Nicola Claire Hunt

Richard Michael Shelton

Dr Liam Michael Grover
email: l.m.grover@bham.ac.uk

An alginate hydrogel matrix for the localised delivery of a fibroblast/keratinocyte co-culture

Keywords: Alginate , Tissue Engineering, Keratinocytes, Fibroblasts, Skin Alginate / Fibroblasts / Keratinocytes / Skin / Tissue engineering A/L, air-liquid interface, ECM, extracellular matrix, H&E, haematoxylin Alginate, Keratinocytes, Fibroblasts, Skin, Tissue engineering Calcein-AM -Calcein Acetoxymethylester

come

An alginate hydrogel matrix for the localised delivery of a fibroblast/keratinocyte co-culture

Introduction

There is a considerable demand for the development of novel methods to replace diseased or damaged tissues. Synthetic materials have been used as tissue replacements for many centuries, but issues remain surrounding the integration of these materials into the body. Tissue engineers seek to produce and condition tissues in vitro with the intention of implanting the resulting construct into the body. The use of the patients' own cells in this process eliminates the chances of immunological rejection and enhances the prospects of complete integration into the patient's body [1].

Skin is composed of two main layers known as the epidermis and the dermis. The epidermis is a keratinocyte-rich layer that comprises four sub-layers, which contain keratinocytes at different stages of differentiation, upon the surface of a basement membrane [START_REF] Fuchs | Skin stem cells: rising to the surface[END_REF]. The epidermis is mainly responsible for providing a barrier function to prevent water loss and infection, which is achieved by the most ventral layer of the epidermis known as the stratum corneum [START_REF] Elias | The skin barrier as an innate immune element[END_REF]. The dermis is rich in ECM, of which the main component is type I collagen and the most prevalent cell type in this layer is the fibroblast [START_REF] Liu | Construction of chitosan-gelatin-hyaluronic acid artificial skin in vitro[END_REF]. In severe wounds where both these tissue layers are lost it is important to replace both layers to achieve effective healing [START_REF] Spiekstra | Woundhealing factors secreted by epidermal keratinocytes and dermal fibroblasts in skin substitutes[END_REF]. The first tissue-engineered skin analogue was developed in 1975, which simply aimed to replace the epidermal layer using stratified keratinocyte cultures [START_REF] Rheinwald | Formation of a keratinizing epithelium in culture by a cloned cell line derived from a teratoma[END_REF]. Since then, a number of other substitutes have been developed which aim to replace either the dermal layer alone or both the epidermal and dermal layers [START_REF] Supp | Engineered skin substitutes: practices and potentials[END_REF][START_REF] Auger | Tissueengineered skin substitutes: from in vitro constructs to in vivo applications[END_REF][START_REF] Clark | Tissue engineering for cutaneous wounds[END_REF]. However, the problem with many of these is the presence of human-or animal-derived components such as de-epidermalised dermis (DED) or collagen, which carry the risk of disease transmission and promoting immune rejection [START_REF] Eisenbud | Skin substitutes and wound healing: Current status and challenges[END_REF][START_REF] Macneil | Progress and opportunities for tissue engineered skin[END_REF]. It has also been suggested that the inclusion of collagen in the initial construct may be detrimental to the healing process in terms of tissue cell migration as collagen appears at the later stages of wound healing [START_REF] Clark | Tissue engineering for cutaneous wounds[END_REF][START_REF] Welch | Temporal relationships of Factin bundle formation, collagen and fibronectin matrix assembly, and Fibronectin receptor expression to wound contraction[END_REF]. There is, therefore, a significant need of development of a bilayered structure capable of assisting in wound healing in which the dermal layer is derived from a non-animal source [START_REF] Spiekstra | Woundhealing factors secreted by epidermal keratinocytes and dermal fibroblasts in skin substitutes[END_REF][START_REF] Macneil | Progress and opportunities for tissue engineered skin[END_REF].

In addition, it is beneficial to have both the keratinocytes and fibroblasts present, as the paracrine interactions between these two cell types are also very important to effective healing [START_REF] Werner | Keratinocyte-fibroblast interactions in wound healing[END_REF][START_REF] Wong | The role of fibroblasts in tissue engineering and regeneration[END_REF]. When co-cultures of these cells are used, however, it is important to mitotically inhibit the fibroblasts to prevent over-growth of the culture. This is typically accomplished by the use of gamma-irradiation or Mitomycin C [START_REF] Stanovic | Human keratinocyte culture in vitro[END_REF][START_REF] Tseng | Comparison between serum-free and fibroblast-cocultured single-cell clonal culture systems: Evidence Deleted: -Deleted[END_REF]. These mitotically inhibited fibroblasts facilitate formation of the epidermis in vitro, but are not actually delivered to the wound site and thus play no role in the replacement of the lost dermis at the wound site. It is therefore desirable to develop a method in which the mitotic inhibition is reversible, so that autologous fibroblasts can be delivered to the wound along with the keratinocytes to help maintain the keratinocyte culture at the wound bed and facilitate replacement of the dermis. The fibroblasts also secrete angiogenic mediators that encourage the neo-vascularisation of the site [START_REF] Eming | Regulation of angiogenesis: Wound healing as a model[END_REF], which is essential for survival of the tissue-engineered construct.

In order to mimic the 3-D organisation of cells in a range of tissues, typically cells are seeded onto a scaffold composed of a macro-porous sponge-like material. Although such structures provide a certain amount of 3-D organisation, the cells still adhere to a 2-D surface. It has been demonstrated that cells cultured on 2-D substrates exhibit distinct phenotypes when compared with cells in the ECM [START_REF] Pedersen | Mechanobiology in the third dimension[END_REF]. In order to produce a tissue representative of that found in vivo, it is therefore desirable to culture the cells in an environment that mimics both the structure and spatial resolution of the ECM.

Consequently, there is currently a focus on the encapsulation of cells in hydrogels [START_REF] Drury | Hydrogels for tissue engineering: scaffold design variables and applications[END_REF][START_REF] Jen | Review: Hydrogels for cell immobilization[END_REF]. The highly hydrated structure of hydrogels allows for good mass transport properties [START_REF] Rowley | Alginate hydrogels as synthetic extracellular matrix materials[END_REF] so that nutrients, waste products, and signalling molecules can effectively perfuse the scaffold to maintain cell viability and thereby facilitate paracrine interactions.

Alginate is a polysaccharide that is derived from brown algae, which can be crosslinked by calcium in a mild gelling reaction to form a hydrogel, and is suitable for cell encapsulation [START_REF] Rowley | Alginate hydrogels as synthetic extracellular matrix materials[END_REF]. Alginate hydrogel has been successfully used for the encapsulation of a variety of cell types including hepatocytes, chondrocytes, osteoblasts and bone marrow stromal cells [START_REF] Khattak | Application of colorimetric assays to assess viability, growth and metabolism of hydrogelencapsulated cells[END_REF][START_REF] Alsberg | Cellinteractive alginate hydrogels for bone tissue engineering[END_REF][START_REF] Smith | 3D culture of bonederived cells immobilised in alginate following light-triggered gelation[END_REF][START_REF] Chang | Injection molding of chondrocyte/alginate constructs in the shape of facial implants[END_REF]. Alginate is approved by the Food and Drug Administration (FDA) for medical applications and has been used in wound dressings for around 20 years with no apparent toxicity. A number of wound dressings, including SeaSorb and Kaltostat, are currently commercially available [START_REF] Boateng | Wound healing dressings and drug delivery systems: A review[END_REF].

In this study, the use of an alginate hydrogel matrix to support the growth of fibroblasts and keratinocytes to facilitate healing of severe skin wounds has been examined. Primary rat keratinocytes and 3T3 fibroblasts were used as models for human autologous keratinocytes and fibroblasts, respectively. This alginate hydrogel seeded with both autologous keratinocytes and fibroblasts could be applied to a wound bed and should be capable of fully replacing both the epidermal and dermal component of the lost skin. The proliferation of the encapsulated fibroblasts should be inhibited to allow for the keratinocytes growing uninhibited upon the alginate surface to produce a stratified epidermal layer, whilst still receiving growth factors and cytokines that are supplied by fibroblasts to promote growth [START_REF] Auger | Tissueengineered skin substitutes: from in vitro constructs to in vivo applications[END_REF][START_REF] Clark | Tissue engineering for cutaneous wounds[END_REF][START_REF] Mansbridge | Threedimensional fibroblast culture implant for the treatment of diabetic foot ulcers: Metabolic activity and therapeutic range[END_REF]. This should be achieved by using a high-concentration alginate, where the encapsulated cells are mono-dispersed throughout the hydrogel, to mechanically confine the fibroblasts. This alginate scaffold should then degrade to allow for the proliferation of the encapsulated fibroblasts and secretion of ECM to replace the alginate hydrogel matrix and the migration of other cell types into the scaffold to restore functional and structural properties of the skin [START_REF] Mansbridge | Threedimensional fibroblast culture implant for the treatment of diabetic foot ulcers: Metabolic activity and therapeutic range[END_REF]. Unless otherwise specified all chemicals were supplied by Sigma Aldrich, Poole, UK.

Materials and methods

Encapsulation of NIH 3T3 murine fibroblasts

Low viscosity alginate cultures were maintained at 37ºC with 5% CO 2 and 100% relative humidity and media was changed three times weekly.

Keratinocyte cultures

Keratinocytes were obtained from neonatal rat sacrificed by cervical dislocation. The keratinocytes were isolated using 0.25% trypsin-0.02% EDTA and mechanical 2.25% v/v HEPES, 2% v/v L-glutamine as monolayers, with the support of a fibroblast feeder layer prepared by treatment with 4 mM Mitomycin C for 2 h at 37ºC, until a sufficient number of keratinocytes were obtained, as determined by counting using a haemocytometer. The keratinocytes were seeded at a density of 0.2 × 10 6 cells/cm 2 onto the surface of alginate containing fibroblasts. The keratinocytes were cultured for a further 7 days on the alginate scaffold before being raised to the air-liquid (A/L) interface to allow for stratification of the keratinocytes. All keratinocyte cultures were maintained at 37ºC with 5% CO 2 and 100% relative humidity.

Live/dead staining

Calcein acetoxymethylester (calcein AM) and propidium iodide (PI) (Invitrogen, Paisley, UK) were used to stain live cells green and dead cells red, respectively, when visualised using fluorescence microscopy (Carl Zeiss, Axiolab, UK). The 1-mm thick sections were taken from the centre of alginate beads containing 3T3 cells using a razor blade and immersed in 0.2 µg/mL calcein-AM for 15 min and 2.5 µg/mLl PI for 5 min in DMEM at 37ºC before examination with a fluorescence microscope (Carl Zeiss) and capture of images (Canon Powershot GS, Surrey, UK).

MTT assay for fibroblast growth

A stock solution of 5 mg/mL MTT in PBS was added to supplemented DMEM at a concentration of 10% v/v. Samples of alginate/3T3 beads were removed from the media, washed in DMEM and immersed in the resulting 10% solution of MTT for 18 h. The MTT solution was then removed and replaced with HCl-isopropanol and left for 2.5 h to allow the formazan produced to dissolve and diffuse out of the alginate. The optical absorbance of the solution at 620 nm was measured using a spectrophotometer (Cecil, Cambridge, UK). This amount of formazan produced by cellular reduction of MTT was determined from the A at 620 nm according to a calibration of different cell concentrations in alginate, as determined by counting on a haemocytometer, and the A at 620 nm after application of the MTT assay.

Histology

An adult rat was humanely sacrificed by cervical dislocation before skin removal.

Alginate encapsulated cells and skin samples were fixed in formalin buffer for 24 h before progressive dehydration in cassettes (Thermo Shandon, Citadel 1000, UK).

The dehydrated samples were then embedded in paraffin wax (Sakura, Tissue Tek, TEC) and stored at room temperature. Samples were then cooled to 5°C and 5-µm sections were taken using a microtome (Leica RM 2035), which were attached to glass slides and dried at 60°C in an oven for 1 h before removal of the wax in xylene and staining with Haematoxylin Van Gieson (HVG) (Merck, Nottingham, UK)

according to a standard protocol to demonstrate presence of collagen [START_REF] Jones | Connective Tissue Stains[END_REF] or

Haematoxylin and Eosin (H&E) (Surgipath Europe, Peterborough, UK) to demonstrate tissue architecture [START_REF] Gamble | The Haemotoxyllins and Eosin[END_REF]. Coverslips were then applied to the slides using XAM (BDH Laboratory Supplies, Poole, UK) and images of the slides obtained with a light microscope (Carl Zeiss) and captured (Canon Powershot GS, Surrey, UK). The images were then corrected using Image J software (NIH, MD) to remove any uneven illumination from the image.

Statistical analysis

Statistical significance (p <0.05) between test groups was determined by one-way analysis of variance (ANOVA) and Tukey post-hoc test (SPSS v.17, Chicago, IL).

Results

To evaluate the effect of long-term encapsulation of fibroblasts in 5% w/v alginate hydrogel, the encapsulated cells were stained in situ with a Calcein-AM/PI live/dead stain. The encapsulated fibroblasts were showed to remain viable for the duration of the study (28 days post encapsulation) in 5% w/v alginate (Fig. 1). The cells were evenly dispersed throughout the alginate matrix and after 28 days there had been no evident change in either cell viability or number. The formation of cell spheroids was not detectable in any of the samples. Cell number, as quantified using the MTT assay (Table 1), did not change significantly over the duration of the study (p >0.05), with very little fluctuation from the initial cell seeding density (7.5 × 10 5 mL).

((Figure 1)) ((Table 1))

To determine whether the encapsulated cells had deposited ECM during the encapsulation period histological sections of alginate-encapsulated fibroblasts were stained with HVG and compared with the normal dermis of rat skin. In Fig. 2a the distribution of cells and collagen in the dermis of normal rat skin can be seen. The nuclei of cells stained blue-black due to the haematoxylin, demonstrating the cell distribution, whilst the Van Gieson solution stains the collagen pink. It can be seen that the dermis has a low cell content and that the cells are present in small clusters or individual units and the main component of the dermis is collagen, which is present in small bundles of around 10-35-µm diameter. In Fig. 2b the cells encapsulated within the alginate matrix remained as dispersed units within the hydrogel after 21 days of encapsulation and had not proliferated or migrated within the gel to form cell clusters as seen in the dermis of normal skin so the cell concentration was lower in the hydrogel than in normal rat skin. The haematoxylin also stained the alginate fibres in the hydrogel blue-black due to their negative charge and the positive charge of the haematoxylin [START_REF] Jones | Connective Tissue Stains[END_REF][START_REF] Gamble | The Haemotoxyllins and Eosin[END_REF]. After 21 days, there was no evidence of collagen, which indicated that the encapsulated fibroblasts fail to secrete ECM during this time.

((Figure 2))

The dry mass of the tissue engineered dermis consisting of 5% w/v alginate and fibroblasts was shown to decrease over 33 days, so that by day 33 around 80% of the original mass remained (Fig. 3). It is evident that by reducing the alginate concentration to 2% w/v the rate of degradation was increased and that by day 33 only around 50% of the original alginate remained. The cells appeared to play very little part in this degradation, as with both 2 and 5% w/v alginate, the degradation profiles are extremely similar for both acellular and cell-seeded samples. All four samples showed a significant decrease in dry mass between days 0 and 33 (p <0.05).

Degradation of the alginate hydrogel has been shown to facilitate the release of entrapped material [START_REF] Gombotz | Protein release from alginate matrices[END_REF]. In the present study, it was observed that as the alginate degraded the cells were released from the alginate matrix and attached to suitable substrates such as glass or tissue culture plastic in which the alginate-encapsulated cells were being cultured, as has been seen for fibroblasts released form a different alginate hydrogel after 14 days cultivation [START_REF] Smith | 3D culture of bonederived cells immobilised in alginate following light-triggered gelation[END_REF]. HVG staining illustrated that cells released from alginate hydrogel were able to secrete collagen on the surface of the glass slide to which the cells had attached, as before they were encapsulated (Fig. 4).

((Figure 3)) ((Figure 4))

The alginate-fibroblast dermal scaffold was seeded with primary neonatal keratinocytes and after 7 days the keratinocyte culture was raised to the A/L interface.

After 14 days at the A/L interface the resulting epidermal layer was examined.

Staining with H&E (Fig. 5) illustrated that the keratinocytes had stratified to form a 3-4 cell-thick layer, but no stratum corneum was observed.

((Figure 5))

Discussion

Encapsulated fibroblasts were shown to remain viable within the alginate hydrogel for up to 28 days post encapsulation. The long-term viability of other encapsulated cells has been demonstrated, for example with chondrocytes [START_REF] Schagemann | Morphology and function of ovine articular cartilage chondrocytes in 3-D hydrogel culture[END_REF] and human embryonic stem cells [START_REF] Siti-Ismail | The benefit of human embryonic stem cell encapsulation for prolonged feeder-free maintenance[END_REF] and two other fibroblast cell lines [START_REF] Chang | Growth of recombinant fibroblasts in alginate microcapsules[END_REF]. In the present study, the viability of cells in the centre of both 2 and 5% w/v alginate hydrogel beads of 3-mm diameter was observed (Fig. 1) and illustrated that there was no critical necrosis and that oxygen tension was high enough to support cell survival, in contrast with what has been found with other alginates and cell types [START_REF] Khattak | Enhancing oxygen tension and cellular function in alginate cell encapsulation devices through the use of perfluorocarbons[END_REF]. The non-aggregated fibroblasts remained viable within un-modified alginate throughout the duration of the study, in contrast with previous reports [START_REF] Khattak | Application of colorimetric assays to assess viability, growth and metabolism of hydrogelencapsulated cells[END_REF][START_REF] Schagemann | Morphology and function of ovine articular cartilage chondrocytes in 3-D hydrogel culture[END_REF][START_REF] Siti-Ismail | The benefit of human embryonic stem cell encapsulation for prolonged feeder-free maintenance[END_REF]. The encapsulated fibroblasts, however, did not proliferate throughout the 28 days (Table 1) and the cells remained as dispersed units and failed to form cell aggregates (Figs. 1 and3), which have been seen to form when embryonic stem cells were encapsulated in alginate [START_REF] Schagemann | Morphology and function of ovine articular cartilage chondrocytes in 3-D hydrogel culture[END_REF].

It is proposed that the lack of mitotic activity is due in part to the use of a relatively low cell-seeding density [START_REF] Dvir-Ginzberg | Liver tissue engineering within alginate scaffolds: Effects of cell-seeding density on hepatocyte viability, morphology, and function[END_REF], the encapsulation of dispersed cells rather than spheroids [START_REF] Bazou | Long-term viability and proliferation of alginate-encapsulated 3-D HepG2 aggregates formed in an ultrasound trap[END_REF], the use of a non-liquefied core [START_REF] Zhang | Proliferation, viability, and metabolism of human tumor and normal cells cultured in microcapsule[END_REF] and the use of high concentrations of alginate, which resulted in the fibroblasts being mechanically confined from each other by cross-linked chains of alginate. These results demonstrate that encapsulation in alginate effectively mitotically inhibits the fibroblasts without the need for treatment with Mitomycin C or gamma-irradiation, to allow for co-culturing of keratinocytes with the fibroblasts.

Evaluation by HVG after 21 days encapsulation of fibroblasts in 5% w/v alginate revealed that there was no collagen present, indicating that the encapsulation inhibited the fibroblasts production of ECM. It is proposed that this is because the fibroblasts sensed the confinement of the 3-D environment through cytoskeletal arrangement [START_REF] Discher | Tissue cells feel and respond to the stiffness of their substrate[END_REF], which altered cell behaviour in comparison with that exhibited in the 2-D environment [START_REF] Pedersen | Mechanobiology in the third dimension[END_REF]. In the body, fibroblasts only begin to secrete large quantities of ECM after injury [START_REF] Welch | Temporal relationships of Factin bundle formation, collagen and fibronectin matrix assembly, and Fibronectin receptor expression to wound contraction[END_REF], when the cells are released from the 3-D confinement of the damaged ECM.

The present study has shown that calcium cross-linked hydrogel degrades over time by acellular mechanisms. This is due in part to the fact that mammalian cells do not possess alginases to hydrolyse the alginate molecules [START_REF] Shoichet | Stability of hydrogels used in cell encapsulation: An in vitro comparison of alginate and agarose[END_REF] and the degradation is possibly due to the calcium cross-links being replaced by monovalent cations in the media, such as sodium or potassium, which cannot cross-link the structure or chelation by phosphate anions [40]. As the matrix degraded, the encapsulated cells were released [START_REF] Smith | 3D culture of bonederived cells immobilised in alginate following light-triggered gelation[END_REF], other cells present at the wound site should be able to migrate into the alginate hydrogel scaffold to assist the healing process and integration of the engineered tissue with the surrounding host tissue [41]. It was shown that degradation could be tailored simply by varying the alginate concentration (Fig. 3).

When the fibroblasts were released from alginate hydrogel they were able to adhere to glass and resume the normal activity that was observed upon 2-D culture, whereby the fibroblasts proliferate to cover the glass substrate and secrete ECM (Fig. 4), similar to when the dermis of normal skin is wounded [START_REF] Clark | Tissue engineering for cutaneous wounds[END_REF][START_REF] Welch | Temporal relationships of Factin bundle formation, collagen and fibronectin matrix assembly, and Fibronectin receptor expression to wound contraction[END_REF]. When the construct is implanted into the body the fibroblasts should eventually replace the degrading alginate with natural ECM. As the alginate scaffold degrades, other cells at the wound site should also be capable of migrating into the scaffold and this should facilitate reinnervation and angiogenesis of the site, which is important for the success of the treatment.

It was possible to culture keratinocytes on the surface of the alginate/fibroblast dermal layer, suggesting that this was an effective way of providing the keratinocytes with a fibroblast feeder-layer compatible with application to the wound site. Unlike the feeder-layer produced by irradiation of the cells or treatment with Mitomycin C, which ultimately causes cell death, the fibroblasts in the present study did not need to be continuously replaced, as encapsulated fibroblasts remained viable throughout the duration of the experiment (Table 1). Histological evaluation with H&E staining illustrated that the keratinocytes formed stratified layers in areas where the keratinocytes had attached, but the stratum corneum was not evident (Fig. 5). Calcium is a key regulator in keratinocyte maturation [42], but when keratinocytes were grown on the surface of the alginate hydrogel-based dermal analogue no terminally differentiated keratinocytes were observed, suggesting that the amount of calcium released by the degrading alginate scaffold was not significant to induce terminal differentiation of all the keratinocytes (Fig. 5). Improvement of cell attachment to the alginate could be achieved by modifying the alginate with adhesion-promoting ligands [START_REF] Rowley | Alginate hydrogels as synthetic extracellular matrix materials[END_REF]43], or by using an alginate with a higher content of guluronic acid In conclusion, alginate hydrogel may prove to be a desirable scaffold for tissueengineering skin. This tissue-engineered skin may have applications in the clinical setting for treatment of chronic skin wounds and in the pharmaceutical industry for high-throughput testing of drugs and pharmaceuticals. Further work should seek to evaluate how the construct behaves in vivo when seeded with autologous fibroblasts and keratinocytes.

We wish to thanks the EC for funding this project within the 6 th Framework (NanoBioTact -NC Hunt). We also wish to thank Sue Finney for her help with histology.

Deleted:

dissociation and cultured in 3 : 1

 31 DMEM:Ham's F12 supplemented with 50 mM hydrocortisone, 10 µg/mLl insulin, 25 ng/mL EGF, 10% v/v FBS (PAA), 1% v/v P/S,

2 . 5

 25 Determining alginate degradationDegradation profiles were obtained over 33 days for acellular and fibroblast encapsulating samples of both 2 and 5% w/w alginate beads of 3 ± 0.2-mm diameter under the normal culture conditions described above. The degradation was assessed by measuring the alginate mass after dehydration in a vacuum freeze-drier (Edwards, EF03, Sussex, UK) for 16 h. Samples were measured in triplicate and the mean and standard deviation for each data point were shown. The release of fibroblasts from the alginate samples was determined by visual examination of the culture dish using a light microscope 33 days after introduction of the alginate samples.

  residues [44], which should facilitate formation of a full-thickness epithelium over the entire surface of the alginate and upon implantation into the body. The additional growth factors and cytokines present at a wound should also facilitate formation of a more complete epidermal layer [45].

Figure 1 .Figure 2 .

 12 Figure1. Fibroblasts stained in situ with calcein AM/PI live/dead stain in 2% w/v and 5% w/v alginate at 3, 14 and 28 days post encapsulation. The evidence of spheroid formation or cell proliferation is visible. The viability of cells throughout the experiment was illustrated by the presence of mainly green cells and very few red cells. This indicates that the diffusion of nutrients and waste products through the matrix was sufficient to maintain the cells' viability and that the alginate scaffold was not necrotic.

Figure 3 .

 3 Figure 3. Degradation profiles of acellular and cell-seeded alginate. Samples were either of 2or 5% w/v alginate concentration and inclusion of cells in the scaffolds had no appreciable effect on the degradation profile. It is also apparent that the 2% w/v alginate degrades faster than the 5% w/v alginate.

Figure 4 .

 4 Figure 4. HVG staining of fibroblasts cells grown on glass coverslips. (a) Before encapsulation this reveals that fibroblasts cells secrete collagen. (b) When encapsulated fibroblasts are released from 5% w/v alginate after 21 days encapsulation the fibroblasts retain the ability to secrete collagen.

Figure 5 .

 5 Figure 5. Keratinocytes cultured on the surface of the alginate/fibroblast dermal analogue produce a stratified layer around 3-4 cells thick (a), as is seen in normal epidermis of skin (b). The presence of cell nuclei indicates that these are basal and/or suprasal cells which can undergo further differentiation to form the stratum corneum, which is not seen on the surface of the stratified layer (a).

Figure 4 -Figure 5 .Figure 1 ÂFigure 2 ÂFigure 3 -Figure 4 ÂFigure 5 .

 4512345 Figure 4 -Haemotoxylin Van Gieson staining of fibroblasts cells grown on glass coverslips. a) Before encapsulation this reveals that fibroblasts cells secrete collagen. b) When encapsulated fibroblasts are released from 5% w/v alginate after 21 days encapsulation the fibroblasts retain the ability to secrete collagen. ¶ ¶ Page Break Figure 5. Keratinocytes cultured on the surface of the alginate/ fibroblast dermal analogue produce a stratified layer around 3-4 cells thick (a), as is seen in normal epidermis of skin (b). The presence of cell nuclei indicates that these are basal and/or suprasal cells which can undergo further differentiation to form the stratum corneum, which is not seen on the surface of the stratified layer (a). ¶ ¶

Table Deleted :

 Deleted . Eiselt, P., Yeh, J., Shea, L. D., Mooney, D. J., Porous carriers for biomedical applications based on alginate hydrogels. Biomaterials 2000, 21, 1921-1927. Barrientos, S., Stojadinovic, O., Golinko, M. S., Brem, H. et al., Growth factors and cytokines in wound healing. Wound Repair Regen. 2008, 16, 585-601.

	showing that epithelial anti-apoptotic activity is present in 3T3 fibroblast-[40] Smidrød O, Skja˚k-Braek G., Alginate as immobilization matrix for cells.
	The authors have declared no conflict of interest. conditioned media. Curr. Eye Res. 1996, 15, 973-984. TIBTECH. 1990, 8, 71-8.
	((Funded by • EC, 6 th Framework (NanoBioTact))) [41] [42] Lansdown, A. B. G., Calcium: a potential central regulator in wound healing	Deleted: hydrogel
	in the skin. Wound Repair Regen. 2002, 10, 271-285.
	[43] Lawson, M. A., Barralet, J. E., Wang, L., Shelton, R. M. et al. , Adhesion and
		Deleted:
	growth of bone marrow stromal cells on modified alginate hydrogels. Tissue Eng.
		Deleted: adhesion
	Deleted: use Deleted: of Deleted: ¶ To conclude Deleted: tissue Deleted: tissue engineered Deleted: and also Deleted: Deleted: through-put Deleted: F o r P R e v i e w F o r P R e v i e w F o r P R e v i e w F o r P R e v i e w F o r P R e v i e w F o r P R e v i e w 2004, 10, 1480-1491. [44] Wang, L., Shelton, R. M., Cooper, P. R., Lawson, M. et al., Evaluation of sodium alginate for bone marrow cell tissue engineering. Biomaterials 2003, 24, 3475-3481. e e r e e r e e r e e r e e r e r e [45]

Table 1 .

 1 Mean number of viable fibroblasts per ml alginate and SD at each time point as determined by the MTT assay. The viable cell number remains relatively constant throughout the duration of the experiment.

			Deleted: -
			Deleted: Number
		Cell number ± SD (×10 5 )
	Day
		2% w/v alginate	5% w/v alginate
	3	7.5±0.64	7.2±0.78.
	14	7.6±0.68	7.8±0.78
	28	7.8±0.80	7.6±0.70
			Deleted: ¶
			Page Break
			¶
			Figure 2 -Haematoxylin Van Gieson
			staining a) of normal dermis of skin
			shows that there is a large presence of
			collagen in bundles of 10-35µm diameter,
			the main component of the ECM. b) of
			5% w/v alginate hydrogel 21 days post
			encapsulation of fibroblasts demonstrated
			no collagen. ¶
			¶
			Page Break
			Figure 3 -Degradation profiles of
			acellular and cell-seeded alginate.
			Samples were either of 2% or 5% w/v
			alginate concentration and inclusion of
			cells in the scaffolds had no appreciable
			effect on the degradation profile. It is also
			apparent that the 2% w/v alginate
			degrades faster than the 5% w/v alginate. ¶
			¶
			¶
			¶
			¶
			¶
			¶
			Page Break

Wiley-VCH Biotechnology Journal

Acknowledgments ¶ Deleted: of


John Villadsen
email: jv@kt.dtu.dk

The Sugar Industry-the Cradle of modern Bio-Industry

Keywords: Cane and beet sugar, Slavery and free labor, Vacuum pans, Crystallizers

Continuous sugar extraction. Sugar as a platform chemical

At first all the West European colonies were settled by white people, growing tobacco for export and tropical crops for subsistence. But within a period of 20 years from the 1640´s the small farms of the Caribbean islands had been converted into large sugar plantations. Dutch technicians who had developed the incipient sugar industry in Brazil were evicted as a result of the wars between Holland and Spain, and they gladly moved their expertise from Brazil to the British and French possessions in the Caribbean.

In the thirty years of transience from a sustainable economy, primarily based on selfsufficiency, to an economy based on a monoculture of sugar the Windward Islands experienced a dramatic ethnographic change, from societies predominantly of European extraction to societies where Negroes outnumbered Whites by up to 10 to 1.

Barbados is an example of the change. The colonization of Barbados in 1629 was financed by private merchant companies just as was the first colonization of Virginia and the Carolinas. They had access to sufficient capital on the North European markets to start sugar production on a scale that soon out-competed the poorly financed Spanish plantations. The small island of Barbados (460 km 2 ) became "the Jewel in the Crown" of Great Britain in the late 17 th century, creating enormous profits to planters and to refineries in England. In the 1650´s, the densely populated little island, on 745 large plantations worked by about 85000 slaves, remitted sugar to England worth over 3 million £ [1, p.112] . Land increased fabulously in price, from 400 £ for a plantation of 200 ha in 1640 to 14 000 £ in 1648. The trade with the island employed 400 ships, and in England small towns such as Bristol and Liverpool developed into thriving commercial centers for sugar refinery and for trade on the Triangular Route between England, West Africa and the colonies in the Caribbean. Goods manufactured in England during the first industrial revolution were sent to Africa, slaves to the Caribbean, and sugar to the increasingly sophisticated refineries in England. Nantes and Bordeaux in France owed their phenomenal growth in the 18 th century to the same trade and industries as Bristol and Liverpool 1 .

For almost two centuries, from 1620 and until after the Napoleonic wars, Mercantilism dominated European economic thinking. The Navigation Act of the 1650´s was an attempt by England to restrict the commerce with the New World to British ships. It spawned 30 years of wars between the Dutch and their British and French competitors until The Dutch finally acceded defeat in the late 1690´s. Thereafter, and until after the Revolutionary wars 1793-1815 France and Great Britain continued the struggle for trade dominance, using the Caribbean sugar islands as pawns and trophies. This struggle was enacted on the background of rivalries, between the individual possessions of the two nations. When a hurricane hit Antigua in the Leeward Islands, Barbados rejoiced because

References [START_REF] Williams | From Columbus to Castro. The history of the Caribbean[END_REF] and [START_REF] Hansen | Slavernes øer[END_REF] have greatly influenced the opinion of the present author as regards the initial rewards and the ultimate disaster of European activities in the Caribbean. Greed, cynicism and sanguinity on the part of planters and their European financers was finally punished when new technology exposed the inadequacy of the colonial economies. A shameful trail of human degradation and suffering is all that is left. Eric Williams (1911Williams ( -1981)), a renowned economic historian, was the first prime minister of independent Trinidad and Tobago, from 1962 to 1981. the price of Barbadian sugar increased. When in 1763 France finally conceded defeat in the Seven Years War it was happy to cede "the snowy acres of Canada" while retaining possession of the sugar islands, Guadeloupe, Martinique and St Lucia. But in reality the outcome of this particular land grab was mediated by the political lobby of planters from Barbados who feared an added competition on the protected British market from sugar grown in "new" British colonies in the West Indies [1, p.130]. In 1752 200 000 ha was cultivated in Jamaica, but 800 000 ha of easily cultivable land lay idle because the absentee planters living on English country estates saw no purpose of increasing production at the risk of a slump in sales price. During the last half of the 18 th century the French sugar cultivation on the 27500 km 2 virgin soils of their colony St Domingue, acquired from Spain in 1731 by partitioning Hispaniola at the present boundary between Haiti and the Dominican Republic, amounted to much more than the total sugar production from all British West Indian colonies. Rather than responding by increasing production in Jamaica or in their new possessions Trinidad, captured from Spain in 1797, and British Guyana, captured from the Dutch in 1799, the planters proposed the Government to invade St Domingue and destroy its sugar production. The British invasion of St Domingue ended in a costly fiasco, and a subsequent attempt by Napoleon to restore St Domingue to slavery based sugar production ended in a humiliating defeat of crack French troops to the insurgent slaves who managed to create in 1804 the first independent state, Haiti, in the Caribbean. Denmark, a midget in comparison with Great Britain and France, entered the fray when it bought St Croix from the French in 1733 (as a "reward" for cynically supporting the French in the war of the Polish Succession against Austria). The previous Danish possessions of St Thomas and St Jan were unsuited for intensive sugar production, but the gently rolling hills of St Croix were ideal [START_REF] Hansen | Slavernes øer[END_REF]. Expanding on previous French, Spanish and English cultivations on the island, by 1754 virtually its whole surface of 212 km 2 had been planted with cane (figure 1). At the end of the 18 th century sugar from St Croix was refined at 13 places in Copenhagen, and refined sugar accounted for more than 50 % of the total exports from Copenhagen to the Baltic Sea countries [3, p.408-9]. Although on a much smaller scale than that of Great Britain, the Danish slave trade from Africa to the Danish West Indies and the transport of sugar from the islands was the foundation of a fabulous mercantile adventure in the late 18 th century. Danish pretentions to play for high stakes in the trade game did, however, lead to severe frictions with Great Britain and eventually led to the disasters that hit Denmark during the Napoleonic wars, resulting in bankruptcy of the state and the loss of Norway.

In fact the old European sugar empires in the Caribbean were all doomed at the entry to the 19 th century. With few exceptions (one far sighted Jamaican planter introduced the steam engine in 1768) their production methods were still those of the 17 th century, based on animal, water or wind power, and with a "free" and inexhaustible supply of manpower, the slaves, to compensate for a lack of interest in technology development.

The imminent threat to the sugar islands was the abolishment of the slave trade (1807) in Great Britain which was slowly enforced around 1830. After emancipation of the slaves (Great Britain in 1833, France, Denmark and Holland in the wake of the 1848 revolutions) indentured labor from India and the Dutch East Indian colonies was conjured up to save the Caribbean sugar production. But the cost of transportation of workers and their families on 5 year contracts, the unwillingness of the 2. The growth and triumph of beet sugar during the 19 th century.

At the end of the Napoleonic wars the infant European sugar industry suffered a heavy setback with the resumption of import of colonial sugar. But the commitment was too large for the enterprise to fail. In France by 1811 32000 ha was planted with sugar beets of a new and better variety than Achards. In 1813, the year before the downfall of Napoleon, a climax was reached with a production of 3500 ton refined sugar in 300 refineries, of which the most prominent was Établissements Say, one of the predecessor companies of the present day French sugar giant Eridania Béghin-Say [START_REF] Anon | History of the company Erdania Béghin-Say[END_REF].

A yearly production of 3500 t sugar was not much compared to a French colonial production of 150000 t before the wars, but slowly the French sugar production increased due to better cultivation methods for sugar beets and to the adaption of rational sugar extraction methods which soon made the colonial production look antiquated. Between 1800 and 1840 the sugar content of the beet increased from 2-3 % to almost 10%. Tropical sugar had to be shipped to Europe at the high freight raters that prevailed before the advent of large tonnage steam powered ships led to a dramatic reduction of transportation costs. It had to be refined in Europe to satisfy the demands of increasingly Finally, the slave worked plantations in the West Indies operated under an increasing threat of riots and revolts that sometimes flared up and led to loss of property and life. Conversely, the work force of the sugar growers in North Western France became increasingly self confident, spurred by better education, exposure to "modern" technology, and the emerging trade unions. In 1839, when about 500 highly capitalized factories in France produced an annual 48 000 ton beet sugar [1, p.385], the domestic sugar interests simply became more important to the state than the colonial economies. Their spokesmen in the Chambre des Députés could outvote the deputies from the Départements d´outre mèr who represented the colonial planters, and taxation of domestic sugar was relaxed.

The British continued to import subsidized sugar from their colonies, from their Cuban business associates, and slowly also from Brazil. British beet sugar production was never seriously considered until the U-boat blockade of World War 1. It developed slowly between the World Wars, and it saved the country from sugar "famine" between 1939 and 1945. Now [START_REF]The European Sugar Sector[END_REF], the United Kingdom ranks fourth in European sugar beet production.

After 1840 Germany became a leader in the development of European sugar production. A protected beet sugar industry which was not ham-strung by obligations to colonial empires, rapidly spread from Prussia to other North German states, for example to the Kingdom of Hannover [START_REF] Wallbaum | Die Rübenzuckerindustrie in Hannover: Zur Entstehung und Page 16[END_REF]. Everywhere sugar beet cultivation led to a rapid development of the rural economies. At a time when the hoe was still used for tilling in many West Indian plantations worked by emancipated slaves or measly paid East Indians, the heavy plough or even steam driven ploughs were introduced. The beet pulp, the remainder of the sugar beet after extraction of the sugar, was dried and became an excellent cattle feed, rich in proteins, carbohydrates and minerals. This byproduct was almost as valuable as the sugar, and when cheap grain from North America and Russia threatened to disrupt the traditional rural economy it greatly assisted the development in North West Europe of cattle production for meat, and especially for dairy products. . The birth of a modern rural economy improved the national coherence and it employed previously underemployed rural workers in the sugar factories and in the subsidiary rural industries. The scale of production was small compared to the scale of modern sugar factories, but thousands became enrolled in the Industrial Society, creating a mutual dependence through trade and manufacture between country and large cities. Under the umbrella of heavy import tariffs the German sugar industry boomed after 1860, and with a production of more than 2 million t in1871 the domestic market was covered, and a gigantic export business started. Originally, taxation of domestic sugar was intended as an important source of internal revenue, but since the taxation on beet sugar was based on a hypothetic yield of sugar from the beet it became a powerful incentive for improvement of the whole sugar sector in the 1860´s. This was the driving force for an enormous capitalization of the German sugar industry. Between 1870 and 1895 it was "the tail that wagged the dog", and the policies of the Kaiserreich became increasingly subservient to the large industries, not least the sugar industry [START_REF] Vascik | Sugar Barons and Bureaucrats: Unravelling the relationship between economic interest and government in modern Germany 1799-1945[END_REF]. Internal sugar price was increased through taxation, and the revenue was used to subsidize export of sugar at cut-rate prices.

Around 1900 Europe, spearheaded by Germany, but with participation from Russia and Austria, sent more than 7 M ton sugar on the market, about 2/3 of the world sugar production. At this time the sugar economies of British and French overseas dominions were bankrupt. Indian sugar was seriously hurt, and even the capital intensive Cuban sugar production was suffering-in part, however, due to the disastrous destructions wrought on the country by the wars of independence in the late 1890´s. The United States, recipient of more than one third of German sugar export [START_REF] Vascik | Sugar Barons and Bureaucrats: Unravelling the relationship between economic interest and government in modern Germany 1799-1945[END_REF], started to impose import duties on sugar that more than equaled the subsidy given to export sugar from Germany. There was a public outcry against German policy, such as "Big German Sugar Trust-said to threaten the industry of all other countries" [9]. These sentiments, although grossly overstating the facts, contributed to poison international relations in the years leading up to World War 1.

The United States had its own slowly developing beet sugar industry to protect, and the US also had to tender for the old slave grown sugar from Louisiana, Hawaiian sugar, grown and harvested by coolies from the Philippines, and sugar from the new dominion, Puerto Rico. Semi-independent Cuba, run by US capital, also sent huge quantities of sugar to the US market.

In the end international opinion, led by an outraged British free-trade-economy (with "special obligations" towards the British Empire!), forced the Brussels convention of 1902 on all the sugar growing countries [START_REF] Vascik | Sugar Barons and Bureaucrats: Unravelling the relationship between economic interest and government in modern Germany 1799-1945[END_REF][START_REF] Batista | Politica tarifárica britanica e evolucao das exportacoes brasilieiras na primeira metade do século XIX[END_REF]. All export subsidies were abolished and import duties reduced. The result was a collapse of the German sugar industry which had exported more than 50 % of its production, but Russian and Austrian sugar was hit equally hard. On the positive side the German government broke the Sugar Trust (die Zucker Kartellen) which had held the strings of German fiscal policy, and which had become a general threat to the very important German export to the British market.

The policies of the large European sugar producers, especially Germany, had some very interesting repercussions for the sugar industry in the smaller countries, Holland, Sweden and Denmark. Denmark is an illustrative example. Here beet sugar production was introduced in 1872 as part of the belated industrial revolution that followed in the wake of the calamitous war of 1864 with Germany and Austria. In the midst of a deep set national disillusion a few far-sighted Danish businessmen, led by the industrialist and international banker Carl Frederik Tietgen (1829-1901), built up a Danish industrial empire modeled on British limited companies and heavily financed internationally. The reasons for its survival during the onslaught of cheap, heavily subsidized sugar from Germany are manifold, but important factors were the corps d´ésprit of well educated Danish growers, a close cooperation between industry and Danish academic institutions, and finally the remarkable resilience of DDS which was vertically and horizontally integrated in the whole pre-World War 1 Danish industrial structure.

The contributions of Danish technology to the development of sugar production, not only from beet but also from cane, will be included in the next section. 70 years of continued technological development made Danish sugar production the most effective in the world during the last half of the 20 th century. It was an important contributor to the spread of Danish Engineering and later Danish Biotechnology to the rest of the world.

Decline of beet sugar through the globalization of trade at the end of the 20 th century.

In the decade from the Brussels convention to the 1 st World War Central European-and to a certain degree French sugar industry-was in disarray. The German sugar trust fell apart, infighting started between overproducing companies, and bankruptcies were common in an industry which had lost its cutting edge technological supremacy after 30 years of subsidies.

On the eve of World War 1 Germany produced only 2.7 M ton sugar, and the drain of workers from agriculture to the trenches from 1914 to 1918 spelt catastrophe for all the Central Powers and for Russia. French sugar was grown and processed in the belt of Northern France which was wrecked during the fighting on the Western Front. Thus in the harvest of 1919 Germany produced only 742000 ton beet sugar [START_REF][END_REF], and the picture was as bleak for the other belligerents of the World War. Neutral nations such as Denmark, Holland and Sweden could profit from the demise of the large European sugar producers, but the primary beneficiaries were the overseas producers. While beet sugar production decreased from 8.75 M tons in 1914 to 3.6 M tons in 1919 cane sugar increased from 9.8 M tons to 12.2 M tons in 1919 [START_REF][END_REF]. Cuba, Brazil, Peru, India, Dutch East India, Australia (Queensland), South Africa, and small islands in the Indian Ocean (Mauritius) and in the Pacific (Fiji) led the resurgence of cane sugar production. The World Crisis of the 1930´s hit all Western economies. Attempts by the Nazi regime to revive the German sugar industry mostly failed [START_REF] Vascik | Sugar Barons and Bureaucrats: Unravelling the relationship between economic interest and government in modern Germany 1799-1945[END_REF], and US beet (and cane) sugar industry was almost suffocated by importation of cheap sugar from the American financed "sphere of influence" in the Caribbean and on mainland Latin America [12 p.742]. Danish sugar industry was saved by a far sighted government policy that guaranteed sugar growers a reasonable income even during the hardships of the 1930´s and yet gave a small, but adequate margin for the shareholders of DDS. The sugar 2

Unfortunately this key reference to the establishment and early growth of the Danish sugar industry (DDS) is not translated. It is rich in detail and pictures and authored by people who participated during the first, difficult period. When peace came to Europe in 1945 it found the sugar industry crippled once more, especially in Germany which had lost a large portion of its sugar growing areas to Poland, and where the resuscitated sugar industry of DDR remained undercapitalized and inefficient, also after the reunification of Germany in 1991.

Towards the end of the 20 th century the global sugar production reached an astonishing 130 M tons per year from a level of about 20 M tons in 1920. The population increase in the Third World (India, China and Brazil) and increasing per capita sugar consumption were the obvious reasons. The main producers were, however, not the same as those of the early 20 th century. Brazil and India, each with 18 million tons were the leading producers. China with 8 million tons was an emerging giant, and the European Union with a production slightly below 18 million tons fought to keep its position. The US produced less than China and had to import sugar [START_REF] Zimmermann | International competitiveness in the sugar production[END_REF].

The reason for the dramatic resurgence of cane sugar is threefold: The cost of land for sugar growth is small in the big tropical countries, the obtainable yield of sugar per ha is higher and the growth season longer, and finally the cost of labor is much smaller. The full advantage of sugar cane cultivation was obtained by heavy capitalization of the tropical sugar industry and by transfer of all the technological improvements achieved in 150 years of European beet sugar production. The only exception is Cuba with an illustrious history of profitable sugar production since the mid 19 th century, and a world leader in sugar export before the revolution in 1959. Its attempt to increase the annual sugar production to 10 M tons in the nationalized sugar industry failed abysmally [START_REF] Hagelberg | Historical overview of Cuba´s cost of sugar production: Implications for the future[END_REF] despite a massive, loan-financed capital injection after 1962. Since the trade agreements with the USSR were abolished after 1991 Cuban sugar has fought to survive at a level of about 4 M tons per year. Sugar production in the second half of the 20 th century has not only seen competition between beet and cane sugar. Since World War 2 artificial sweeteners have gained some popularity (especially with diabetes patients) despite the questionable health issue of ingesting organic chemicals instead of a "natural product", sugar. Especially aspartame, cyclamates and sorbitol have obtained about 15 % of the market share for sweeteners since their introduction in the 1960´s. They are supposedly risk free compared to saccharine, and they appeal to the present day "less-sugar" philosophy.

Of much more serious consequence to the sugar industry is the development of "isoglucose" (a term used by the EU) by liquefaction of corn starch (HFCS, High Fructose Corn Syrup). In 2006 HFCS is on a par with sucrose in the US [START_REF]The European Sugar Sector[END_REF] and covers a third of the sugar consumption in Japan, Korea and Canada. In Europe production of and use of HFSC for soft drinks and in the bakery has until now been modest with an annual quota of 300 000 tons, but it may increase as a result of the drastic decline of the EU sugar subsidies after 2007-unless, of course, the price of grain increases immoderately due to the lingering food crisis.

From the start the European Common Market subsidized European agriculture, and in particular the sugar industry with import duties and even with export bounties that amounted to a light version of the sugar regime before the Brussels agreement of 1902. A quota system by which each member state of the EU was allowed to produce a certain amount of sugar at guaranteed sales price was instituted in 1968 and continued basically unchanged until 2006. "A fair income for EU producers and self-sufficiency" [START_REF]The European Sugar Sector[END_REF] was the guiding principle. There was an A-quota, determined by an estimate of domestic consumption, and a B-quota aimed at export opportunities. From 1993 to 2007 the guaranteed minimum price to growers was 46.72 and 32.42 € per ton beet used to produce respectively A and B quota sugar. The "intervention", i.e. guaranteed minimum price to producers, was 631.9 and 523.7 € per ton for white and "raw" sugar respectively. Although the net sugar export from the EU (2.8 M tons in 2004/5) is minute in comparison with an export from Brazil of more than 11 M tons, the subsidy by a domestic price 3 times higher than the world market price has long been untenable. In 2005 a case was brought to the WTO by Brazil, Australia and Thailand. A WTO panel ruled that the so called C sugar exports at world market price was in fact subsidized by revenues from production under A and B quotas while sugar from third party countries was denied access through import duties. This is the background for the new sugar regime in the EU. The A and B quota sugar distinction disappears, and over a 4 year period from 2007/8 to 2010/11 the intervention price for both white and raw sugar is lowered by 36 % to 404 € /ton for white sugar, and the price to growers will decrease to 26 € /ton beet. At the same time the preferential treatment of sugar import from the former colonial empires in Africa, the Caribbean and the Pacific (ACP countries) is abolished, and import duties from third party countries are significantly lowered.

The immediate effect has been in the ACP countries. Thus, since 2007 sugar production has ceased in Trinidad and Tobago, a traumatic experience for a nation that had "cane is my life" as its motto. Luckily in Trinidad and Tobago the oil and gas industry has long since replaced sugar as a major source of national income, but Jamaica and the French islands have nothing to replace sugar except the uncertain tourist industry.

The repercussions for the 325000 sugar growers in the EU are also significant, and with subsidies from the EU the growers have to reduce the cultivation area. Since the late 1980´s the sugar producers have prepared for an inevitable change by consolidating their positions, basically by acquiring sugar quotas from less efficient countries in the EU. The case of Denmark is typical: In 1989 DDS bought the single Danish coop-sugar factory, and within a few years Danisco, the successor company to DDS, bought Swedish and Finnish sugar, and had acquired the derelict sugar production in Mecklenburg-Vorpommern. The 8 factories of this Bundesland were reduced to one, state-of-the-art factory in Anklam where the capacity has increased from 3000 to more than 10000 t sliced sugar beets per day. With a later acquisition of Lithuanian sugar Danisco produced 1 M ton sugar in 2007, more than twice the sugar quota 391000 t for Denmark. Finally, in July of 2008 Nordzucker in Germany bought the sugar division of Danisco, a clear sign that consolidation of European sugar production is still in progress.

The turbulence in the sugar industry exemplifies a revolution of the whole agricultural sector. The position of "sugar" in the 21 st century and the opportunities that are unfolding for the best of the traditional sugar producing companies naturally relies on strategic corporate decisions, but the fund of technical and scientific knowledge accumulated in the sugar companies is the foundation on which success will grow. 11 Some of the technological developments that came from the sugar industry through the past centuries will be reviewed below. In particular the entrepreneurship of the sugar industry will be a strong asset when sugar joins with the fermentation industry to take a leading role in a future that is much less dependent on oil.

The legacy of the sugar industry to the world of tomorrow With an annual sugar production hovering around 160 million tons the global sugar industry is certainly not in recession. The European share in the global production will continue to decrease, but as discussed in the previous section there has been enough time for the beet sugar producers to change their focus. The foremost asset of the sugar industry is now its high technological standard, a standard that has been continuously improved through 170 years of sharp competition.

The historical contribution of the sugar industry is that it fundamentally changed rural life. Large sugar factories emerged in small country towns, and they employed thousands of workers who might eventually use their experience to establish new industries. Roads and railways contributed to improved communications in rural areas which experienced a rapid accumulation of capital and an improved status on the national scene. It was on the large acreage farms owned by well educated, progressive farmers that the economic benefits of sugar production were reaped. The social structure in the democratic countries of Northern Europe did, however, prevent the formation of a class of impoverished rural workers, and cooperatives could sometimes compete on equal terms with public companies backed by large financial institutions. This was, and perhaps still is, the fundamental difference between European sugar producers and cane sugar producers in India, in Cuba or Brazil where absentee owners from the US or Europe did not encourage local participation at a level higher than measly paid unskilled work. Sugar cane growth gave subsistence living for thousands, but the trickle-down effect of the wealth generated to the owners materialized at a snails pace. Unions were not fighting for the rights of workers, and the misery of migratory workers exploded in frequent and bloody riots. During the first 80 to 100 years of European sugar production, the situation was of course not much better, and migratory workers from especially Poland flocked to till the fields for cultivation of sugar beets and to do the arduous work of weeding the rows of beet plants. Still, European (and North American) science contributed significantly to lessen the toil of sugar production. The sugar beet naturally produces a multiple-germ seed ball which results in several seedlings emerging from one seed ball. Removing all but one seedling from a plant in order to get one large, healthy beet drastically reduced the cost of labor in the field and gradually made the intolerable use of underpaid workers obsolete.

In the US and in several European countries intensive research programs were carried out to produce single germ seeds. Since the early 1940´s mono-germ seeds (discovered by two Russian plant scientist, Dr and Mrs V.F. Savitsky who worked in the US), combined with mechanical seed planting are milestones in this progress. Other agricultural scientific programs such as the production since 1950 of polyploid seeds by Maribo Seeds, a subsidiary of DDS, have led to beets with high sugar content (18-20%), larger beet yield per ha (up to 50 tons/ha), and also to beets with resistance to a number of plant infections. One could have hoped that the Round-up resistant sugar beets, first successfully used in pilot scale by Maribo Seeds during the 1980´s, would have been met with political acceptance in Europe. The total use of herbicides is diminished, the plant protection and fertilizer programs are optimized, and relatively innocuous chemicals can be used. Plant growers in the US and elsewhere appear to have obtained significant advantages by GMO techniques, also in the cultivation of sugar.

The huge efforts of agricultural scientists to improve the value of the crop have run in parallel with efforts to improve the productivity and yield in the sugar factories.

In the highly competitive environment of the mid-19 th century where beet sugar producers fought, not only amongst themselves but also with large cane sugar producers such as Cuba, a number of immensely useful inventions were made.

The open pan evaporation that preceded crystallization of sugar had a history dating back to the Antioquity. The high temperatures used, and the almost complete lack of process control led to vast losses in white sugar, and the cost of wood rose unacceptably, even to the point of bankrupting some of the almost denuded tropical sugar islands.. Also of course, open-pan evaporation was decidedly unhealthy for the workers who stirred the broth in a foul smelling environment. The closed, steam heated pan evaporators working under partial vacuum were first developed in the sugar industry around 1810, and by 1850 vacuum pans were widely used. The first patent on a multi-effect evaporator system in which steam from the first stage is used at a lower pressure to evaporate almost the same amount of water in the next stage, was granted in 1846 to a French-American engineer, Norbert Rillieux [START_REF]Evaporating Pan" to N.Rillieux[END_REF].

The sugar industry contributed heavily to the development of industrial centrifuges in the 1880´ s, hereby reducing the time to collect sugar crystals from the viscous supernatant from hours to minutes. A number of patents originating from the sugar industry for filtration equipment demonstrate the role that sugar played for another key unit-operation in chemical and biochemical industry. Add to these specific pieces of equipment all the inventions made for transportation of beets in the factory and for cutting up the beets in regular sized slices that could withstand the extraction process and at the same time permit almost complete extraction of the sugar. Finally, numerous inventions in cost effective heat exchangers and evaporators, an early and detailed application of in-depth process monitoring, process control and process optimization, and scores of ingenious inventions that could give small, but still significant improvements in the large scale.

Most of these inventions were made in the beet sugar industry, first in Europe and somewhat later, but often greatly improved in the US. Cane sugar inherited these technological advances during the 20 th century and, combined with favorable growth conditions, and with what some people claim to be access to "unlimited new growth areas", the road towards dominance of the cane sugar industry is free. The progress in sugar chemistry, biotechnology and in mechanical skills is evident from the thousands of scientific papers and monographs that have been published, often by people who worked in the industry and who patiently struggled to improve the processes for which they were responsible. Two examples will be given of individuals who made lasting contributions both to the sugar industry and to the engineering sciences in general. The examples are taken from In 1940 the DDS engineer A. Brieghel-Müller gave the theoretical background of the lime treatment of raw sugar juice in his doctoral thesis [START_REF] Brieghel-Müller | Über Filtrationsuntersuchungen[END_REF]. Raw juice contains invertsugar (glucose + fructose), amino acids, proteins and other N-containing compounds that will give rise to discoloration of the final sugar. Polysaccharides (pectin from frost damaged beets) will lead to severe filtration problems. "Liming", i.e. the treatment of the juice with Ca(OH) 2 , will eliminate many of the impurities as coagulates. But due to the complicated nature of the juice, liming is not just one process, but it consists a series of steps: Pre-liming, main liming, saturation with CO 2 etc. The process has been studied, at least since the 1870´s, and a catalogue can been compiled of many hundred juice purification methods, often one for each factory. Brieghel-Müller´s contribution was that he provided the chemical and physical-chemical foundation for the empirically derived recipes. As a life-long employee at DDS he materially contributed to make Danish sugar the purest sugar on the market, with little loss of either quantity or quality. 10 years later another DDS engineer, Henning Brünniche-Olsen, was granted patent on the horizontal sugar extractor [START_REF]An apparatus for continuously mixing together a liquid and one or more substances, GB patent N o 674.794[END_REF] that revolutionized the sugar industry. When cane is milled in the classical way a very large fraction of the sugar remains in the bagasse. Beets cannot be milled at all. Hence batch extraction, and later continuous extraction of sugar was developed in the beet sugar industry. Brünniche-Olsens invention perfected the continuous, counter current extraction of sugar from beet slices (cosettes). The complicated, but robust machinery is able to displace 97 wt % of the sugar in the beet with the same amount of water, giving an extract to the evaporators with about 14 wt % sugar, i.e. a mass flow that is only about 25 wt % higher than the beet treated in the extractor.

The DDS-extractor was an immediate commercial success. Its very high yield of sugar and very low energy cost (high initial sugar concentration in the juice) made it the extractor of choice in about 50 % of sugar factories world-wide. Variants of the continuous extractor, such as a vertical extractor (more complicated, but with a capacity of 10000 t beet per day compared with 3500 t per day for the standard DDS extractor) were also marketed, and in a period from about 1955 to 1980 engineers from DDS were retrofitting sugar factories all over the world, for cane as well as for beet sugar. In an attempt to combine sugar production with cultivation of other farm products a mini-sugar factory (300 t /day) with a DDS extractor was developed for small scale cane sugar production in Third World Countries. It was delivered in crates at the site, ready for immediate assembly, a remarkably simple solution for e.g. rural districts in Tanzania.

As was the case with Brieghel-Müller´s work, the DDS encouraged publication both of the scientific background of the invention and of many technical details of the DDS extractor. The doctoral thesis of Brünniche-Olsen [START_REF] Brünniche-Olsen | Solid-Liquid Extraction[END_REF] The whole adventure of bio-fuels started in the sugar industry. The technical expertise required to run a sugar factory profitably was seamlessly transferred to another large scale production, ethanol by fermentation. Behind the smoke-screen of politically motivated subsidies to the bio-ethanol industry one finds a true story of massive cuts in production costs that may really make bio-ethanol cheaper than oil-based transportation fuels. The sugar companies have not only understood to design and operate the fermentation/downstream processes, but they have also embraced the technologies of Molecular Biology. Thereby the vital upstream design of a yeast or a bacterial strain to give both a high selectivity for the desired product and a high productivity per unit capital are considered on a par with the fermentation and the downstream processes. The highest potential for application of this fund of knowledge may not necessarily be in the food industries where regulations will hamper the use of GMO, at least in some parts of the world. The "old" chemical industry could be the most natural partner for a "new" sugar industry. Together the two partners possess expertise in all the fields needed for success: Chemical Process Technology, Food and Ingredients production, and finally in Modern Biology. They wish to establish production of bio-ethanol, not just develop an enzyme based process route. At the end of the day this might prove a better strategy than that of other companies which (perhaps out of necessity because they lack the financial strength) prefer to sell or license individual processes to a final producer of the commodity.

Very recently [22] another alliance was announced, this time between Danisco and Goodyear. The goal is to produce poly-isoprene for the tire and rubber industry.

The press release states that by 2013 the two companies will have an industrial process to produce bio-based isoprene as an alternative to petroleum-based production of this key compound for the rubber industry.

As strange as it may seem to a layman that one can produce "tires by a fermentation process" the biochemical role of isoprene (2-methyl-1,3-butadiene) in cells is of course well known. The companies are at the present mute about their process which is probably still in the process of being patented, but one may reasonably assume that the mevalonate 15 pathway has been amplified, and that one of the intermediates such as isopentenylpyrophosphate is being polymerized to the desired polymer, 4-poly-isoprene. This would be analogous to the mechanism by which high molecular weight hyaluronic acid is synthesized and excreted by bacteria.

Conclusion

At the entry to the 21 st century the sugar industry appears to have a bright, but unforeseen future which is not necessarily based on producing sugar, but rather on the application of its accumulated technological base, pared with acquisition of new expertise in Biotechnology. The Biorefinery is a highly publicized synomym for all the commodity products that can be made from carbohydrates. Bio-fuels can and will eventually be made from cheap lignocellulosic biomass, but for the more sensitive and higher value added products such as polymers sugar is the right raw material since the risk of incurring high downstream costs are significantly lower than from a complex raw material.

Here is the challenge and the opportunity for the sugar industry all over the world. [22] Danisco, (and Genencor) press release, September 16/17, 2008.

Figure captions

Figure 1 "Kort over Eylandet S t Croix udi Amerika" ("Map of the island of S t Croix in America")

Copenhagen 1754 (Royal Library, Copenhagen 4340111-0-1754/3). Reproduced with permission.

The intense sugar cultivation [START_REF] Hansen | Slavernes øer[END_REF] on this small (212 km 2 ) island in the Danish West Indies is clearly seen from the figure. Fredrichstead and Christianstead are now tourist attractions in the American Virgin Islands. Sugar cultivation ceased many years ago.

Figure 2

The first manual of sugar beet production, [START_REF] Achard | Der neueste deutsche Stellvertreter des indischen Zucker, oder der Zucker aus Runkelrüben-der wichtigste und wohlthätigste Entdeckung des 18. Jahrhundert[END_REF].

The title translates to "The newest substitute for (West) Indian sugar, or sugar from beetsthe most important discovery of the 18 th century" Royal Library, Copenhagen. Reproduced with permission.

  The sugar industry: The cradle of modern Bio-Industry ((Biotech Highlight))

  to keep 1/3 of all profit derived from reduction of production costs. This prepared the way for accumulation of capital in the industry, and the technical know-how accumulated in DDS was the key to the expansion of Danish sugar technology to the rest of the world after 1950.

13 the

 13 Danish sugar industry, but certainly the examples could also have been taken from any of the other front-runners in the sugar industry.

  Here the successful outcome of the co-operation between DuPont, Genencor and Tate & Lyle to produce, since 2006/7, 45000 tons/year of the intermediate 1,3-propane diol for the new polyester Sorona® has probably opened up for many similar alliances. The polylactic acid polymer production of Cargill is another example of a sugar based agro-company that has invested heavily in traditional chemical industry. The 2008 alliance between DuPont and Danisco [21] by which the two companies will invest heavily in the race to develop a commercially attractive second generation bioethanol production is made possible by the acquisition of Genencor by Danisco in 2005.

  Danisco, Annual report 2007/8, Copenhagen, May 2008.

  themselves to death in the sugar plantations, and the steady attrition due to illness and death of the imported labor proved the failure of substituting cheap labor for capital and modern technology. The East Indian immigration from around 1835 has shaped the multicultural societies of the modern Caribbean nations with all the richness of their culture, but with a latent threat to their internal coherence. It did, however, not save the sugar economies of the British, French or Dutch Caribbean dominions.

	laborers to work Having argued their case for fifty years, planters from the Indian subcontinent finally
	convinced Parliament, against fierce opposition from the West Indian lobby, that Indian
	sugar should be imported to England with the same preferential status as Caribbean
	sugar. This opened up for the import of huge quantities of cheap cane sugar from British
	India. In the Caribbean a massive influx of Anglo-Saxon capital to the Spanish colony of
	Cuba rapidly transformed Cuban sugar production. The sugar estates were extensive with
	areas up to several thousand hectares, and the sugar was extracted with the use of steam
	power in huge central factories that dwarfed the factories of Jamaica and Barbados. Thus F around 1850 sugar production (223 000 tons per year [1, p.361]) in Cuba surpassed the total production from the British West Indies, and by 1894 the Cuban production of 1.05 o M tons [1, p.366] was more than twice as large as that from the rest of the Caribbean r where even the large and fertile sugar colonies, British Guiana and Trinidad, had started
	their decline. The deadliest threat to tropical sugar was, however, the emergence of European sugar. P
	The first sample of refined sugar from sugar beets was presented to the Prussian king in e 1799 by Franz Karl Achard, director of the Prussian Academy of Science in Berlin, and grown on his own estates in Silesia. The first manual of beet-sugar growth was also from e 1799, figure 2 [4]. It is alleged that the British Government offered Achard a bribe of 30 r 000 £ to report a failure of his process [1, p.243], but events soon proved Achard right to uphold his scientific integrity. From 1807 the British blockade of Napoleonic Europe led R w e i v to a rapid establishment of large scale sugar production in Germany and especially in e France.
	5

  Tietgen created a Danish food and transport empire. Danish railways, a fleet of ocean going freighters and a harbor for export of Danish meat and dairy products, Esbjerg in southern Jutland, to compensate for the harbors in Schleswig-Holstein lost in the 1864 war. His Great Nordic Telegraph Company eventually monopolized the European telegraph communications with China and Japan, and finally, The Danish Sugar Company (De Danske Sukkerfabrikker, DDS) became a lighthouse for Danish Chemical Technology. With the democratic constitution of 1849 the Danish government had lost all interest in the Danish West Indies. The revenue from importation of Danish colonial sugar became inconsequential in the revitalized Danish economy, and the tropical islands were soon offered for sale. The post Civil War US showed an interest in acquiring Caribbean possessions, but Congress did not close the deal until 1917 when the completion of the Panama Canal in 1914 required a deep protective zone. enough to supply the Danish population with domestic sugar, even when the per person consumption increased from about 10 to 40 kg per year[START_REF] Drachmann | Aktieselskabet De Danske Sukkerfabrikker 1872-1922[END_REF] 2 .

	1890 this was
	F o
	r
	P
	e e r
	R e v i e w
	8

After a modest start (9000 ton beets on 375 ha, and a sugar production of 1000 tons) in 1874 the Danish sugar production reached 135000 tons grown on 6700 ha in 1912. After

  is an early example of a mature application of Transport Phenomena and Mathematical Modeling in Biochemical Engineering. A series of extraction experiments on cosettes give the parameters for a PDE model of the extractor, and the results are applied to design of full scale extractors. Several manuals or textbooks on sugar technology were later published by DDS[START_REF] Anon | but the principal author is R.Frik-Madsen) Om sukker og sukkerfabrikation hos DDS[END_REF], most of them authored by the CSO, Dr Rud Frik-Madsen, inventor of an UF/RO system that developed into a division of DDS. A remarkable feature of these books is that they also less successful DDS technologies, and give rather detailed advice on how improvements have been made or should be made. This publication activity of a major industrial company suggests that the company has total faith in the superiority of its approach. It is willing to share its current knowledge with other sugar producers, and perhaps through a demonstration of technological strength obtain further contracts for transfer of knowledge. It is the preparedness for change that is the conclusion drawn from the technical examples discussed above. The sugar industry is ready to enter other fields, not necessarily based on sugar grown and produced domestically, but based on the accumulated knowledge within the industry.

	freely discuss,
	F o
	r
	P
	e e r
	R e v i e w
	14

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