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.
In this study, we have investigated the efficacy of inorganic nanotubes as reinforcing agents to improve the mechanical properties of poly(propylene fumarate) (PPF) composites as a function of nanomaterial loading concentration (0.01-0.2 wt%). Tungsten disulfide nanotubes (WSNTs) were used as reinforcing agents in the experimental groups. Single- and multi- walled carbon nanotubes (SWCNTs and MWCNTs) were used as positive controls, and crosslinked PPF composites were used as baseline control. Mechanical testing (compression and three-point bending) shows a significant enhancement (up to 28-190%) in the mechanical properties (compressive modulus, compressive yield strength, flexural modulus, and flexural yield strength) of WSNT reinforced PPF nanocomposites compared to the baseline control. In comparison to positive controls, at various concentrations, significant improvements in the mechanical properties of WSNT nanocomposites were also observed. In general, the inorganic nanotubes (WSNTs) showed a better (up to 127%) or equivalent mechanical reinforcement compared to carbon nanotubes (SWCNTs and MWCNTs). Sol fraction analysis showed significant increases in the crosslinking density of PPF in the presence of WSNTs (0.01-0.2 wt%). Transmission electron microscopy (TEM) analysis on thin sections of crosslinked nanocomposites showed the presence of WSNTs as individual nanotubes in the PPF matrix, whereas SWCNTs and MWCNTs existed as micron sized aggregates. The trend in the surface area of nanostructures obtained by BET surface area analysis was SWCNTs > MWCNTs > WSNTs. The BET surface area analysis, TEM analysis, and sol fraction analysis results taken together suggest that chemical composition (inorganic vs. carbon nanomaterials), presence of functional groups (such as sulfide and oxysulfide), and individual dispersion of the nanomaterials in the polymer matrix (absence of aggregation of the reinforcing agent) are the key parameters affecting the mechanical properties of nanostructure-reinforced PPF composites, and the reason for the observed increases in the mechanical properties compared to the baseline and positive controls.
polymer nanocomposites; carbon nanotubes; tungsten nanotubes; mechanical properties; bone tissue engineering
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.
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.
Composite chitosan-nano-hydroxyapatite microspheres and scaffolds prepared using a co-precipitation method have shown potential for use in bone regeneration. The goal of this research was to improve the functional properties of the composite scaffolds by modifying the fabrication parameters. The effects of degree of deacetylation (DDA), drying method, hydroxyapatite content and an acid wash on scaffold properties were investigated. Freeze-dried 61% DDA scaffolds degraded faster (3.5 ± 0.5% mass loss) than air-dried 61% DDA scaffolds and 80% DDA scaffolds, but had a lower compressive modulus of 0.12 ± 0.01 MPa. Air-dried 80% DDA scaffolds displayed the highest compressive modulus (3.79 ± 0.51 MPa) and these scaffolds were chosen as the best candidate for use in bone regeneration. Increasing the amount of hydroxyapatite in the air-dried 80% DDA scaffolds did not further increase the compressive modulus of the scaffolds. An acid wash procedure at pH 6.1 was found to increase the degradation of air-dried 80% DDA scaffolds from 1.3 ± 0.1% to 4.4 ± 0.4%. All of the formulations tested supported the proliferation of SAOS-2 cells.
bone regeneration; chitosan; hydroxyapatite; tissue engineering
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.
osteogenic signaling; hydroxyapatite; BMP-2; RT-PCR; bone marrow stromal cells; poly(propylene fumarate)
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.
Poly(propylene fumarate) (PPF); Hydroxyapatite (HA); Nanocomposite; Protein adsorption; Osteoblast response
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.
In this work, a series of copolymers of polypropylene fumarate-co-polycaprolactone (PPF-co-PCL) were synthesized via a three-step polycondensation reaction of oligomeric polypropylene fumarate (PPF) with polycaprolactone (PCL). The effects of PPF precursor molecular weight, PCL precursor molecular weight, and PCL fraction in the copolymer (PCL feed ratio) on the maximum crosslinking temperature, gelation time, and mechanical properties of the crosslinked copolymers were investigated. The maximum crosslinking temperature fell between 38.2±0.3 and 47.2±0.4 °C, which increased with increasing PCL precursor molecular weight. The gelation time was between 4.2±0.2 and 8.5±0.7 min, and decreased with increasing PCL precursor molecular weight. The compressive moduli ranged from 44±1.8 to 142±7.4 MPa, with enhanced moduli at higher PPF precursor molecular weight and lower PCL feed ratio. The compressive toughness was in the range of 4.1±0.3 and 17.1±1.3 KJ/m3. Our data suggest that the crosslinking and mechanical properties of PPF-co-PCL can be modulated by varying the composition. Therefore the PPF-co-PCL copolymers may offer increased versatility as an injectable, in situ polymerizable biomaterial than the individual polymers of PPF and PCL.
Polypropylene fumarate; polycaprolactone; injectable biomaterials; in situ polymerizable
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), pre-osteoblasts (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 crosslinking 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 non-cytotoxic 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 non-cytotoxic materials (HDPE).
cytotoxicity; poly(propylene fumarate); hMSC; MC3T3
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.
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.
Bone tissue engineering; Nanocomposite; Biocompatibility; Nanobiomaterials; Micro-computed tomography
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.
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.
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.
Poly(propylene fumarate) (PPF); Hydroxyapatite (HA); Nanocomposite; Solid freeform fabrication (SFF); Pre-osteoblast responses
There is a need to develop synthetic scaffolds for repairing large defects in load-bearing bones. Bioactive glasses have attractive properties as a scaffold material for bone repair, but data on their mechanical properties are limited. The objective of the present study was to comprehensively evaluate the mechanical properties of strong porous scaffolds of silicate 13-93 bioactive glass fabricated by robocasting. As-fabricated scaffolds with a grid-like microstructure (porosity = 47%; filament diameter = 330 μm; pore width = 300) were tested in compressive and flexural loading to determine their strength, elastic modulus, Weibull modulus, fatigue resistance, and fracture toughness. Scaffolds were also tested in compression after they were immersed in simulated body fluid (SBF) in vitro or implanted in a rat subcutaneous model in vivo. As fabricated, the scaffolds had a strength = 86 ± 9 MPa, elastic modulus = 13 ± 2 GPa, and a Weibull modulus = 12 when tested in compression. In flexural loading, the strength, elastic modulus, and Weibull modulus were 11 ± 3 MPa, 13 ± 2 GPa, and 6, respectively. In compression, the as-fabricated scaffolds had a mean fatigue life of ~106 cycles when tested in air at room temperature or in phosphate-buffered saline at 37 °C under cyclic stresses of 1–10 MPa or 2–20 MPa. The compressive strength of the scaffolds decreased markedly during the first 2 weeks of immersion in SBF or implantation in vivo, but more slowly thereafter. The brittle mechanical response of the scaffolds in vitro changed to an elasto-plastic response after implantation for longer than 2–4 weeks in vivo. In addition to providing critically needed data for designing bioactive glass scaffolds, the results are promising for the application of these strong porous scaffolds in loaded bone repair.
Polymerization of high internal phase emulsions (polyHIPEs) is a relatively new method for the production of high porosity scaffolds. The tunable architecture of these polyHIPE foams make them attractive candidates for tissue engineered bone grafts. Previously studied polyHIPE systems require either toxic diluents or high cure temperatures which prohibit their use as an injectable bone graft. In contrast, we have developed an injectable polyHIPE that cures at physiological temperatures to a rigid, high-porosity foam. First, a biodegradable macromer, propylene fumarate dimethacrylate (PFDMA), was synthesized that has appropriate viscosity and hydrophobicity for emulsification. The process of surfactant selection is detailed with particular focus on the key structural features of both polymer (log P values, hydrogen bond acceptor sites) and surfactant (HLB values, hydrogen bond donor sites) that enable stable HIPE formation. Incubation of HIPEs at 37°C was used to initiate radical crosslinking of the unsaturated double bond of the methacrylate groups to polymerize the continuous phase and lock in the emulsion geometry. The resulting polyHIPEs exhibited ~75% porosity, pore sizes ranging from 4 to 29 μm, and an average compressive modulus and strength of 33 and 5 MPa, respectively. These findings highlight the great potential of these scaffolds as injectable, tissue engineered bone grafts.
With the aim of fabricating multifunctional fibers with enhanced mechanical properties, electrical conductivity and electrochemical performance, we develop wet-spinning of composite formulation based on functionalized PEG-SWNT and PEDOT:PSS. The method of addition and loading are directly correlated to the quality and the ease of spinnability of the formulation and to the mechanical and electrical properties of the resultant fibers. Both the fiber modulus (Y) and strength (σ) scaled linearly with PEG-SWNT volume fraction (Vf). A remarkable reinforcement rate of dY/dVf = 417 GPa and dσ/dVf = 4 GPa were obtained when PEG-SWNTs at Vf ≤ 0.02. Further increase of PEG-SWNTs loading (i.e. up to Vf 0.12) resulted in further enhancements up to 22.8 GPa and 254 MPa in Modulus and ultimate stress, respectively. We also show the enhancement of electrochemical supercapacitor performance of composite fibers. These outstanding mechanical, electrical and electrochemical performances place these fibers among the best performing multifunctional composite fibers.
The fibrils in the bone matrix are glued together by ECM proteins to form laminated structures (osteons) to provide elasticity and a supportive substrate for osteogenesis. The objective of this work was to investigate material properties and osteogenic differentiation of bone marrow stromal (BMS) cells seeded on osteon-mimetic fiber-reinforced hydrogel/apatite composites. Layers of electrospun poly(L-lactide) (L-PLA) fiber mesh coated with a poly(lactide-co-ethylene oxide fumarate) (PLEOF) hydrogel precursor solution were stacked and pressed together, and crosslinked to produce a laminated fiber-reinforced composite. Hydroxyapatite (HA) nanocrystals were added to the precursor solution to produce an osteoconductive matrix for BMS cells. Acrylamide-terminated RGD peptide (Ac-GRGD) was conjugated to the PLEOF/HA hydrogel phase to promote focal point adhesion of BMS cells. Laminates were characterized with respect to Young’s modulus, degradation kinetics, and osteogenic differentiation of BMS cells. The moduli of the laminates under dry and wet conditions were significantly higher than those of the fiber mesh and PLEOF/HA hydrogel, and within the range of values reported for wet human cancellous bone. At days 14 and 21, ALPase activity of the laminates was significantly higher than those of the fiber mesh and hydrogel. Lamination significantly increased the extent of mineralization of BMS cells and laminates with HA and conjugated with RGD (Lam-RGD-HA) had 2.7-, 3.5-, and 2.8-fold higher calcium content (compared to laminates without HA or RGD) after 7, 14, and 21 days, respectively. The Lam-RGD-HA group had significantly higher expression of osteopontin (OP) and osteocalcin (OC) compared to the hydrogel or laminates without HA or RGD, consistent with the higher ALPase activity and calcium content of Lam-RGD-HA. Laminated osteon-mimetic structures have the potential to provide mechanical strength to the regenerating region as well as supporting the differentiation of progenitor cells to the osteogenic lineage.
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.
osteogenesis; human mesenchymal stem cells (hMSCs); silk; composite; matrix stiffness
Reconstruction of large bone defects remains problematic in orthopedic and craniofacial clinical practice. Autografts are limited in supply and are associated with donor site morbidity while other materials show poor integration with the host’s own bone. This lack of integration is often due to the absence of periosteum, the outer layer of bone that contains osteoprogenitor cells and is critical for the growth and remodeling of bone tissue. In this study we developed a one-step platform to electrospin nanofibrous scaffolds from chitosan, which also contain hydroxyapatite nanoparticles and are crosslinked with genipin. We hypothesized that the resulting composite scaffolds represent a microenvironment that emulates the physical, mineralized structure and mechanical properties of non-weight bearing bone extracellular matrix while promoting osteoblast differentiation and maturation similar to the periosteum. The ultrastructure and physicochemical properties of the scaffolds were studied using scanning electron microscopy and spectroscopic techniques. The average fiber diameters of the electrospun scaffolds were 227±154 nm as spun, and increased to 335±119 nm after crosslinking with genipin. Analysis by X-ray diffraction, Fourier transformed infrared spectroscopy and energy dispersive spectroscopy confirmed the presence of characteristic features of hydroxyapatite in the composite chitosan fibers. The Young’s modulus of the composite fibrous scaffolds was 142±13 MPa, which is similar to that of the natural periosteum. Both pure chitosan scaffolds and composite hydroxyapatite-containing chitosan scaffolds supported adhesion, proliferation and osteogenic differentiation of mouse 7F2 osteoblast-like cells. Expression and enzymatic activity of alkaline phosphatase, an early osteogenic marker, were higher in cells cultured on the composite scaffolds as compared to pure chitosan scaffolds, reaching a significant, 2.4 fold, difference by day 14 (p<0.05). Similarly, cells cultured on hydroxyapatite-containing scaffolds had the highest rate of osteonectin mRNA expression over 2 weeks, indicating enhanced osteoinductivity of the composite scaffolds. Our results suggest that crosslinking electrospun hydroxyapatite-containing chitosan with genipin yields bio-composite scaffolds, which combine non-weight-bearing bone mechanical properties with a periosteum-like environment and facilitate the proliferation, differentiation and maturation of osteoblast-like cells. We propose that these scaffolds might be useful for the repair and regeneration of maxillofacial defects and injuries.
Chitosan; hydroxyapatite; genipin; electrospinning; bone tissue engineering; osteoblast differentiation
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.
Bone Tissue Engineering; Bone Morphogenetic Protein; Bone Regeneration; Intramedullary Rod; Composite Scaffold
Bioglass has been used for bone-filling material in bone tissue engineering, but its lean mechanical strength limits its applications in load-bearing positions. Carbon nanotubes (CNTs), with their high aspect ratio and excellent mechanical properties, have the potential to strengthen and toughen bioactive glass material without offsetting its bioactivity. Therefore, in this research, multiwall carbon nanotube (MWCNT)/45S5 Bioglass composite scaffolds have been successfully prepared by means of freeze casting process. 45S5 Bioglass was synthesized by the sol-gel processing method. The obtained material was characterized with X-ray powder diffraction (XRD). The mechanical properties of the scaffolds, such as compression strength and elastic modulus, were measured. Finally, compared with the scaffolds prepared by 100% 45S5 Bioglass powders, the addition of 0.25 wt.% MWCNTs increases the compressive strength and elastic modulus of 45S5 Bioglass scaffolds from 2.08 to 4.56 MPa (a 119% increase) and 111.50 to 266.59 MPa (a 139% increase), respectively.
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.
Photo-crosslinking; Polymer blends; Poly(propylene fumarate) (PPF); Poly(caprolactone fumarate) (PCLF); Controlled physical properties; Cell responses
Many musculoskeletal tissues exhibit significant anisotropic mechanical properties reflective of a highly oriented underlying extracellular matrix. For tissue engineering, recreating this organization of the native tissue remains a challenge. To address this issue, this study explored the fabrication of biodegradable nanofibrous scaffolds composed of aligned fibers via electrospinning onto a rotating target, and characterized their mechanical anisotropy as a function of the production parameters. The characterization showed that nanofiber organization was dependent on the rotation speed of the target; randomly oriented fibers (33% fiber alignment) were produced on a stationary shaft, whereas highly oriented fibers (94% fiber alignment) were produced when rotation speed was increased to 9.3 m/sec. Non-aligned scaffolds had an isotropic tensile modulus of 2.1 ± 0.4 MPa, compared to highly anisotropic scaffolds whose modulus was 11.6 ± 3.1 MPa in the presumed fiber direction, suggesting that fiber alignment has a profound effect on the mechanical properties of scaffolds. Mechanical anisotropy was most pronounced at higher rotation speeds, with a greater than 33-fold enhancement of the Young’s modulus in the fiber direction compared to perpendicular to the fiber direction at a rotation speed reached 8 m/sec. In cell culture, both the organization of actin filaments of human mesenchymal stem cells and the cellular alignment of meniscal fibroblasts were dictated by the prevailing nanofiber orientation. This study demonstrates that controllable and anisotropic mechanical properties of nanofibrous scaffolds can be achieved by dictating nanofiber organization through intelligent scaffold design.
Tissue Engineering; Tensile Properties; Anisotropy; Mechanical Testing; Biodegradable Scaffolds