By adding HA nanoparticles to PPF, the mechanical properties of crosslinked PPF/HA nanocomposites have not been increased due to the initially high modulus of crosslinked PPF. However, hydrophilicity and serum protein adsorption on the surface of nanocomposites have been significantly increased, resulting in enhanced cell attachment, spreading, and proliferation after 4 days of cell seeding. These results indicate that crosslinkable PPF/HA nanocomposites are useful for hard tissue replacement because of excellent mechanical strength and osteoconductivity.
PMCID: PMC2430424  PMID: 18403013
Poly(propylene fumarate) (PPF); Hydroxyapatite (HA); Nanocomposite; Protein adsorption; Osteoblast response
20.  Retention of in vitro and in vivo BMP-2 bioactivity in sustained delivery vehicles for bone tissue engineering 
Biomaterials  2008;29(22):3245-3252.
In this study, we investigated the in vitro and in vivo biologic activity of bone morphogenetic protein 2 (BMP-2) released from four sustained delivery vehicles for bone regeneration. BMP-2 was incorporated in 1) a gelatin hydrogel, 2) poly(lactic-co-glycolic acid) (PLGA) microspheres embedded in a gelatin hydrogel, 3) microspheres embedded in a poly(propylene fumarate) (PPF) scaffold and 4) microspheres embedded in a PPF scaffold surrounded by a gelatin hydrogel. A fraction of the incorporated BMP-2 was radiolabeled with 125I to determine its in vitro and in vivo release profiles. The release and bioactivity of BMP-2 were tested weekly over a period of 12 weeks in preosteoblast W20-17 cell line culture and in a rat subcutaneous implantation model. Outcome parameters for in vitro and in vivo bioactivity of the released BMP-2 were alkaline phosphatase (AP) induction and bone formation, respectively. The four implant types showed different in vitro release profiles over the 12-week period, which changed significantly upon implantation. The AP induction by BMP-2 released from gelatin implants showed a loss in bioactivity after 6 weeks in culture, while the BMP-2 released from the other implants continued to show bioactivity over the full 12-week period. Micro-CT and histological analysis of the delivery vehicles after 6 weeks of implantation showed significantly more bone in the microsphere/PPF scaffold composites (implant 3, p < 0.02). After 12 weeks, the amount of newly formed bone in the microsphere/PPF scaffolds remained significantly higher than in the gelatin and microsphere/gelatin hydrogels (p < 0.001), however there was no statistical difference compared to the microsphere/PPF/gelatin composite. Overall, the results from this study show that BMP-2 could be incorporated into various bone tissue engineering composites for sustained release over a prolonged period of time with retention of bioactivity.
PMCID: PMC2577841  PMID: 18472153
21.  Enhanced mechanical performance and biological evaluation of a PLGA coated β-TCP composite scaffold for load-bearing applications 
European polymer journal  2011;47(8):1569-1577.
Porous β-tricalcium phosphate (β-TCP) has been used for bone repair and replacement in clinics due to its excellent biocompatibility, osteoconductivity, and biodegradability. However, the application of β-TCP has been limited by its brittleness. Here, we demonstrated that an interconnected porous β-TCP scaffold infiltrated with a thin layer of poly (lactic-co-glycolic acid) (PLGA) polymer showed improved mechanical performance compared to an uncoated β-TCP scaffold while retaining its excellent interconnectivity and biocompatibility. The infiltration of PLGA significantly increased the compressive strength of β-TCP scaffolds from 2.90 MPa to 4.19 MPa, bending strength from 1.46 MPa to 2.41 MPa, and toughness from 0.17 MPa to 1.44 MPa, while retaining an interconnected porous structure with a porosity of 80.65%. These remarkable improvements in the mechanical properties of PLGA-coated β-TCP scaffolds are due to the combination of the systematic coating of struts, interpenetrating structural characteristics, and crack bridging. The in vitro biological evaluation demonstrated that rat bone marrow stromal cells (rBMSCs) adhered well, proliferated, and expressed alkaline phosphatase (ALP) activity on both the PLGA-coated β-TCP and the β-TCP. These results suggest a new strategy for fabricating interconnected macroporous scaffolds with significantly enhanced mechanical strength for potential load-bearing bone tissue regeneration.
PMCID: PMC3164545  PMID: 21892228
Poly (lactic-co-glycolic acid); β-tricalcium phosphate; Functional composites; Scaffold; Bone tissue engineering
22.  Ingrowth of Human Mesenchymal Stem Cells into Porous Silk Particle Reinforced Silk Composite Scaffolds: An In Vitro Study 
Acta biomaterialia  2010;7(1):144-151.
Silk fibroin protein is biodegradable and biocompatible, exhibiting excellent mechanical properties for various biomedical applications. However, porous 3D silk fibroin scaffolds, or silk sponges, usually fall short in matching the initial mechanical requirements for bone tissue engineering. In the present study, silk sponge matrices were reinforced with silk microparticles to generate protein-protein composite scaffolds with desirable mechanical properties for in vitro osteogenic tissue formation. It was found that increasing the silk microparticle loading led to a substantial increase in the scaffold compressive modulus from 0.3 MPa (nonreinforced) to 1.9 MPa for 1:2 (matrix:particle) reinforcement loading by dry mass. Biochemical, gene expression, and histological assays were employed to study the possible effects of increasing composite scaffold stiffness, due to microparticle reinforcement, on in vitro osteogenic differentiation of human mesenchymal stem cells (hMSCs). Increasing silk microparticle loading increased the osteogenic capability of hMSCs in the presence of bone morphogenic protein-2 (BMP-2) and other osteogenic factors in static culture for up to six weeks. The calcium adsorption increased dramatically with increasing loading, as observed from biochemical assays, histological staining, and microCT (μCT) analysis. Specifically, calcium content in the scaffolds increased by 0.57, 0.71, and 1.27 mg (per μg of DNA) from 3 to 6 weeks for matrix to particle dry mass loading ratios of 1:0, 1:1 and 1:2, respectively. In addition, μCT imaging revealed that at 6 weeks, bone volume fraction increased from 0.78% for nonreinforced to 7.1% and 6.7% for 1:1 and 1:2 loading, respectively. Our results support the hypothesis that scaffold stiffness may strongly influence the 3D in vitro differentiation capabilities of hMSCs, providing a means to improve osteogenic outcomes.
PMCID: PMC2967589  PMID: 20656075
osteogenesis; human mesenchymal stem cells (hMSCs); silk; composite; matrix stiffness
23.  The influence of stereolithographic scaffold architecture and composition on osteogenic signal expression with rat bone marrow stromal cells 
Biomaterials  2011;32(15):3750-3763.
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.
PMCID: PMC3075725  PMID: 21396709
Osteogenic signal expression; Stereolithography; Stiffness; Pore geometry; Bone marrow stromal cells; Poly(propylene fumarate)
24.  Enhanced Cell Ingrowth and Proliferation through Three-Dimensional Nanocomposite Scaffolds with Controlled Pore Structures 
Biomacromolecules  2010;11(3):682-689.
We present enhanced cell ingrowth and proliferation through crosslinked three-dimensional (3D) nanocomposite scaffolds fabricated using poly(propylene fumarate) (PPF) and hydroxyapatite (HA) nanoparticles. Scaffolds with controlled internal pore structures were produced from computer-aided design (CAD) models and solid freeform fabrication (SFF) technique, while those with random pore structures were fabricated by NaCl leaching technique for comparison. The morphology and mechanical properties of scaffolds were characterized using scanning electron microscopy (SEM) and mechanical testing, respectively. Pore interconnectivity of scaffolds was assessed using X-ray micro-computed tomography (micro-CT) and 3D imaging analysis. In vitro cell studies have been performed using MC3T3-E1 mouse preosteoblasts and cultured scaffolds in a rotating-wall-vessel bioreactor for 4 and 7 days to assess cell attachment, viability, ingrowth depth, and proliferation. The mechanical properties of crosslinked nanocomposite scaffolds were not significantly different after adding HA or varying pore structures. However, pore interconnectivity of PPF/HA nanocomposite scaffolds with controlled pore structures has been significantly increased, resulting in enhanced cell ingrowth depth 7 days after cell seeding. Cell attachment and proliferation are also higher in PPF/HA nanocomposite scaffolds. These results suggest that crosslinked PPF/HA nanocomposite scaffolds with controlled pore structures may lead to promising bone tissue engineering scaffolds with excellent cell proliferation and ingrowth.
PMCID: PMC2839506  PMID: 20112899
Poly(propylene fumarate) (PPF); Hydroxyapatite (HA); Nanocomposite; Solid freeform fabrication (SFF); Pre-osteoblast responses
25.  Photo-crosslinked Hybrid Polymer Networks Consisting of Poly(propylene fumarate) (PPF) and Poly(caprolactone fumarate) (PCLF): Controlled Physical Properties and Regulated Bone and Nerve Cell Responses 
Biomacromolecules  2008;9(4):1229-1241.
Aiming to achieve suitable polymeric biomaterials with controlled physical properties for hard and soft tissue replacements, we have developed a series of blends consisting of two photo-crosslinkable polymers: polypropylene fumarate (PPF) and polycaprolactone fumarate (PCLF). Physical properties of both uncrosslinked and UV crosslinked PPF/PCLF blends with PPF composition ranging from 0% to 100% have been investigated extensively. It has been found that the physical properties such as thermal, rheological, and mechanical properties could be modulated efficiently by varying the PPF composition in the blends. Thermal properties including glass transition temperature (Tg) and melting temperature (Tm) have been correlated with their rheological and mechanical properties. Surface characteristics such as surface morphology, hydrophilicity and the capability of adsorbing serum protein from culture medium have also been examined for the crosslinked polymer and blend discs. For potential applications in bone and nerve tissue engineering, in vitro cell studies including cytotoxicity, cell adhesion, and proliferation on crosslinked discs with controlled physical properties have been performed using rat bone marrow stromal cells and SPL201 cells, respectively. In addition, the role of mechanical properties such as surface stiffness in modulating cell responses has been emphasized using this model blend system.
PMCID: PMC2888142  PMID: 18307311
Photo-crosslinking; Polymer blends; Poly(propylene fumarate) (PPF); Poly(caprolactone fumarate) (PCLF); Controlled physical properties; Cell responses

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