Biomaterials (v.29, #15)

Electro-spinning of pure collagen nano-fibres – Just an expensive way to make gelatin? by Dimitrios I. Zeugolis; Shih T. Khew; Elijah S.Y. Yew; Andrew K. Ekaputra; Yen W. Tong; Lin-Yue L. Yung; Dietmar W. Hutmacher; Colin Sheppard; Michael Raghunath (2293-2305).
Scaffolds manufactured from biological materials promise better clinical functionality, providing that characteristic features are preserved. Collagen, a prominent biopolymer, is used extensively for tissue engineering applications, because its signature biological and physico-chemical properties are retained in in vitro preparations. We show here for the first time that the very properties that have established collagen as the leading natural biomaterial are lost when it is electro-spun into nano-fibres out of fluoroalcohols such as 1,1,1,3,3,3-hexafluoro-2-propanol or 2,2,2-trifluoroethanol. We further identify the use of fluoroalcohols as the major culprit in the process. The resultant nano-scaffolds lack the unique ultra-structural axial periodicity that confirms quarter-staggered supramolecular assemblies and the capacity to generate second harmonic signals, representing the typical crystalline triple-helical structure. They were also characterised by low denaturation temperatures, similar to those obtained from gelatin preparations (p  > 0.05). Likewise, circular dichroism spectra revealed extensive denaturation of the electro-spun collagen. Using pepsin digestion in combination with quantitative SDS-PAGE, we corroborate great losses of up to 99% of triple-helical collagen. In conclusion, electro-spinning of collagen out of fluoroalcohols effectively denatures this biopolymer, and thus appears to defeat its purpose, namely to create biomimetic scaffolds emulating the collagen structure and function of the extracellular matrix.
Keywords: Collagen denaturation; Gelatin; Denaturation temperature; Second harmonic generation; Transmission electron microscopy; Circular dichroism;

The successful applications of magnesium-based alloys as degradable orthopaedic implants are mainly inhibited due to their high degradation rates in physiological environment and consequent loss in the mechanical integrity. This study examines the degradation behaviour and the mechanical integrity of calcium-containing magnesium alloys using electrochemical techniques and slow strain rate test (SSRT) method, respectively, in modified-simulated body fluid (m-SBF). Potentiodynamic polarisation and electrochemical impedance spectroscopy (EIS) results showed that calcium addition enhances the general and pitting corrosion resistances of magnesium alloys significantly. The corrosion current was significantly lower in AZ91Ca alloy than that in AZ91 alloy. Furthermore, AZ91Ca alloy exhibited a five-fold increase in the surface film resistance than AZ91 alloy. The SSRT results showed that the ultimate tensile strength and elongation to fracture of AZ91Ca alloy in m-SBF decreased only marginally (∼15% and 20%, respectively) in comparison with these properties in air. The fracture morphologies of the failed samples are discussed in the paper. The in vitro study suggests that calcium-containing magnesium alloys to be a promising candidate for their applications in degradable orthopaedic implants, and it is worthwhile to further investigate the in vivo corrosion behaviour of these alloys.
Keywords: Magnesium; Calcium; Biodegradation; Corrosion; Fracture mechanism;

Amino alcohol-based degradable poly(ester amide) elastomers by Christopher J. Bettinger; Joost P. Bruggeman; Jeffrey T. Borenstein; Robert S. Langer (2315-2325).
Currently available synthetic biodegradable elastomers are primarily composed of crosslinked aliphatic polyesters, which suffer from deficiencies including (1) high crosslink densities, which results in exceedingly high stiffness, (2) rapid degradation upon implantation, or (3) limited chemical moieties for chemical modification. Herein, we have developed poly(1,3-diamino-2-hydroxypropane-co-polyol sebacate)s, a new class of synthetic, biodegradable elastomeric poly(ester amide)s composed of crosslinked networks based on an amino alcohol. These crosslinked networks feature tensile Young's modulus on the order of 1 MPa and reversable elongations up to 92%. These polymers exhibit in vitro and in vivo biocompatibility. These polymers have projected degradation half-lives up to 20 months in vivo.
Keywords: Biodegradable; Elastomer; Tissue engineering;

Modulation of the cross-talk between macrophages and osteoblasts by titanium-based particles by Gema Vallés; Enrique Gil-Garay; Luis Munuera; Nuria Vilaboa (2326-2335).
Titanium (Ti) and its alloys have widespread uses as implant materials for orthopaedic and dental applications. To improve their surface characteristics, modifications that give rise to an outer ceramic layer of rutile have been developed. It is expected that after a long period of service, rutile particles will arise from these modified surfaces. Rutile particles have recently been proposed as reinforcement agents of substrates designed for bone tissue engineering applications. In this study, the ability of Ti and rutile particles to modulate secretion of soluble factors involved in bone turnover has been assayed in an in vitro co-culture system of macrophages and human osteoblasts that allows the exchange of soluble factors between both cell types without direct cell contact. Exposure of co-cultured macrophages to sub-cytotoxic doses of Ti or rutile particles did not modify the osteoblastic expression of surface RANKL or the secretion of OPG into the media. Both IL-6 and PGE2 levels increased to a similar extent after treatment with rutile or Ti particles. M-CSF and GM-CSF levels were lower after treatment with rutile particles than with Ti. Experiments employing neutralising antibodies indicate that exposure of co-cultured macrophages to both Ti-based particles induces the release of M-CSF, GM-CSF, IL-6 and PGE2 through up-regulation of IL-1β and TNF-α. We comparatively examined the response of co-cultured macrophages, osteoblasts or both types of cells after exposure to particles. The results indicate that interactions of osteoblasts with particles can modulate the extent of the response initiated by macrophages. Maximal levels of secretions of all tested factors were reached after exposure of co-cultured cells to Ti particles, which is suggestive of the lower bioreactivity of rutile particles.
Keywords: Macrophage; Osteoblast; Cytokine; Titanium; Titanium oxide; Osteolysis;

Microvascular maturity elicited in tissue treated with cytokine-loaded hyaluronan-based hydrogels by Luke W. Hosack; Matthew A. Firpo; J. Anna Scott; Glenn D. Prestwich; Robert A. Peattie (2336-2347).
Hydrogels composed of crosslinked, chemically modified hyaluronic acid (HA), gelatin (Gtn) and heparin (Hp) were preloaded with vascular endothelial growth factor (VEGF), angiopoietin-1 (Ang-1), keratinocyte growth factor (KGF) or platelet derived growth factor (PDGF) either individually or in combination with VEGF and implanted into the Balb/c mouse ear pinna. At 7 and 14 days post-surgery, elicited vascular maturity levels were quantified using immunohistochemical (IHC) staining techniques and reported as a vascular maturity index (VMI). At both time points, it was discovered that the dual cytokine combinations elicited greater maturity levels than that of cytokine administered individually. For example, VEGF and KGF-containing HA:Hp implants at day 7 yielded VMI values of −0.1375 and −0.092, respectively, whereas their combination resulted in a VMI of 0.176 (p  < 0.007). At day 7, only one of the seven HA:Hp experimental cases yielded a positive VMI (VEGF + KGF), whereas four of the seven HA:Hp cases yielded positive VMI values at day 14, indicating a sustained maturity response. The same general trends were found to exist in tissue treated with HA:Hp:Gtn experimental implants. Differences in elicited maturity also existed between tissue treated with HA:Hp and HA-containing hydrogels (VMI = 0.176 for HA:Hp-VEGF + KGF vs. −0.064 for HA-VEGF + KGF, p  < 0.012), and these differences are thought to result from differences in characteristic cytokine release rates. This result also suggests that the presentation of multiple growth factors (GFs) on immobilized Hp may actively contribute to cytokine related signal transduction, a characteristic that may be exploited in the future to improve the efficacy of cytokine-loaded implants towards tissue regeneration therapeutic strategies.
Keywords: Angiogenesis; Animal model; Immunohistochemistry; Cytokine; Glycosaminoglycan; Controlled drug release;

The potential to improve cell infiltration in composite fiber-aligned electrospun scaffolds by the selective removal of sacrificial fibers by Brendon M. Baker; Albert O. Gee; Robert B. Metter; Ashwin S. Nathan; Ross A. Marklein; Jason A. Burdick; Robert L. Mauck (2348-2358).
Aligned electrospun scaffolds are promising tools for engineering fibrous musculoskeletal tissues, as they reproduce the mechanical anisotropy of these tissues and can direct ordered neo-tissue formation. However, these scaffolds suffer from a slow cellular infiltration rate, likely due in part to their dense fiber packing. We hypothesized that cell ingress could be expedited in scaffolds by increasing porosity, while at the same time preserving overall scaffold anisotropy. To test this hypothesis, poly(ɛ-caprolactone) (a slow-degrading polyester) and poly(ethylene oxide) (a water-soluble polymer) were co-electrospun from two separate spinnerets to form dual-polymer composite fiber-aligned scaffolds. Adjusting fabrication parameters produced aligned scaffolds with a full range of sacrificial (PEO) fiber contents. Tensile properties of scaffolds were functions of the ratio of PCL to PEO in the composite scaffolds, and were altered in a predictable fashion with removal of the PEO component. When seeded with mesenchymal stem cells (MSCs), increases in the starting sacrificial fraction (and porosity) improved cell infiltration and distribution after three weeks in culture. In pure PCL scaffolds, cells lined the scaffold periphery, while scaffolds containing >50% sacrificial PEO content had cells present throughout the scaffold. These findings indicate that cell infiltration can be expedited in dense fibrous assemblies with the removal of sacrificial fibers. This strategy may enhance in vitro and in vivo formation and maturation of functional constructs for fibrous tissue engineering.
Keywords: Tissue engineering; Electrospinning; Fibrous scaffolds; Cellular infiltration; Mesenchymal stem cells;

Non-invasive optical characterization of biomaterial mineralization by Sharad Gupta; Martin Hunter; Peggy Cebe; Jonathan M. Levitt; David L. Kaplan; Irene Georgakoudi (2359-2369).
Current approaches to study biomaterial mineralization are invasive and prevent dynamic characterization of this process within the same sample. Polarized light scattering spectroscopy (LSS) may offer a non-invasive alternative for assessing the levels of mineralization as well as some aspects of the organization of the mineral deposits. Specifically, we used LSS to characterize the formation of hydroxyapatite deposits on three types of silk films (water-annealed, methanol-treated and polyaspartic acid (PAA)-mixed) following 1, 3, 5 and 7 cycles of mineralization. We found that the total light scattering intensity provided a quantitative measure of the degree of mineralization as confirmed by thermal gravimetric analysis (TGA). The PAA-mixed silk films yielded the highest level of mineral deposition and the water-annealed ones the least, consistent with the β sheet content of the films prior to the onset of mineralization. The wavelength dependence of the singly backscattered light was consistent with a self-affine fractal morphology of the deposited films within scales in the range of 150–300 nm; this was confirmed by Fourier analysis of scanning electron microscopy (SEM) images of the corresponding films. The deposits of minerals in the water-annealed films were predominantly flake-like, with positively correlated density fluctuations (Hurst parameter, H  > 0.5), whereas methanol-treated and PAA-mixed silk films resulted in densely-packed, bulk mineral deposits with negatively correlated density fluctuations (H  < 0.5). Therefore, LSS could serve as a valuable tool for understanding the role of biomaterial properties in mineral formation, and, ultimately, for optimizing biomaterial designs that yield mineral deposits with the desired organization.
Keywords: Silk; Fibroin; Tissue engineering; Polarization; Light scattering;

A thiol–acrylate photopolymerization was used to incorporate enzymatically cleavable peptide sequences into PEG hydrogels to induce chondrogenic differentiation of encapsulated human mesenchymal stem cells (hMSCs). An adhesive sequence, RGD, was designed with an MMP-13 specific cleavable linker. RGD promotes survival of hMSCs encapsulated in PEG gels and has shown to induce early stages of chondrogenesis, while its persistence can limit complete differentiation. Therefore, an MMP-13 cleavage site was incorporated into the peptide sequence to release RGD mimicking the native differentiation timeline. Active MMP-13 production of encapsulated hMSCs was seen to increase from day 9 to 14 and only in chondrogenic differentiating cultures. Seeded hMSCs attached to the material prior to enzymatic cleavage, but a significant population of the cells detach after cleavage and release of RGD. Finally, hMSCs encapsulated in RGD-releasing gels produce 10 times as much glycosaminoglycan as cells with uncleavable RGD functionalities, by day 21 of culture. Furthermore, 75% of the cells stain positive for collagen type II deposition where RGD is cleavable, as compared to 19% for cultures where RGD persists. Collectively, these data provide evidence that temporal regulation of integrin-binding peptides is important in the design of niches in differentiating hMSCs to chondrocytes.
Keywords: Human mesenchymal stem cells; RGD; Collagenase-3; Chondrogenesis;

A cartilage ECM-derived 3-D porous acellular matrix scaffold for in vivo cartilage tissue engineering with PKH26-labeled chondrogenic bone marrow-derived mesenchymal stem cells by Qiang Yang; Jiang Peng; Quanyi Guo; Jingxiang Huang; Li Zhang; Jun Yao; Fei Yang; Shenguo Wang; Wenjing Xu; Aiyuan Wang; Shibi Lu (2378-2387).
We developed a natural, acellular, 3-D interconnected porous scaffold derived from cartilage extracellular matrix (ECM). Human cartilage was physically shattered, then decellularized sequentially with use of hypotonic buffer, TritonX-100, and a nuclease solution and made into a suspension. The scaffold was fabricated by simple freeze-drying and cross-linking techniques. On histology, scaffolds showed most of the ECM components after removal of the cell fragments, and scanning electron microscopy revealed a 3-D interconnected porous structure. Cellular viability assay revealed no cytotoxic effects. In vitro study showed that the novel scaffold could provide a suitable 3-D environment to support the adheration, proliferation and differentiation of bone marrow-derived mesenchymal stem cells (BMSCs) to chondrocytes in culture with chondrogenic medium after 21 days. Chondrogenically induced BMSCs labeled with fluorescent dye PKH26 were then grown on scaffolds and implanted subcutaneously into nude mice. Four weeks later, cartilage-like tissue formed, with positive staining for Safranin O, tuoluidine blue and collagen II. Cells in the samples seemed to confirm that they originated from the labeled BMSCs, as confirmed by in vivo fluorescent imaging and immunofluorescence examination. In conclusion, the cartilage ECM-derived porous scaffold shows potential as biomaterial for cartilage tissue engineering, and PKH26 fluorescent labeling and in vivo fluorescent imaging can be useful for cell tracking and analyzing cell-scaffold constructs in vivo.
Keywords: Cartilage tissue engineering; ECM (extracellular matrix); Fluorescence; Decellularization; Scaffolds;

The immobilization of basic fibroblast growth factor on plasma-treated poly(lactide-co-glycolide) by Hong Shen; Xixue Hu; Jianzhong Bei; Shenguo Wang (2388-2399).
In this study, possibility of the method of immobilization of basic fibroblast growth factor (bFGF) on polylactone-type polymer scaffolds via plasma treatment was investigated. To introduce acid carboxylic functional groups on the surface of the polymer matrix, poly(lactide-co-glycolide) (PLGA) film was treated with carbon dioxide (CO2) plasma and then incubated in a phosphate buffer saline (PBS, pH 7.4) solution of bFGF. The bFGF binding efficiency to the CO2 plasma-treated PLGA (PT-PLGA) films under different treating parameters was investigated and compared. It was found bFGF binding efficiency to PLGA was enhanced by CO2 plasma treatment. The binding efficiency of bFGF to PLGA was variational with CO2 plasma treating time and it reached a maximum after a treating time of 20 min under the power of 20 W. The changes of surface chemistry and surface topography induced by CO2 plasma treatment played main roles in improving binding efficiency. Bound bFGF was released continuously from the films for up to 7 days in vitro. The stability of bFGF immobilized on PLGA film via CO2 plasma treatment was tested further under dynamic conditions by a Parallel Plate Flow Chamber. Mouse 3T3 fibroblasts were cultured on the bFGF bound PLGA with a prior plasma treatment (20 W, 20 min) (PT-PLGA/bFGF) film, which showed that bFGF released from PT-PLGA/bFGF film was bioactive. Adhesion and growth of cells on PLGA scaffolds were greatly improved by immobilization of bFGF on them. Therefore, the method of CO2 plasma treatment combining bFGF anchorage not only was usable in delivering bFGF, but also could be applied extensively for surface modification of scaffolds in tissue engineering.
Keywords: Basic fibroblast growth factor (bFGF); Deliver vehicle; PLGA scaffold; CO2 plasma treatment; Surface modification; Tissue engineering;

Photocrosslinked anhydride systems for long-term protein release by Ashley A. Weiner; Eileen A. Bock; Margaret E. Gipson; V. Prasad Shastri (2400-2407).
Injectable delivery systems are attractive as vehicles for localized delivery of therapeutics especially in the context of regenerative medicine. In this study, the potential of photocrosslinked polyanhydride (PA) networks as an encapsulation matrix for long-term delivery of macromolecules was studied. The in vitro release of two model proteins (horseradish peroxidase (HRP) and bovine serum albumin labeled with fluorescein isothiocyanate (FITC-BSA)) was evaluated from crosslinked networks composed of sebacic acid dimethacrylate (MSA), 1,6-bis-carboxyphenoxyhexane dimethacrylate (MCPH), and poly(ethylene glycol) diacrylate (PEGDA), supplemented with calcium carbonate. Prior to incorporation into the networks, proteins were formulated by dilution in a cyclodextrin excipient followed by gelatin-based wet granulation. Protein release was quantified by activity assay (HRP) or fluorescence (FITC-BSA). Each protein was readily released from the networks with a unique release behavior. Most importantly, release of protein with retention of activity was achieved for durations ranging from 1 week to 4 months. The released HRP was additionally visualized using SDS-PAGE. In general, a more hydrophobic network resulted in slower rates of protein release. Incorporation of PEGDA into the matrices was critical for maintenance of integrity during degradation. These results suggest that this system may be useful as an injectable delivery system for long-term delivery of macromolecules.
Keywords: Photopolymerization; Drug delivery; Injectable; Polyanhydrides; Protein; Controlled release;

Peptides as functional biomaterials offer the possibility of incorporating various biological activities required for different biomedical applications. Here, we take advantage of this property of peptide materials and design a DNA delivery vector equipped with multiple functions critical to efficient gene transfection. The Tat peptide, a cationic cell-penetrating peptide, is known to enhance the cellular uptake of a large variety of molecules such as drugs and proteins. However, the application of the Tat peptide in DNA delivery is limited by the inability to release DNA in endosomes and the instability of peptide/DNA complexes. We incorporate in the Tat sequence histidine and cysteine residues that are able to promote endosomal escape of DNA and protect DNA in the extracellular environment. We observe up to 7000-fold improvement in gene transfection efficiency by a modified Tat peptide covalently fused with 10 histidine residues (Tat-10H) over the original Tat peptide. After incorporating two cysteine residues into the Tat-10H design, the resulting bis(cysteinyl) histidine-rich peptide is more effective than the Tat-10H peptide, because interpeptide disulfide bonds form by air oxidation upon binding to DNA, leading to enhanced stability of peptide/DNA complexes. These findings demonstrate the feasibility of using multi-functional peptide materials to extend the applications of the Tat vector to efficient gene delivery.
Keywords: Gene transfer; Self assembly; Nanoparticle; Peptide;

Polyethyleneimine-mediated gene delivery into human adipose derived stem cells by Hyun Hee Ahn; Jung Hwa Lee; Kyung Sook Kim; Ju Young Lee; Moon Suk Kim; Gilson Khang; Il Woo Lee; Hai Bang Lee (2415-2422).
In this study, we examined the use of polyethyleneimine (PEI) as a carrier for gene delivery in human adipose tissue-derived stem cells (hADSCs). These multipotent cells can form bone, cartilage, adipose, and other connective tissues. In primary culture, hADSCs are fibroblastic in appearance in primary culture, and they show a high rate of proliferation for at least five passages. Immunophenotyping showed that these cells are positive for the mesenchymal stem cell markers CD29 and CD44 but negative for the hematopoietic cell surface markers CD34, CD45, and c-kit. PEI and Lipofectamine were compared as gene carriers for hADSCs. DNA completely bound PEI at a negative-to-positive (N/P) charge ratio of 4. The PEI–DNA complexes were spherical with smooth surfaces. As the proportion of PEI was increased, the size of the PEI–DNA complexes decreased from 990 to 130 nm, the positive surface charge decreased, and the cytotoxicity increased. Flow cytometry revealed that the transfection efficiency using PEI at N/P charge ratios of 4 and 8 was higher than that of Lipofectamine. The highest transfection efficiency (19%) was obtained at an N/P charge ratio of 8. After transfection, the enhanced green fluorescent protein (EGFP) started to localize in the nuclei of hADSCs at 4 h 30 m and localize over cytoplasm from 9 h 30 m. In conclusion, PEI acts as an effective gene carrier for hADSCs.
Keywords: Human adipose tissue-derived stem cells; Gene carrier; Polyethylenimine; Transfection;

Protein adsorption–desorption on nanoscale surface plays a key role in biomaterials, cell adhesion, biosensors, biofuel cells and biomineralization. Silicate-substituted hydroxyapatite (SiHA) is one of the most interesting bioceramics in the field of bioactive hard tissue implants. In this paper, the adsorption–desorption behaviors of leucine-rich amelogenin protein (LRAP) on a series of SiHA (100) surfaces were investigated using the molecular dynamics (MD), steered molecular dynamics (SMD) simulations and density functional theory (DFT) calculations. It was found that the silicate ions on SiHA (100) surface cause a shield effect, which was composed of the charge repulsion and the steric hindrance of silicates. These findings suggest that surface engineering technologies can be potentially used to directly control/manufacture the nanoscale surface texture and the composition of material surfaces, thereby to mediate the interaction of proteins with biomaterials.
Keywords: Silicate-substituted hydroxyapatite (SiHA); Leucine-rich amelogenin protein (LRAP); Steered molecular dynamics (SMD); Shield effect; Adsorption;