Potential of electromagnetic and ultrasound stimulations for bone regeneration

15 Potential of electromagnetic and ultrasound stimulations for bone regeneration L. FASSINA , University of Pavia, Italy, P. DUBRUEL , University of Ghent, Belgium, G. MAGENES, University of Pavia, Italy and S. VAN VLIERBERGHE , University of Ghent, Belgium

DOI: 10.1533/9780857098104.3.445 Abstract: Gelatin-based cryogels have been seeded with human SAOS-2 osteoblasts. In order to overcome the drawbacks associated with in vitro culture systems, such as limited diffusion and inhomogeneous cell–matrix distribution, we have described the application of electromagnetic and ultrasound stimulation to physically enhance the cell culture in vitro. The results indicate that the physical stimulation of cell-seeded gelatin-based cryogels upregulates the bone matrix production. Key words: gelatin-based cryogel, SAOS-2 osteoblasts, electromagnetic stimulation, ultrasound stimulation, bone matrix production.

15.1

Introduction

A frequently applied approach in the domain of tissue engineering is the development of porous scaffolds, often containing bioactive compounds (e.g. proteins) (Ellis and Yannas, 1996; Freyman et al., 2001; Pek et al., 2004; Zaleskas et al., 2004). Autologous or allogenic cells can be seeded and cultured on these materials, resulting in newly formed tissue in vitro (Nehrer et al., 1997) or in vivo (Lee et al., 2003; Nehrer et al., 1997). In the past, a large number of materials, synthetic as well as natural, have been proposed as cell carriers. The most frequently applied synthetic polymers include poly(caprolactone), poly(DL-lactic acid), poly(DL-glycolic acid) and derivatives thereof (Chen et al., 2001; Desmet et al., 2010; Whang et al., 1995). Common natural cell matrices include chitosan (Lee et al., 2004; O’Brien et al., 2005), collagen (O’Brien et al., 2005; Schoof et al., 2001) and gelatin (Kang et al., 1999; Ren et al., 2001; Ulubayram et al., 2002). For the present chapter, gelatin was selected, since it is a self-assembling, non-toxic, biodegradable, inexpensive and non-immunogenic material (Ulubayram et al., 2002). It has already been widely applied in medicine as a wound dressing, and as an adhesive and absorbent pad for surgical use 445 © 2014 Elsevier Ltd

446

Biomaterials for Bone Regeneration

(Choi et al., 2001). Moreover, in previous studies on gelatin-based sponges, their potential in the field of tissue engineering has already been demonstrated (Dubruel et al., 2007; VanVlierberghe et al., 2008). In general, gelatin is processed into a hydrogel at ambient temperature. Interestingly, in addition to hydrogel formation at ambient temperature, hydrogels can also be synthesized by applying a cryogenic treatment. Lozinsky has referred to porous hydrogels produced by a cryogenic treatment, using the term ‘cryogels’ (Lozinsky, 2002). The process of cryogelation for the production of porous materials has already been widely applied for a series of purposes (Bajpai and Saini, 2009; Bolgen et al., 2009; Vrana et al., 2009). Gelatin cryogels were already prepared as cell carriers for a panel of human cells (Dubruel et al., 2007; VanVlierberghe et al., 2007a). A well-defined ‘curtain-like’ pore architecture was induced by applying a cryogenic treatment on scaffolds containing both gelatin and chondroitin sulphate (VanVlierberghe et al., 2008). In order to improve the biomimetic properties of gelatin-based cryogels, a physical stimulus (i.e. a pulsed electromagnetic wave or ultrasound treatment) was applied to cell-seeded scaffolds. Previous research already indicated the potential of the application of a physical stimulus to cell-seeded biomaterials including polyurethane, titanium and hydroxyapatite scaffolds (Fassina, 2006, 2007b, c, 2008a, b, 2009, 2010b; Icaro et al., 2006). Fassina et al. showed that the physical stimulus applied resulted in the upregulation of extracellular matrix (ECM) deposition onto the biomaterial surface. As consequence, a natural bioactive coating composed of ECM was obtained avoiding the above-mentioned approach where the biomimetic properties of cell carriers are improved only by a protein coating applied before the cell seeding. Now, we combine for the first time polymeric scaffolds (i.e. gelatin-based cryogels) with a physical stimulus (i.e. a pulsed electromagnetic wave or ultrasound treatment) in order to introduce a ‘natural’, autologous, biointeractive surface coating. Physical stimuli such as pulsed electromagnetic fields (PEMFs) or lowintensity ultrasounds have been used in clinical settings in order to accelerate the healing process of fresh fractures and non-unions (Carpentier et al., 2011; Massari et al., 2009). In particular, electromagnetic fields have been widely used in orthopaedics for decades (Midura et al., 2005; Walker et al., 2007). Electromagnetic therapy is approved for bone disorders including pseudarthrosis and osteoporosis (Ciombor and Aaron, 1993; Huang et al., 2008; Otter et al., 1998); moreover, this treatment reportedly aids the healing of osteotomies (Midura et al., 2005). Its clinical effectiveness was initially thought to be due to (i) an accelerated production of bone matrix by weak induced electric currents (Friedenberg and Brighton, 1966) and to (ii) a downregulated bone matrix

Potential of electromagnetic and ultrasound stimulations

447

loss (de Haas et al., 1980; Grace et al., 1998). In recent studies, it has been reported that electromagnetic exposure could enhance cell proliferation and accelerate osteogenesis (Sun et al., 2010; Tsai et al., 2007). On the other hand, low-intensity ultrasound stimulus accelerates the fracture healing in animal models (Wang et al., 1994) and in clinical studies (Cook et al., 1997; Heckman et al., 1994). Ultrasound exposure increases the influx of calcium into bone cells, and, consequently, it impacts the levels of nitric oxide, PGE2, c-fos, COX-2, osteopontin, and osteocalcin (Chen et al., 2003; Hadjiargyrou et al., 2002; Kokubu et al., 1999; Reher et al., 2002). The preceding phenomena could be explained by the signal transduction model of Pavalko (Pavalko et al., 2003), a model involving stretch-activated calcium channels, plasma membrane integrins, protein kinases, and the actin cytoskeleton (Lee et al., 2000). The plasma membrane integrins act as links between ECM, cytoskeleton proteins, and actin filaments; the ultrasound stimuli are transferred to the adherent cells through their adhesive contacts with the surrounding ECM, where they increase the cell surface expression of the integrins and, consequently, they cause a reorganization of the actin cytoskeleton with the formation of stress fibers (Yang et al., 2005).

15.2

Materials to enhance the in vitro cell culture

In this section, we report the methods to obtain a biocompatible hydrogel and to physically enhance the cell cultures.

15.2.1

Hydrogel synthesis and characterization

A porous gelatin-based cryogel with an average pore size of 135 μm diameter was applied. Methacrylamide-modified gelatin type B was used as starting material. The gelatin applied was isolated from bovine skin by an alkaline process (Rousselot). The material possessed an approximate iso-electric point of 5 and Bloom strength of 257. The synthesis of methacrylamidemodified gelatin was performed as described earlier (Van Den Bulcke et al., 2000). Part of the amine functions of gelatin were reacted with methacrylic anhydride. A derivative with a degree of substitution of 60%, based on the lysine and hydroxylysine units, was used (Van Den Bulcke et al., 2000). In a subsequent step, the modified gelatin was used for the production of 10% (w/v) hydrogels (VanVlierberghe et al., 2007b). Shortly, the hydrogels were obtained by dissolving 1 g gelatin type B, previously modified with methacrylamide side groups, in 10 mL double distilled water at 40°C, containing 2 mol% photo-initiator Irgacure® 2959 (Ciba Specialty Chemicals N.V.), as calculated to the amount of methacrylamide side chains. The solution was then injected into the mould of a cryo-unit, after which the solution was allowed to gel for 1 h at room temperature. In a final step,

448

Biomaterials for Bone Regeneration

the hydrogel was exposed to UV-light (276 nm, 10 mW/cm2, Vilber Lourmat) for 2 h. Next, a cryogenic treatment was applied as described in detail in a previous paper (VanVlierberghe et al., 2007b). The hydrogels were cooled from 21°C to −30°C at a cooling rate of −0.15°C/min. After incubating the sample for 1 h at the final freezing temperature, the frozen hydrogel was transferred to a freeze-dryer to remove the ice crystals, resulting in a porous scaffold. The hydrogels were sterilized using ethylene oxide (cold cycle, 37°C) prior to cell seeding. The visualization of the porous structure was performed using micro-computed tomography (micro-CT) analysis and scanning electron microscopy (SEM). For the micro-CT analysis, a ‘Skyscan 1072’ X-ray micro-tomograph was used, as described in detail previously (VanVlierberghe et al., 2008). Briefly, the system consisted of an X-ray shadow microscopic system and a computer with tomographic reconstruction software. The porous gelatin cryogel was scanned at a voltage of 130 kV and a current of 76 μA. For the SEM analysis, a Fei Quanta 200F (field emission gun) SEM was used to image the gold-sputtered sample.

15.2.2

Cell seeding

The human osteosarcoma cell line SAOS-2 (sarcoma osteogenic-2) was obtained from the American Type Culture Collection (HTB85, ATCC). The cells were cultured in McCoy’s 5A modified medium with l-glutamine and HEPES (4-(2-hydroxyethyl)piperazine-1-ethanesulfonic acid) (Cambrex Bio Science), supplemented with 15% foetal bovine serum, 2% sodium pyruvate, 1% antibiotics, 10−8 M dexamethasone, and 10 mM β-glycerophosphate (Sigma-Aldrich). Ascorbic acid, another osteogenic supplement, is a component of McCoy’s 5A modified medium. The cells were cultured at 37°C in the presence of 5% CO2. A cell suspension of 4 × 105 cells in 100 μL was seeded on the top of each cryogel and, after 0.5 h, 1 mL of culture medium was added to submerge the scaffolds with medium. The cells were allowed to attach overnight.

15.2.3

Cell culture inside an electromagnetic bioreactor

The electromagnetic bioreactor consisted of a carrying structure custommachined in a polymethylmethacrylate tube. The windowed tube carried a well-plate and two solenoids whose planes were parallel (Fassina, 2006). The gelatin scaffolds were at a distance of 5 cm from each solenoid plane. The solenoids were powered by a Biostim SPT pulse generator (Igea, Carpi, Italy) (i.e. a generator of PEMFs). Given the position of the solenoids and the characteristics of the pulse generator, the electromagnetic stimulus possessed

Potential of electromagnetic and ultrasound stimulations

449

the following parameters: intensity of the magnetic field = 2 ± 0.2 mT, amplitude of the induced electric tension = 5 ± 1 mV, signal frequency = 75 ± 2 Hz, and pulse duration = 1.3 ms. The electromagnetic bioreactor was placed into a standard cell culture incubator at 37°C in the presence of 5% CO2. The electromagnetic culture was stimulated by the PEMF 24 h/day for a total of 22 days. The culture medium was changed on days 4, 7, 10, 13, 16, and 19.

15.2.4

Ultrasound stimulus

An ultrasound stimulus (Fassina, 2009, 2010a) was applied through the culture medium by a FAST ultrasound generator (Igea, Carpi, Italy) to the seeded gelatin cryogels. The mechanical wave had the following characteristics: signal frequency equal to 1.5 ± 0.03 MHz, duty cycle of 200 ± 4 μs, repetition rate equal to 1 ± 0.02 kHz, and average power of 149 ± 3 mW. The ultrasound culture was placed in a standard cell culture incubator with an environment of 37°C and 5% CO2, and was stimulated for 20 min/day over 22 days. The culture medium was changed on days 4, 7, 10, 13, 16, and 19.

15.2.5

Control culture

The control culture was placed into a standard cell culture incubator. The duration of the control culture was 22 days and the culture medium was changed on days 4, 7, 10, 13, 16, and 19.

15.3

Processing techniques

In this section, we report the methods to quantify the main characteristics of the cell cultures at the end of the incubation period.

15.3.1

Determination of DNA content

The cells were lysed by a freeze–thaw method in sterile deionized distilled water, and the released DNA content was evaluated using a fluorometric method (PicoGreen, Molecular Probes). A standard DNA curve, obtained from a known amount of osteoblasts, was applied in order to express the results as cell number per scaffold (Fassina, 2005).

15.3.2

Extraction of the bone matrix

At the end of the culture period, the cultured scaffolds were washed with sterile PBS (phosphate buffered saline) (137 mM NaCl, 2.7 mM KCl, 4.3 mM Na2HPO4, 1.4 mM KH2PO4, pH = 7.4) four times for 10 min in order

450

Biomaterials for Bone Regeneration

to remove the culture medium, and then incubated for 24 h at 37°C with 1 mL of sterile sample buffer (1.5 M Tris-HCl, 60% [w/v] sucrose, 0.8% [w/v] Na-dodecyl-sulphate, pH = 8.0). At the end of the incubation period, the sample buffer aliquots were removed and the total protein concentration in the three culture systems was evaluated by the BCA (bicinchoninic acid) Protein Assay Kit (Pierce Biotechnology). The calibration curves to measure the extracted proteins were performed by an ELISA assay (enzyme-linked immunosorbent assay) (Fassina, 2006; Saino et al., 2010). The results are expressed as fg/(cell × scaffold).

15.3.3

Statistics

The results are expressed as mean ± standard deviation. In order to compare the results between the three culture systems, one-way analysis of variance (ANOVA) with post hoc Bonferroni test was applied, electing a significance level of 0.05.

15.4

Applications

In this section, the experimental results are discussed.

15.4.1

Hydrogel development and characterization

The porous biomaterial functioning as a cell support was prepared by a cryogenic treatment of crosslinked methacrylamide-modified gelatin. First, the modified gelatin was synthesized by reacting part of the lysine and hydroxylysine units of gelatin type B with methacrylic anhydride, as described in detail in a previous paper (Van Den Bulcke et al., 2000). The degree of substitution of the gelatin used was 60% (determination via 1H-nuclear magnetic resonance (1H-NMR) spectroscopy), based on the gelatin primary amine functions. Next, hydrogels were formed by gelation of an aqueous methacrylamide-modified gelatin solution, followed by radical cross-linking using a UV-active photoinitiator. Finally, the chemically crosslinked hydrogels were subjected to cryogenic treatment. The pore size, morphology, and porosity of the 3D scaffold developed were analysed by micro-CT and SEM analysis. The pore size, as studied by SEM analysis (Fig. 15.1a), was in the range of 135 μm. Interestingly, in addition to a non-destructive visualization of the scaffold architecture (Fig. 15.1b), micro-CT analysis also enables determining the porosity distribution throughout the entire scaffold. Figure 15.2 indicates that the porosity of the gelatin-based cryogel developed ranges from 77% up to 84%. After that, the gelatin-based cryogels were evaluated for their potential to support the proliferation of SAOS-2 osteoblasts and their ECM production under static conditions and physical stimuli.

Potential of electromagnetic and ultrasound stimulations (a)

451

(b)

1 mm

15.1 SEM image (a) and micro-CT image (b) showing the pore size and morphology of the gelatin-based cryogels developed.

85

Porosity (%)

83 81 79 77 75 500

1500

2500

3500

4500

5500

Scaffold depth (μm)

15.2 Scaffold porosity as a function of the depth obtained via micro-CT analysis.

Therefore, the human SAOS-2 osteoblasts were seeded onto the surface of the gelatin-based cryogels, followed by culturing them, with or without physical stimuli, for 22 days. These culture methods enabled studying the SAOS-2 cells as they modified the biomaterial surface through cell proliferation and through the production of ECM deposited onto the gelatin-based cryogels. The cell–matrix distribution was compared between the three culture systems.

15.4.2

Evaluation of cell proliferation

The evaluation of the cell proliferation is based on the measure of the DNA content at the end of the culture period. For the static culture, the cell number per disc increased to 21.4 × 105 ± 5.7 × 105, while, for the electromagnetic and ultrasound cultures, the cell numbers were 19.1 × 105 ± 2.9 × 105 and 23.1 × 105 ± 4.5 × 105 (p > 0.05), respectively.

452

15.4.3

Biomaterials for Bone Regeneration

Extracellular matrix (ECM) extraction

In order to evaluate the amount of bone ECM secreted onto the gelatin cryogels, an ELISA of the extracted matrix was performed. The results indicated that the physical stimuli significantly increased the production and the deposition of various ECM proteins onto the surface of the gelatin biomaterial (p < 0.05) (Table 15.1). In order to improve the biomimetic properties of gelatin-based cryogels, physical stimuli were applied after cell seeding. The results indicated that they did not affect the cell proliferation. However, the physical stimuli did result in an upregulation of the production of some ECM proteins, such as type-I collagen, osteopontin, and osteocalcin (Table 15.1). Interestingly, the above-mentioned proteins are fundamental constituents of the physiological bone matrix. In particular, type-I collagen is the most important and abundant structural protein of the bone matrix. Osteopontin is an extracellular glycosylated bone phosphoprotein secreted at the early stages of the osteogenesis before the onset of the mineralization; the protein binds calcium and is likely to be involved in the regulation of the hydroxyapatite crystal growth; moreover, it promotes cell attachment through specific interaction with the vitronectin receptor. Osteocalcin is secreted after the onset of mineralization, and binds to bone minerals. The obtained results can be clarified using Pavalko’s signalling model (Pavalko et al., 2003). The physical stimuli bring about both an increase of the Ca2+ flux into the osteoblast cytosol and the release of the intracellular Ca2+. According to the model, the increase of the cytosolic Ca2+ concentration is the starting point of signalling pathways targeting specific bone matrix genes. The use of a cell line already indicated the potential of the stimuli in combination with the gelatin-based cryogels developed. Interestingly, we anticipate that, upon fine-tuning the parameters of the physical stimuli, autologous bone marrow stromal cells could also be applied instead of SAOS-2 osteoblasts in order to realize full immunocompatibility with the treated patient.

Table 15.1 Amount of extracellular matrix (ECM) constituents onto gelatin discs Total ECM production in fg/(cell × scaffold)

Osteocalcin Osteopontin Type-I collagen Note: p < 0.05.

Control culture

Electromagnetic culture

Ultrasound culture

1.23 ± 0.12 6.12 ± 0.53 29.00 ± 6.21

2.30 ± 0.22 9.60 ± 0.75 48.70 ± 7.56

3.60 ± 0.27 14.80 ± 0.45 44.05 ± 4.51

Potential of electromagnetic and ultrasound stimulations

453

In the present chapter, a gelatin-based cryogel was combined with physical stimuli in order to develop tissue-engineering constructs enabling bone repair. The results clearly indicated that the physical stimuli resulted in the upregulation of ECM proteins. Elaborating an idea of Castner and Ratner (2002), we physically enhanced the coating of gelatin with osteoblasts and with ECM: we followed a particular biomimetic strategy whereby the seeded cells built a new biocompatible surface over the biomaterial, making it very useful for the biointegration. The idea of Castner and Ratner and a discussion of the concept of biocompatibility follow. When a biomaterial is implanted in a biological environment, a non-specific and non-physiological layer of adsorbed proteins mediates the interaction of the surrounding host cells with the material surface. The body interprets this protein layer as a foreign invader that must be walled off in an avascular and tough collagen bag. Therefore, the biomedical surfaces must be developed so that the host tissue can recognize them as ‘self’. Castner and Ratner think of the biocompatible surfaces of the biomaterials that heal as the surfaces as having the character of a ‘clean, fresh wound’: these ‘self-surfaces’ could experience a physiological inflammatory reaction around the biomaterials, leading to normal healing, leading in turn to physiological osteointegration in bone tissue engineering. In the present chapter, we have followed a particular biomimetic strategy: we obtained a surface coating of the biomaterial, over which the seeded and physically stimulated osteoblasts built a new biocompatible surface made of cell–matrix layers, that is, a physiological surface with the character of a ‘clean, fresh wound’.

15.5

Future trends

Using the preceding biomimetic tissue-engineering approach (Fassina, 2006, 2007a, b, c, 2008a, b, 2009, 2010b, c, 2012; Icaro et al., 2006; Saino et al., 2010, 2011a, b), gelatin-based cryogels could be combined with differentiated cells and their ECM proteins as implants for bone repair in clinical applications. In conclusion, we theorize that the obtained cultured self-surface could be used fresh, that is, rich in autologous cells and matrix, or after sterilization with ethylene oxide, that is, rich only in autologous matrix. In future work, we intend to use our constructs, which are rich in autologous matrix, as a simple, storable, tissue-engineering product for the bone repair.

15.6

Acknowledgements

The authors would like to acknowledge the Research Foundation – Flanders (FWO) for the research grant assigned to S. Van Vlierberghe. The authors

454

Biomaterials for Bone Regeneration

would also like to thank the PolExGene consortium. PolExGene is a STREP project (contract number 019114) funded under the EU sixth Framework Programme. This work was also supported by the INAIL Grant 2010 to A. L. and by the INAIL Grant 2010 to G. R. We are grateful to Dr R. Cadossi and Dr S. Setti (Igea, Carpi, Italy).

15.7

References

Bajpai, A.K. and Saini, R. (2009). Designing of macroporous biocompatible cryogels of PVA-haemoglobin and their water sorption study. Journal of Materials Science: Materials in Medicine, 20 (10) 2063–2074, available from: PM:19455407. Bolgen, N., Vargel, I., Korkusuz, P., Guzel, E., Plieva, F., Galaev, I., Matiasson, B. and Piskin, E. (2009). Tissue responses to novel tissue engineering biodegradable cryogel scaffolds: an animal model. Journal of Biomedical Materials Research Part A, 91 (1) 60–68, available from: PM:18690660. Carpentier, B., Layrolle, P. and Legallais, C. (2011). Bioreactors for bone tissue engineering. The International Journal of Artificial Organs, 34 (3) 259–270, available from: PM:21374561. Castner, D.G. and Ratner, B.D. (2002). Biomedical surface science: Foundations to frontiers. Surface Science, 500 (1–3) 28–60, available from: ISI:000175303400003. Chen, G.P., Ushida, T. and Tateishi, T. (2001). Development of biodegradable porous scaffolds for tissue engineering. Materials Science and Engineering C – Biomimetic and Supramolecular Systems, 17 (1–2) 63–69, available from: ISI:000171888300012. Chen, Y.J., Wang, C.J., Yang, K.D., Chang, P.R., Huang, H.C., Huang, Y.T., Sun, Y.C. and Wang, F.S. (2003). Pertussis toxin-sensitive Galphai protein and ERKdependent pathways mediate ultrasound promotion of osteogenic transcription in human osteoblasts. FEBS Letters, 554 (1–2) 154–158, available from: PM:14596931. Choi, Y.S., Lee, S.B., Hong, S.R., Lee, Y.M., Song, K.W. and Park, M.H. (2001). Studies on gelatin-based sponges. Part III: a comparative study of crosslinked gelatin/alginate, gelatin/hyaluronate and chitosan/hyaluronate sponges and their application as a wound dressing in full-thickness skin defect of rat. Journal of Materials Science: Materials in Medicine, 12 (1) 67–73, available from: PM:15348379. Ciombor, D.M. and Aaron, R.K. (1993). Influence of electromagnetic fields on endochondral bone formation. Journal of Cellular Biochemistry, 52 (1) 37–41, available from: PM:8320273. Cook, S.D., Ryaby, J.P., McCabe, J., Frey, J.J., Heckman, J.D. and Kristiansen, T.K. (1997). Acceleration of tibia and distal radius fracture healing in patients who smoke. Clinical Orthopaedics Related Research (337) 198–207, available from: PM:9137191. de Haas, W.G., Watson, J. and Morrison, D.M. (1980). Non-invasive treatment of ununited fractures of the tibia using electrical stimulation. The Journal of Bone & Joint Surgery British, 62-B (4) 465–470, available from: PM:6968752. Desmet, T., Billiet, T., Berneel, E., Cornelissen, R., Schaubroeck, D., Schacht, E. and Dubruel, P. (2010). Post-plasma grafting of AEMA as a versatile tool to

Potential of electromagnetic and ultrasound stimulations

455

biofunctionalise polyesters for tissue engineering. Macromolecular Bioscience, 10 (12) 1484–1494, available from: PM:20857390. Dubruel, P., Unger, R., VanVlierberghe, S., Cnudde, V., Jacobs, P., Schacht, E. and Kirkpatrick, C.J. (2007). Porous gelatin hydrogels: 2. In vitro cell interaction study. Biomacromolecules, 8 (2) 338–344. Ellis, D.L. and Yannas, I.V. (1996). Recent advances in tissue synthesis in vivo by use of collagen-glycosaminoglycan copolymers. Biomaterials, 17 (3) 291–299, available from: PM:8745326. Fassina, L., Saino, E., De Angelis, M.G., Magenes, G., Benazzo, F. and Visai, L. (2010a). Low-power ultrasounds as a tool to culture human osteoblasts inside cancellous hydroxyapatite. Bioinorganic Chemistry and Applications, Article ID 456240, available from: PM:20379359. Fassina, L., Saino, E., Sbarra, M.S., Visai, L., Cusella De Angelis, M.G., Mazzini, G., Benazzo, F. and Magenes, G. (2009). Ultrasonic and electromagnetic enhancement of a culture of human SAOS-2 osteoblasts seeded onto a titanium plasma-spray surface. Tissue Engineering Part C, 15 (2) 233–242, available from: PM:19119923. Fassina, L., Saino, E., Sbarra, M.S., Visai, L., De Angelis, M.G., Magenes, G. and Benazzo, F. (2010b). In vitro electromagnetically stimulated SAOS-2 osteoblasts inside porous hydroxyapatite. Journal of Biomedical Materials Research Part A, 93 (4) 1272–1279, available from: PM:19827111. Fassina, L., Saino, E., Visai, L., Avanzini, M.A., Cusella De Angelis, M.G., Benazzo, F., VanVlierberghe, S., Dubruel, P. and Magenes, G. (2010c). Use of a gelatin cryogel as biomaterial scaffold in the differentiation process of human bone marrow stromal cells. Conference Proceedings IEEE Engineering in Medicine and Biology Society, 2010, 247–250, available from: PM:21096747. Fassina, L., Saino, E., Visai, L., De Angelis, M.G., Benazzo, F. and Magenes, G. (2007a). Enhanced in vitro culture of human SAOS-2 osteoblasts on a sand-blasted titanium surface modified with plastic deformation. Conference Proceedings IEEE Engineering in Medicine and Biology Society, 2007, 6411–6414, available from: PM:18003489. Fassina, L., Saino, E., Visai, L. and Magenes, G. (2007b). Physically enhanced coating of a titanium plasma-spray surface with human SAOS-2 osteoblasts and extracellular matrix. Conference Proceedings IEEE Engineering in Medicine and Biology Society, 2007, 6415–6418, available from: PM:18003490. Fassina, L., Saino, E., Visai, L. and Magenes, G. (2008a). Electromagnetically enhanced coating of a sintered titanium grid with human SAOS-2 osteoblasts and extracellular matrix. Conference Proceedings IEEE Engineering in Medicine and Biology Society, 2008, 3582–3585, available from: PM:19163483. Fassina, L., Saino, E., Visai, L., Schelfhout, J., Dierick, M., Van, H.L., Dubruel, P., Benazzo, F., Magenes, G. and Van, V.S. (2012). Electromagnetic stimulation to optimize the bone regeneration capacity of gelatin-based cryogels. International Journal of Immunopathology and Pharmacology, 25 (1) 165–174, available from: PM:22507329. Fassina, L., Saino, E., Visai, L., Silvani, G., Cusella De Angelis, M.G., Mazzini, G., Benazzo, F. and Magenes, G. (2008b). Electromagnetic enhancement of a culture of human SAOS-2 osteoblasts seeded onto titanium fiber-mesh scaffolds. Journal of Biomedical Materials Research Part A, 87 (3) 750–759, available from: PM:18200542.

456

Biomaterials for Bone Regeneration

Fassina, L., Visai, L., Asti, L., Benazzo, F., Speziale, P., Tanzi, M.C. and Magenes, G. (2005). Calcified matrix production by SAOS-2 cells inside a polyurethane porous scaffold, using a perfusion bioreactor. Tissue Engineering, 11 (5–6) 685– 700, available from: PM:15998210. Fassina, L., Visai, L., Benazzo, F., Benedetti, L., Calligaro, A., De Angelis, M.G., Farina, A., Maliardi, V. and Magenes, G. (2006). Effects of electromagnetic stimulation on calcified matrix production by SAOS-2 cells over a polyurethane porous scaffold. Tissue Engineering, 12 (7) 1985–1999, available from: PM:16889527. Fassina, L., Visai, L., De Angelis, M.G., Benazzo, F. and Magenes, G. (2007c). Surface modification of a porous polyurethane through a culture of human osteoblasts and an electromagnetic bioreactor. Technology and Health Care, 15 (1) 33–45, available from: PM:17264411. Freyman, T.M., Yannas, I.V., Yokoo, R. and Gibson, L.J. (2001). Fibroblast contraction of a collagen-GAG matrix. Biomaterials, 22 (21) 2883–2891, available from: PM:11561894. Friedenberg, Z.B. and Brighton, C.T. (1966). Bioelectric potentials in bone. The Journal of Bone & Joint Surgery America, 48 (5) 915–923, available from: PM:5942807. Grace, K.L., Revell, W.J. and Brookes, M. (1998). The effects of pulsed electromagnetism on fresh fracture healing: osteochondral repair in the rat femoral groove. Orthopedics, 21 (3) 297–302, available from: PM:9547814. Hadjiargyrou, M., Lombardo, F., Zhao, S., Ahrens, W., Joo, J., Ahn, H., Jurman, M., White, D.W. and Rubin, C.T. (2002). Transcriptional profiling of bone regeneration. Insight into the molecular complexity of wound repair. The Journal of Biological Chemistry, 277 (33) 30177–30182, available from: PM:12055193. Heckman, J.D., Ryaby, J.P., McCabe, J., Frey, J.J. and Kilcoyne, R.F. (1994). Acceleration of tibial fracture-healing by non-invasive, low-intensity pulsed ultrasound. The Journal of Bone & Joint Surgery America, 76 (1) 26–34, available from: PM:8288661. Huang, L.Q., He, H.C., He, C.Q., Chen, J. and Yang, L. (2008). Clinical update of pulsed electromagnetic fields on osteoporosis. Chinese Medical Journal (England), 121 (20) 2095–2099, available from: PM:19080282. Icaro, C.A., Casasco, M., Riva, F., Farina, A., Fassina, L., Visai, L. and Casasco, A. (2006). Stimulation of osteoblast growth by an electromagnetic field in a model of bone-like construct. European Journal of Histochemistry, 50 (3) 199–204, available from: PM:16920643. Kang, H.W., Tabata, Y. and Ikada, Y. (1999). Fabrication of porous gelatin scaffolds for tissue engineering. Biomaterials, 20 (14) 1339–1344, available from: PM:10403052. Kokubu, T., Matsui, N., Fujioka, H., Tsunoda, M. and Mizuno, K. (1999). Low intensity pulsed ultrasound exposure increases prostaglandin E2 production via the induction of cyclooxygenase-2 mRNA in mouse osteoblasts. Biochemical Biophysical Research Communications, 256 (2) 284–287, available from: PM:10079177. Lee, H.S., Millward-Sadler, S.J., Wright, M.O., Nuki, G. and Salter, D.M. (2000). Integrin and mechanosensitive ion channel-dependent tyrosine phosphorylation of focal adhesion proteins and beta-catenin in human articular chondrocytes after mechanical stimulation. Journal of Bone and Mineral Research, 15 (8) 1501–1509, available from: PM:10934648.

Potential of electromagnetic and ultrasound stimulations

457

Lee, J.E., Kim, S.E., Kwon, I.C., Ahn, H.J., Cho, H., Lee, S.H., Kim, H.J., Seong, S.C. and Lee, M.C. (2004). Effects of a chitosan scaffold containing TGF-β1 encapsulated chitosan microspheres on in vitro chondrocyte culture. Artificial Organs, 28 (9) 829–839, available from: PM:15320946. Lee, S.B., Jeon, H.W., Lee, Y.W., Lee, Y.M., Song, K.W., Park, M.H., Nam, Y.S. and Ahn, H.C. (2003). Bio-artificial skin composed of gelatin and (1-->3) (1-->6)-β-glucan. Biomaterials, 24 (14) 2503–2511, available from: PM:12695077. Lozinsky, V.I. (2002). Cryogels on the basis of natural and synthetic polymers: Preparation, properties and application. Uspekhi Khimii, 71 (6) 559–585, available from: ISI:000177211100003. Massari, L., Caruso, G., Sollazzo, V. and Setti, S. (2009). Pulsed electromagnetic fields and low intensity pulsed ultrasound in bone tissue. Clinical Cases in Mineral and Bone Metabolism, 6 (2) 149–154, available from: PM:22461165. Midura, R.J., Ibiwoye, M.O., Powell, K.A., Sakai, Y., Doehring, T., Grabiner, M.D., Patterson, T.E., Zborowski, M. and Wolfman, A. (2005). Pulsed electromagnetic field treatments enhance the healing of fibular osteotomies. Journal of Orthopaedic Research, 23 (5) 1035–1046, available from: PM:15936919. Nehrer, S., Breinan, H.A., Ramappa, A., Young, G., Shortkroff, S., Louie, L.K., Sledge, C.B., Yannas, I.V. and Spector, M. (1997). Matrix collagen type and pore size influence behaviour of seeded canine chondrocytes. Biomaterials, 18 (11) 769–776, available from: PM:9177854. O’Brien, F.J., Harley, B.A., Yannas, I.V. and Gibson, L.J. (2005). The effect of pore size on cell adhesion in collagen-GAG scaffolds. Biomaterials, 26 (4) 433–441, available from: PM:15275817. Otter, M.W., McLeod, K.J. and Rubin, C.T. (1998). Effects of electromagnetic fields in experimental fracture repair. Clinical Orthopaedics Related Research, 355 S90–104 available from: PM:9917630. Pavalko, F.M., Norvell, S.M., Burr, D.B., Turner, C.H., Duncan, R.L. and Bidwell, J.P. (2003). A model for mechanotransduction in bone cells: the load-bearing mechanosomes. Journal of Cellular Biochemistry, 88 (1) 104–112, available from: PM:12461779. Pek, Y.S., Spector, M., Yannas, I.V. and Gibson, L.J. (2004). Degradation of a collagen-chondroitin-6-sulfate matrix by collagenase and by chondroitinase. Biomaterials, 25 (3) 473–482, available from: PM:14585696. Reher, P., Harris, M., Whiteman, M., Hai, H.K. and Meghji, S. (2002). Ultrasound stimulates nitric oxide and prostaglandin E2 production by human osteoblasts. Bone, 31 (1) 236–241, available from: PM:12110440. Ren, L., Tsuru, K., Hayakawa, S. and Osaka, A. (2001). Sol-gel preparation and in vitro deposition of apatite on porous gelatin-siloxane hybrids. Journal of NonCrystalline Solids, 285 (1–3) 116-122, available from: ISI:000169410800019. Saino, E., Fassina, L., Van, V.S., Avanzini, M.A., Dubruel, P., Magenes, G., Visai, L. and Benazzo, F. (2011a). Effects of electromagnetic stimulation on osteogenic differentiation of human mesenchymal stromal cells seeded onto gelatin cryogel. International Journal of Immunopathology and Pharmacology, 24 (1 Suppl 2) 1–6 available from: PM:21669129. Saino, E., Grandi, S., Quartarone, E., Maliardi, V., Galli, D., Bloise, N., Fassina, L., De Angelis, M.G., Mustarelli, P., Imbriani, M. and Visai, L. (2011b). In vitro calcified matrix deposition by human osteoblasts onto a zinc-containing bioactive glass. European Cells and Materials, 21, 59–72, available from: PM:21240845.

458

Biomaterials for Bone Regeneration

Saino, E., Maliardi, V., Quartarone, E., Fassina, L., Benedetti, L., De Angelis, M.G., Mustarelli, P., Facchini, A. and Visai, L. (2010). In vitro enhancement of SAOS-2 cell calcified matrix deposition onto radio frequency magnetron sputtered bioglass-coated titanium scaffolds. Tissue Engineering Part A, 16 (3) 995–1008, available from: PM:19839719. Schoof, H., Apel, J., Heschel, I. and Rau, G. (2001). Control of pore structure and size in freeze-dried collagen sponges. Journal of Biomedical Materials Research, 58 (4) 352–357, available from: PM:11410892. Sun, L.Y., Hsieh, D.K., Lin, P.C., Chiu, H.T. and Chiou, T.W. (2010). Pulsed electromagnetic fields accelerate proliferation and osteogenic gene expression in human bone marrow mesenchymal stem cells during osteogenic differentiation. Bioelectromagnetics, 31 (3) 209–219, available from: PM:19866474. Tsai, M.T., Chang, W.H., Chang, K., Hou, R.J. and Wu, T.W. (2007). Pulsed electromagnetic fields affect osteoblast proliferation and differentiation in bone tissue engineering. Bioelectromagnetics, 28 (7) 519–528, available from: PM:17516509. Ulubayram, K., Eroglu, I. and Hasirci, N. (2002). Gelatin microspheres and sponges for delivery of macromolecules. Journal of Biomaterials Applications, 16 (3) 227–241, available from: PM:11939457. Van Den Bulcke, A.I., Bogdanov, B., De, R.N., Schacht, E.H., Cornelissen, M. and Berghmans, H. (2000). Structural and rheological properties of methacrylamide modified gelatin hydrogels. Biomacromolecules, 1 (1) 31–38, available from: PM:11709840. VanVlierberghe, S., Cnudde, V., Dubruel, P., Masschaele, B., Cosijns, A., Paepe, I.D., Jacobs, P.J., Hoorebeke, L.V., Remon, J.P. and Schacht, E. (2007a). Porous gelatin hydrogels: 1. Cryogenic formation and structure analysis. Biomacromolecules, 8 (2) 331–337, available from: PM:17291055. VanVlierberghe, S., Cnudde, V., Dubruel, P., Masschaele, B., Cosijns, A., Paepe, I.D., Jacobs, P.J., Hoorebeke, L.V., Remon, J.P. and Schacht, E. (2007b). Porous gelatin hydrogels: 1. Cryogenic formation and structure analysis. Biomacromolecules, 8 (2) 331–337, available from: PM:17291055. VanVlierberghe, S., Dubruel, P., Lippens, E., Masschaele, B., Van, H.L., Cornelissen, M., Unger, R., Kirkpatrick, C.J. and Schacht, E. (2008). Toward modulating the architecture of hydrogel scaffolds: curtains versus channels. Journal of Materials Science: Materials in Medicine, 19 (4) 1459–1466, available from: PM:18299964. Vrana, N.E., O’Grady, A., Kay, E., Cahill, P.A. and McGuinness, G.B. (2009). Cell encapsulation within PVA-based hydrogels via freeze-thawing: a one-step scaffold formation and cell storage technique. Journal of Tissue Engineering and Regenerative Medicine, 3 (7) 567–572, available from: PM:19598204. Walker, N.A., Denegar, C.R. and Preische, J. (2007). Low-intensity pulsed ultrasound and pulsed electromagnetic field in the treatment of tibial fractures: a systematic review. Journal of Athletic Training, 42 (4) 530–535, available from: PM:18174942. Wang, S.J., Lewallen, D.G., Bolander, M.E., Chao, E.Y., Ilstrup, D.M. and Greenleaf, J.F. (1994). Low intensity ultrasound treatment increases strength in a rat femoral fracture model. Journal of Orthopaedic Research, 12 (1) 40–47, available from: PM:8113941.

Potential of electromagnetic and ultrasound stimulations

459

Whang, K., Thomas, C.H., Healy, K.E. and Nuber, G. (1995). A novel method to fabricate bioabsorbable scaffolds. Polymer, 36 (4) 837–842, available from: ISI:A1995QK67100021. Yang, R.S., Lin, W.L., Chen, Y.Z., Tang, C.H., Huang, T.H., Lu, B.Y. and Fu, W.M. (2005). Regulation by ultrasound treatment on the integrin expression and differentiation of osteoblasts. Bone, 36 (2) 276–283, available from: PM:15780953. Zaleskas, J.M., Kinner, B., Freyman, T.M., Yannas, I.V., Gibson, L.J. and Spector, M. (2004). Contractile forces generated by articular chondrocytes in collagenglycosaminoglycan matrices. Biomaterials, 25 (7–8) 1299–1308, available from: PM:14643604.