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Collagen fibres by thermoplastic and wet spinning
Materials Science and Engineering C 30 (2010) 1266–1271
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Materials Science and Engineering C j o u r n a l h o m e p a g e : w w w. e l s ev i e r. c o m / l o c a t e / m s e c
Collagen fibres by thermoplastic and wet spinning M. Meyer a,⁎, H. Baltzer a, K. Schwikal b a b
Forschungsinstitut fuer Leder und Kunststoffbahnen, Meissner Ring 1-5, D-09599 Freiberg, Germany Thüringisches Institut für Textil-und Kunststoff-Forschung e.V. Breitscheidstr. 97, D-07407 Rudolstadt, Germany
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Article history: Received 11 June 2010 Accepted 8 July 2010 Available online 14 July 2010 Keywords: Collagen Fibre Melt extrusion Wet spinning
a b s t r a c t Collagen threads of high yardage were manufactured by two different techniques being wet spinning of collagen dispersions and melt spinning of thermoplastic collagen. The fibres were characterised according to their structural, textile physical and biochemical properties. The wet spun fibres showed higher physical stability than the thermoplastic spun ones. Both fibre types were cross linked with different agents (formaldehyde, glutaraldehyde, and 1-ethyl-3-(3-dimethylaminopropyl)carbodiimid (EDC)) to increase mechanical stability as well as to lower susceptibility against enzymatic attack. Fibres cross linked by 0.1% glutaraldehyde as well as non cross linked fibres showed no cytotoxic effect against mouse fibroblasts. © 2010 Elsevier B.V. All rights reserved.
1. Introduction Collagen is still one of the most popular biomaterials though synthetic polymers became available for medical purposes in the last decades. Collagen combines very important properties. It is biocompatible, biodegradable, and hemostyptic; its biodegradability is being adjusted by cross linking. In tissue engineering applications the collagen's surface leads to good cell attachment, proliferation and differentiation of cells [1,2]. Therefore, collagen is widely used to manufacture medical devices e.g. membranes and sponges, to coat implants and as a solution for tissue augmentation in plastic surgery and as matrix material for cell culture [2–4]. Collagen is the major component of the extracellular matrix (ECM). Accounting for about 30% of the total body protein in vertebrates it represents the most important part of connective tissue beside water. Collagen comprises a group of different collagen types. Many of them are fibre forming others are building networks. The main fibril forming type in skin, tendon and blood vessels is collagen type I [5]. For most applications collagen is extracted from mammalian tendon or skin (porcine, bovine or equine sources). The usually highly insoluble raw material is treated with acid to obtain acid soluble collagen (ASC) or by pepsin digestion to improve yield (pepsin treated collagen, PSC). The concentration of these highly viscous acidic collagen solutions usually lies between 1% and 3%. In some cases collagen is not extracted from the raw material by dissolution but the raw stock is purified and minced completely
⁎ Corresponding author. E-mail address: michael.meyer@filkfreiberg.de (M. Meyer). 0928-4931/$ – see front matter © 2010 Elsevier B.V. All rights reserved. doi:10.1016/j.msec.2010.07.005
several times resulting in collagen dispersions [3,6]. These dispersions show higher solid contents and higher process yields. Solid collagen devices are usually made from collagen solutions or from purified collagen dispersions by casting and drying at room temperature leading to compact membranes or by lyophilisation leading to porous sponges [7]. These structures are usually undirected. To date it is a challenge to manufacture directed 2D and 3D structures, which could be very interesting for tissue engineering applications as well as for making textile implants. However, the possibility of making directed structures from collagen solutions and dispersions, respectively is limited. Other than synthetic polymers (polyesters, polyamides, polypropylene, and PTFE) or other natural polymers (silk, cotton) collagen is difficult to be formed into fibres of high yardage, because collagen does not consist of isolable long fibres. Some trials were performed in the past to manufacture threads from different collagen sources. Cavarallo et al. [8] described a process of precipitation of ASC, extracted from bovine tendon in solutions by polyethylene glycol 8000 followed by washing in a buffering solution and dewatering in isopropanol. The threads were cross linked to achieve suitable stability. In a series of papers Zeugolis et al. [9–12] described the preparation of threads (30 cm) according to a similar technique, and Vasilev et al. [13] showed that spinning of long collagen fibres was possible by extrusion of 1.5%–3% solutions of ASC in precipitation baths consisting of acetone and ethanol. The stability of the spinning process strongly depended on the collagen concentration. In contrast, Chanukov et al. [14] and Bienkiewicz et al. [15] used collagen dispersions prepared by extensive mincing with similar results. However, to date there are no high yardage collagen threads available. The so far described techniques used aqueous solutions or dispersions of collagen containing dry matter contents of 1% up to 10%.
M. Meyer et al. / Materials Science and Engineering C 30 (2010) 1266–1271
Recently, Meyer et al. [16] published a technique to process partly denatured collagen thermoplastically using a completely dry process. This thermoplastic collagen differs from gelatine by its low solubility in water at N35 °C. However, the thermoplastic collagen is already partly denatured having lost its fibrous structure. This material allows to be processed by the same machines as for processing thermoplastic synthetic polymers using temperature controlled processes. Our investigation aimed at manufacturing collagen threads with high yardage by two different but simple technologies namely wet spinning of collagen dispersion coupled with precipitation of the material as well as thermoplastic melt spinning. The resulting fibres of several dozen meters of length were characterised regarding their processing properties as well as regarding their structural, textile physical and biochemical properties. 2. Experimental part 2.1. Raw material Porcine and bovine skins were used as collagen raw stock obtained from a local abattoir. The skins were washed extensively and soaked in a solution of sodium hydroxide at pH 12.5 overnight. The swollen skins were split two times on a conventional splitting machine (Turner 537) used in tanneries. The middle split was soaked in ammonium sulphate solution to adjust pH by 7 and washed again with water.
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bath designed as a chute containing ethanol/acetone mixtures at different compositions. The threads were picked up at the end of the chute, air dried by a blow drier and wound up on a bobbin.
2.3. Thermoplastic granules and thermoplastic spinning According to the principle technology for preparation of thermoplastic collagen the split skins were denatured thermally by soaking for 10 min in boiling water, drained off excess water and loft dried [16]. The pieces were then ground to powder on a centrifugal mill (Görgens Engineering GmbH, Dormagen, Germany). This powder was mixed with 5 to 15% glycerol and 15 to 50% deionised water in a fast mixer resulting in granules to be fed to an extruder. Melt spinning was performed on a single-screw extruder RCP-0500 (Randcastle Extrusion Systems, New Jersey, US) coupled with a spinning pump, a stretching system consisting of primary rolls, secondary rolls and a heating section and finally take-up rolls. The extrusion temperature was adjusted in the range of 90 to 98 °C depending on the recipe to achieve applicable viscosities. The rotation speed was set between 50 and 120 rpm to get a processing pressure of 50 bar. The spinning pump was adjusted to 10 rpm. The collagen melt was formed to unifilar fibres by extrusion through nozzles with diameters of 0.3 and 0.5 mm.
2.2. Collagen dispersions and wet spinning
2.4. Material testing
The collagen dispersions were prepared from alkaline treated bovine and porcine splits. These splits were treated with 1% H2O2 solution, minced by a meat chopper, acidified to pH 4 by hydrochloric acid and further treated in a colloid mill (Cavitron, special design) several times to get homogenous collagen masses. The dry matter content of these masses was 1 to 2%. This dry matter content was too low to obtain stable threads, however. Therefore, the masses were adjusted to pH 5, concentrated by centrifugation on a tube centrifuge (special design) up to a dry matter content of 10% and acidified again by addition of low volumes of 1 M hydrochloric acid. This mass had to be aged for 24 h minimum by storage at 4 °C. The collagen dispersions were processed to threads by a cylinder spinning system (Biedermann & Wolschendorf OHG; Saalfeld, Germany) with a volume of 6 cm3 and a maximum force of 10 kN. The spinneret design depended on the experimental conditions. Cylindrical and conical nozzles with diameters between 250 and 500 μm were used. The spinning temperature was fixed in all experiments to 26 °C. The spun primary thread was coagulated in a
The fibre parameters such as tensile strength and elongation at break of the fibres were determined with the automatic testing system Fafegraph M (Textechno, Mönchengladbach, Germany) according to DIN EN ISO 5079 with 25 replicates for each sample. The analyses were performed at 20 °C and 60% relative humidity, with a feed of 10 mm/min. The fineness was investigated according to DIN EN ISO 1973. During SEM-measurements the fibres were fixed on sample holders and were subsequently coated with gold using a magnetron sputter coating device Polaron SC7620 (Quorum Technologies Ltd., Ringmer, UK). The measurements were carried out with a Leica S440i (Leica Microsystems GmbH, Wetzlar, Germany) at 20 kV. X-ray diffraction was measured on a Bruker D8 Advance diffractometer using CuKα radiation (λ = 0.154 nm) in transmission. The generator system operated at 40 kV. The manufactured collagen fibres were prepared for measurement by taking some pieces together in a parallel orientation and fixing them with cellulose nitrate. The aqueous dispersion was brought as film on a glass plate and dried on air.
Fig. 1. SEM-measurements of fibres manufactured with a conical spinneret from dispersions of porcine collagen (a) and bovine hide collagen (b) at the same magnification.
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lyophilised and weighed. Insolubility was calculated according to Insolubility [%] = m (residue dry, after enzymatic treatment) [μg] / m (dry, before enzymatic treatment) [μg] × 100. 2.8. Cytotoxicity Cytotoxicity was measured by a screening method, testing the inhibition of proliferation of extracts of the threads according to EN ISO 10993-5. The samples were washed two times with cell culture medium (DMEM-FBS) and further extracted with DMEM-FBS for 7 days at 37 +/− 2 °C. An “eluate” without sample acted as negative control and as positive control a solution of 7.5% DMSO in cell culture medium was used. All eluates were diluted with DMEM-FBS to 55.1, 36.7, 24.5, 16.3, 10.9 and 7.3 vol.%. The diluted eluates were plated out in 96-well plates, seeded with 50 μl of a freshly prepared cell suspension (73.5 × 104 cells of L929 mouse fibroblasts). The cells were incubated for 72 h at 37 +/− 2 °C (5% CO2; 95% air). Finally the protein content was measured photometric using BCA protein quantification test (Absorption A at 550 nm). The eluates showed cytotoxic behaviour, when proliferation inhibition (PI) was N30% calculated according to PI [%] = 100 − ((A550 sample)−(A550 blank))/ (A550 negative control)−(A550 blank)) × 100. 3. Results and discussion Two different collagen materials, namely thermoplastic collagen and collagen dispersion were processed to threads using wet and melt spinning techniques, respectively. 3.1. Collagen dispersion Fig. 2. WAXS reflexes (vertical — black; horizontal — grey) of a film prepared from dispersion before fibre forming (a) and the manufactured fibres (b). Peak 1 reflects the intermolecular lateral packing. Peak 2 results from amorphous scatter. Peak 3 shows the periodic axial rise per residue.
2.5. Cross linking Cross linking was performed by soaking the samples in aqueous solutions of glutaraldehyde and EDC (1-ethyl-3-(3dimethylaminopropyl)-carbodiimide), respectively. With formaldehyde the fibres were cross linked in gas phase by storing them overnight at room temperature in a tightly closed exsiccator which contained some milliliters of fresh formaldehyde solution (30%). This cross linking in gas phase allows to avoid wetting in aqueous solutions during cross linking. 2.6. Differential scanning calorimetrie (DSC) DSC is a common method to get hints about denatured and native parts of collagen as well as to measure cross linking. Samples of 1– 5 mg were put in aluminium pans, an excess of an aqueous solution of 0.9% NaCl was added and the pans were sealed tightly. The thermograms were recorded in a Perkin Elmer DSC 7 using a heating rate of 5 °C/min. 2.7. Enzymatic digestion Fibre samples were digested enzymatically to get hints about their degradation behaviour. For this, 100 μg trypsin was dissolved in 1000 μl H2O deion. Up to 1 mg of each sample was soaked in a 800 μl freshly prepared solution of 0.2 M NH4HCO3 in H2O deion and 200 μl of the trypsin solution was added. The vials were incubated at 37 °C for 2 h and 200 μl of the trypsin solution was added again and incubated for 2 h at 37 °C. Then, the samples were spun down, the supernatant was discarded and washed three times with H2O deion. Finally the centrifugate was
The collagen dispersion was prepared and formed to threads by a wet spinning technique as described above. The resulting wet strands were precipitated in a coagulation bath, dried and wound up on a bobbin. Various parameters influenced the stability of the fibres: the tenacity depended on the collagen source, the isolation method, the swelling behaviour, the solid content of the mass, the composition of the coagulation bath and the spinneret design. Fig. 1 presents SEM-pictures of fibres spun from porcine and bovine dispersions with a conical shaped spinneret. The porcine fibres (Fig. 1a) show a filamentous surface structure and the structural elements are oriented in parallel. On the contrary the bovine hide fibres (Fig. 1b) show structures which are irregular containing separated clew-like forms. The latter fibres were very brittle and broke easily when slightly touched. The native collagen fibrils of bovine skin are thicker and longer than the porcine ones [17]. Furthermore, because of the older age of the cattle compared to the pigs when slaughtered the bovine skin collagen shows a higher natural cross linking degree [18]. Presumably, these structural differences cause the difference in fibre appearance. Therefore, all further wet spinning trials were made with porcine material. Both, the manufactured collagen fibres as well as the film prepared from the dispersion showed the same WAXS reflexes (Fig. 2a, b). As described by Maxwell et al. [19] the reflexes correspond to the different structural units of collagen. Peak 1 (0.86 nm−1) represents
Table 1 Textile physical properties of threads made from collagen dispersions depending on the mixture of the precipitation agents ethanol and acetone (3.8% dry matter content; pH 4.0; cylindrical spinneret). Sample EtOH: Thread properties acetone Thickness Fineness [μm] [tex]
Tenacity [cN/tex]
sd [%]
P1 P2 P3
8.48 8.30 5.75
12.5 20.3 16.5 18.8 25.5 13.1
9:1 8:2 7:3
89 118 140
9.8 11.8 19.7
Elongation sd [%] [%] 19.47 24.3 36.6
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Table 2 Textile physical properties of threads made from collagen dispersions depending on spinneret design and dry matter content (pH 4.0; precipitation agent ethanol:acetone 9:1). Sample
P1 P4 P5
Dry matter content
Spinneret design
Thread properties Thickness [μm]
Fineness [tex]
Tenacity [cN/tex]
sd [%]
Elongation [%]
sd [%]
3.8 11.1 3.8
cylindrical cylindrical conical
89 179 170
9.8 28.1 20.0
8.48 7.12 4.65
12.5 12.5 22.5
20.3 28.5 14.6
19.5 15.9 35.0
scattering related to the intermolecular lateral packing of the collagen molecules. The broad reflex (peak 2) reflects primarily the amorphous scattering of the sample and peak 3 (0.29 nm) corresponds to the periodicity of the axial rise per residue or the distances between the Gly-X-Y repeats, respectively. The intensity of the scattering reflexes of the film manufactured from the collagen dispersion did not vary significantly with different scattering angles. However, the scattering signal of the processed collagen fibre showed different intensities depending on the angle. While peak 1 of the vertical measurement is much higher than that at the horizontal angle peak 3 disappears completely in a horizontal direction. Purslow et al. [24] described the WAXS signals of a single collagen fibril bundle compared with the reflections of a disperse population of collagen fibril bundles. They showed that the angle dependent scattering intensity depends on their orientation. Therefore, especially peak 1 reflecting the lateral packing of the collagen molecules clearly indicates an orientation of the collagen fibril bundles of the dispersion by the spinning procedure in axial direction. The tenacity of the fibres made from collagen dispersions depended on the composition of the coagulation bath (Table 1). By the use of ethanol (technical grade, 96%) no stable fibres could be prepared, whereas respectable stability was found after the addition of moderate amounts of acetone. In our experiments, adding of 10% (P1) and 20% (P2) acetone respectively, resulted in the best fibre stability (8.5 cN/tex or 8.3 cN/tex). However, if the acetone concentration was further increased the stability decreased again to 5.8 cN/tex (P3). In contrast, the elongation at break decreased with increasing acetone content during precipitation. It stood out that the standard deviations showed high values of 12 to 25% for tenacity and elongation reflecting still a low accuracy during manufacturing process. The mechanical properties of the wet spun fibres also depended on the spinneret design (Table 2). A cylindrical shape led to a tenacity which was twice (P1, 8.48 cN/tex) as much as that of a conical design (P5, 4.65 cN/tex), using the same spinning conditions. In addition, elongation at break decreased from 20.3% to 14.6% by using the conical spinneret. In our trials we adjusted fibre thickness and fineness, respectively by varying the dry matter content of the collagen dispersion to be spun.
Expectedly, increasing dry matter content led to higher thickness of the fibres and the tenacity decreased slightly with increasing thickness (P1 and P4, Table 2). These tenacities found in our experiments are in the same range found by other authors (4 to 12 cN/tex) [14,15,20]. Merely Vasilev et al. achieved higher values (20–23 cN/tex; 0,5 tex) by coagulation of acid soluble collagen in acetone/ethanol mixtures for 60 s at a spinning speed of around 1 m/min [13]. For comparison, synthetic fibres, which are used as degradable suture material show tenacities 3 to 6 times higher (30 to 57 cN/tex [21]) than those, which were found for the fibres prepared from collagen dispersions. 3.2. Thermoplastic collagen To obtain threads from thermoplastic collagen the prepared granules were fed into the single-screw extruder, melt on and pressed through the cylindrical spinneret. The fibres were directed over two wind up systems. By using different speeds of the rolls it was possible to achieve a draft of spun threads, being a common technique to improve the stability of polymer threads. In most cases this process was stable enough to wind up an “endless” thread. The monofils from thermoplastic collagen were not as stable as those prepared from the collagen suspensions. Anyhow, high yarded monofils could be manufactured and also used for further processing e.g. textile structures (Fig. 3). In the first trials the threads were embroidered on a polyvinyl alcohol carrier, which is one possible technique to manufacture textile structures from these threads. Dry threads of thermoplastic collagen behaved in a brittle way, but breaking was prevented by the addition of glycerol as plasticizer. With increasing glycerol concentration the tenacity of the threads decreased and elongation increased (Fig. 4). At concentrations of glycerol lower than 15% winding up and drafting of the threads were not possible. The most stable spinning conditions were achieved using glycerol contents between 25% and 35%. Without any draft only low tenacities (0.99–1.12 cN/tex) were obtained. Drafting of the fibres containing 25% glycerol up to a ratio of 1:2 led to an improved tenacity (1.58 cN/tex) and a slightly reduced elongation at break. Furthermore, glycerol is known as a humidifying agent for proteins [22,23]. In general, in our investigation the mechanical properties of
Fig. 3. TC fibres wound up on rolls (left) and embroidered on a polyvinyl alcohol carrier (right).
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M. Meyer et al. / Materials Science and Engineering C 30 (2010) 1266–1271 Table 4 Onset temperatures of DSC thermograms of wet spun and thermoplastically spun threads before and after cross linking (CL) with 1% glutaraldehyde.
P1 TC 1.0
Non CL
CL
38 ± 0.5 °C 32 ± 0.1 °C
56 ± 2 °C 40 ± 1 °C
3.3. Comparative measurements
Fig. 4. Tenacity (●) and elongation at break (▲) of TC fibres depending on the glycerol content.
Fig. 5. Tenacity (●) and elongation at break (▲) of TC fibres depending on the relative humidity of the surrounding atmosphere.
the threads were measured at 60% relative humidity (RH). To measure the dependency of the mechanical parameters on the humidity, the draught TC fibres containing 25% of glycerol were stored at different RH and measured again. Increasing humidity from 33% RH up to 78% RH led to a decrease of a quarter of the stability at low humidity (Fig. 5), the corresponding elongation at break increased from 55% to 93%, showing high standard deviations, however. Cross linking is often used to improve the collagen's mechanical stability and stability against enzymatic digestion and resorption [8]. Therefore, it was tried to increase the mechanical stability of the threads by an additional treatment with various cross linking agents like formaldehyde (FA) and 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide (EDC), respectively (Table 3). The low stability of the fibres could be improved by 3.5 to 4.5 times by cross linking and the corresponding elongation at break was reduced from 125% without cross linking to 39% by cross linking with EDC.
As a degradable biomaterial collagen is resorbed after being implanted or during its use in cell culture experiments. The resorption time depends on the several criteria amongst others nativity and cross link degree of the collagen but also parameters of the environment as pH, temperature, buffering concentration. Samples of both fibre types were investigated by DSC and by enzymatic digestion to correlate the nativity of the collagen, cross link degree and the susceptibility to enzymatic degradation. The nativity of the collagen of the wet spun threads could be confirmed (Tonset = 38 °C) in contrast to TC which showed a very small melting peak at low Tonset = 32 °C reflecting the situation of a highly concentrated gelatin gel. By cross linking with 1% glutaraldehyde the denaturation temperature of the wet spun fibres increased up to Tonset = 56 °C in contrast to the TC fibres reaching just Tonset = 40 °C (Table 4). To learn more about the susceptibility to enzymatic cleavage TC fibres with and without cross linking were digested in a trypsin solution as well as wet spun threads before and after denaturation. The TC samples cross linked by FA and by 1% GA showed only slight degradation over 8 h (Fig. 6), whereas lower concentration of GA led to a higher susceptibility against trypsin. Threads prepared from TC which were not treated by cross linking agents were degraded completely within 2 h. In contrast, one half of the fibres prepared from collagen dispersion could be solved by tryptic digest when the collagen of the fibres was held in native state. If the collagen was denatured by heat before digestion the samples were destroyed within less than 1 h as it was found for TC without cross linking (Fig. 7). Therefore, both, cross linking in the case of TC and preservation of the native structure in the case of the collagen dispersion improve stability against tryptic degradation of the samples. Finally, the threads made of TC and post treated with different cross linking agents as well as those made from collagen dispersion were investigated regarding their cytotoxicity against mouse fibroblasts. Both fibre types (dispersion; TC) which had been cross linked by formaldehyde behaved highly cytotoxic (Inhibition N30% at highest eluate concentration). The fibres which had been cross linked with 1% GA
Table 3 Textile physical properties of TC fibres cross linked by different cross linking agents. EDC — 1-ethyl-3-(3-dimethylaminopropyl)-carbodiimide. Sample Nr.
Properties of threads Post treatment
Thickness Fineness Tenacity [μm] [tex] [cN/tex]
TC1.0 – 315 TC1.1 EDC 401 TC1.2 formaldehyde 333
152 131 127
0.68 2.85 2.52
sd [%]
Elongation sd [%] [%]
17.8 125 51.7 39 41.3 54
17.8 32.7 56.1
Fig. 6. Enzymatic digests of TC fibres depending on the cross linking agent and cross linking degree. ● — formaldehyde; Δ — 1% glutaraldehyde (GA); ▼ — 0.1% GA; ○ — 0.01% GA; ■ — without cross linking.
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Acknowledgements We thank Oliver Kotlarski for the preparation of thermoplastic collagen and his important share in melt extrusion. The German Ministry of Economy is gratefully acknowledged for financial support.
References
Fig. 7. Enzymatic digests of fibres made from collagen dispersion. ○ — native; ● — denatured.
showed cytotoxicity as well (Table 5). However, materials which were treated with lower concentration of GA showed considerably lower inhibition. The fibres treated with a solution of 0.1% GA and lower concentration as well as those without cross linking were not evaluated as cytotoxic.
4. Conclusion Both of the two different technologies wet and melt spinning allowed to form threads of several dozen meters in length. Nevertheless, both techniques show pros and cons. Wet spinning of collagen dispersions is much more laborious compared to dry spinning of thermoplastic collagen because dispersions show low dry matter contents and the collagen needs to be precipitated by an organic solvent. However, by wet spinning fibres were achieved with higher tenacity than those prepared from TC. In contrast processing of thermoplastic collagen is much easier than wet spinning of dispersion. To enhance the fibre properties of the TC threads different strategies were successful as cross linking and stretching of the filaments. The physical parameters to be achieved depend on the raw material to be used as well as the spinning conditions. The quality of the fibres prepared from collagen dispersion is up to the composition of the coagulation bath and the spinneret design, whereas that of TC depends on the glycerol concentration and the draft of the fibres. In addition, by cross linking the stability against trypsin digestion was increased several times. Using a maximum of 0.1% glutaraldehyde in a solution for cross linking acceptable low proliferation inhibition was observed interpreted as low cytotoxic effect. Despite the relatively low mechanical stability, it was possible to embroider the collagen fibres on polymeric carriers. Therefore, the different techniques of fibre forming led to collagen threads suitable to be further processed by textile technologies.
Table 5 Cytotoxicity on mouse fibroblasts of diluted sample eluates. TC — thermoplastic collagen; FA — formaldehyde treatment (gas phase); GA — glutaraldehyde treatment. Sample
Inhibition [%] at eluate concentration
Material
Cross linked
44.4%
29.6%
19.8%
13.2%
8.8%
5.9%
Collagen dispersion Collagen dispersion TC TC TC TC TC
– FA – FA 0.01% GA 0.1% GA 1% GA
3 100 14 100 22 23 66
1 100 9 100 17 18 27
0 92 6 100 14 10 18
0 76 0 100 9 8 12
0 54 0 100 6 4 8
0 17 0 82 1 2 4
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Michael Meyer studied biology at Albert Ludwigs Universität Freiburg, Germany, process engineering at Bergakademie Freiberg, Germany and he finished his doctorate in 2001 in macromolecular chemistry at the Technical University of Dresden, Germany. Since 2002 he is the head of the Department Leather/Biopolymers of the Research Institute of Leather and Plastic Sheeting, Freiberg, Germany. The main focus of his scientific work is R&D of collagen materials and their characterisation especially for the medical device industry.
Hagen Baltzer studied forestry at the Technical University of Dresden and process engineering at Bergakademie Freiberg. To date he works at the Department Leather/ Biopolymers of the Research Institute of Leather and Plastic Sheeting, Freiberg, Germany. He is specialised in process development of collagen raw materials and in manufacturing processes of medical devices.
Katrin Schwikal studied chemistry at the Friedrich Schiller University Jena, where she received her diploma in 2004 and her PhD in 2007 in the group of Prof. Thomas Heinze. Since 2007 she joined the Thuringian Institute of Textile and Plastics Research, Rudolstadt. Her research activities are focused on the characterisation, shaping and functionalization of biopolymers, especially polysaccharides and proteins.
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Materials Science and Engineering C j o u r n a l h o m e p a g e : w w w. e l s ev i e r. c o m / l o c a t e / m s e c
Collagen fibres by thermoplastic and wet spinning M. Meyer a,⁎, H. Baltzer a, K. Schwikal b a b
Forschungsinstitut fuer Leder und Kunststoffbahnen, Meissner Ring 1-5, D-09599 Freiberg, Germany Thüringisches Institut für Textil-und Kunststoff-Forschung e.V. Breitscheidstr. 97, D-07407 Rudolstadt, Germany
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Article history: Received 11 June 2010 Accepted 8 July 2010 Available online 14 July 2010 Keywords: Collagen Fibre Melt extrusion Wet spinning
a b s t r a c t Collagen threads of high yardage were manufactured by two different techniques being wet spinning of collagen dispersions and melt spinning of thermoplastic collagen. The fibres were characterised according to their structural, textile physical and biochemical properties. The wet spun fibres showed higher physical stability than the thermoplastic spun ones. Both fibre types were cross linked with different agents (formaldehyde, glutaraldehyde, and 1-ethyl-3-(3-dimethylaminopropyl)carbodiimid (EDC)) to increase mechanical stability as well as to lower susceptibility against enzymatic attack. Fibres cross linked by 0.1% glutaraldehyde as well as non cross linked fibres showed no cytotoxic effect against mouse fibroblasts. © 2010 Elsevier B.V. All rights reserved.
1. Introduction Collagen is still one of the most popular biomaterials though synthetic polymers became available for medical purposes in the last decades. Collagen combines very important properties. It is biocompatible, biodegradable, and hemostyptic; its biodegradability is being adjusted by cross linking. In tissue engineering applications the collagen's surface leads to good cell attachment, proliferation and differentiation of cells [1,2]. Therefore, collagen is widely used to manufacture medical devices e.g. membranes and sponges, to coat implants and as a solution for tissue augmentation in plastic surgery and as matrix material for cell culture [2–4]. Collagen is the major component of the extracellular matrix (ECM). Accounting for about 30% of the total body protein in vertebrates it represents the most important part of connective tissue beside water. Collagen comprises a group of different collagen types. Many of them are fibre forming others are building networks. The main fibril forming type in skin, tendon and blood vessels is collagen type I [5]. For most applications collagen is extracted from mammalian tendon or skin (porcine, bovine or equine sources). The usually highly insoluble raw material is treated with acid to obtain acid soluble collagen (ASC) or by pepsin digestion to improve yield (pepsin treated collagen, PSC). The concentration of these highly viscous acidic collagen solutions usually lies between 1% and 3%. In some cases collagen is not extracted from the raw material by dissolution but the raw stock is purified and minced completely
⁎ Corresponding author. E-mail address: michael.meyer@filkfreiberg.de (M. Meyer). 0928-4931/$ – see front matter © 2010 Elsevier B.V. All rights reserved. doi:10.1016/j.msec.2010.07.005
several times resulting in collagen dispersions [3,6]. These dispersions show higher solid contents and higher process yields. Solid collagen devices are usually made from collagen solutions or from purified collagen dispersions by casting and drying at room temperature leading to compact membranes or by lyophilisation leading to porous sponges [7]. These structures are usually undirected. To date it is a challenge to manufacture directed 2D and 3D structures, which could be very interesting for tissue engineering applications as well as for making textile implants. However, the possibility of making directed structures from collagen solutions and dispersions, respectively is limited. Other than synthetic polymers (polyesters, polyamides, polypropylene, and PTFE) or other natural polymers (silk, cotton) collagen is difficult to be formed into fibres of high yardage, because collagen does not consist of isolable long fibres. Some trials were performed in the past to manufacture threads from different collagen sources. Cavarallo et al. [8] described a process of precipitation of ASC, extracted from bovine tendon in solutions by polyethylene glycol 8000 followed by washing in a buffering solution and dewatering in isopropanol. The threads were cross linked to achieve suitable stability. In a series of papers Zeugolis et al. [9–12] described the preparation of threads (30 cm) according to a similar technique, and Vasilev et al. [13] showed that spinning of long collagen fibres was possible by extrusion of 1.5%–3% solutions of ASC in precipitation baths consisting of acetone and ethanol. The stability of the spinning process strongly depended on the collagen concentration. In contrast, Chanukov et al. [14] and Bienkiewicz et al. [15] used collagen dispersions prepared by extensive mincing with similar results. However, to date there are no high yardage collagen threads available. The so far described techniques used aqueous solutions or dispersions of collagen containing dry matter contents of 1% up to 10%.
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Recently, Meyer et al. [16] published a technique to process partly denatured collagen thermoplastically using a completely dry process. This thermoplastic collagen differs from gelatine by its low solubility in water at N35 °C. However, the thermoplastic collagen is already partly denatured having lost its fibrous structure. This material allows to be processed by the same machines as for processing thermoplastic synthetic polymers using temperature controlled processes. Our investigation aimed at manufacturing collagen threads with high yardage by two different but simple technologies namely wet spinning of collagen dispersion coupled with precipitation of the material as well as thermoplastic melt spinning. The resulting fibres of several dozen meters of length were characterised regarding their processing properties as well as regarding their structural, textile physical and biochemical properties. 2. Experimental part 2.1. Raw material Porcine and bovine skins were used as collagen raw stock obtained from a local abattoir. The skins were washed extensively and soaked in a solution of sodium hydroxide at pH 12.5 overnight. The swollen skins were split two times on a conventional splitting machine (Turner 537) used in tanneries. The middle split was soaked in ammonium sulphate solution to adjust pH by 7 and washed again with water.
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bath designed as a chute containing ethanol/acetone mixtures at different compositions. The threads were picked up at the end of the chute, air dried by a blow drier and wound up on a bobbin.
2.3. Thermoplastic granules and thermoplastic spinning According to the principle technology for preparation of thermoplastic collagen the split skins were denatured thermally by soaking for 10 min in boiling water, drained off excess water and loft dried [16]. The pieces were then ground to powder on a centrifugal mill (Görgens Engineering GmbH, Dormagen, Germany). This powder was mixed with 5 to 15% glycerol and 15 to 50% deionised water in a fast mixer resulting in granules to be fed to an extruder. Melt spinning was performed on a single-screw extruder RCP-0500 (Randcastle Extrusion Systems, New Jersey, US) coupled with a spinning pump, a stretching system consisting of primary rolls, secondary rolls and a heating section and finally take-up rolls. The extrusion temperature was adjusted in the range of 90 to 98 °C depending on the recipe to achieve applicable viscosities. The rotation speed was set between 50 and 120 rpm to get a processing pressure of 50 bar. The spinning pump was adjusted to 10 rpm. The collagen melt was formed to unifilar fibres by extrusion through nozzles with diameters of 0.3 and 0.5 mm.
2.2. Collagen dispersions and wet spinning
2.4. Material testing
The collagen dispersions were prepared from alkaline treated bovine and porcine splits. These splits were treated with 1% H2O2 solution, minced by a meat chopper, acidified to pH 4 by hydrochloric acid and further treated in a colloid mill (Cavitron, special design) several times to get homogenous collagen masses. The dry matter content of these masses was 1 to 2%. This dry matter content was too low to obtain stable threads, however. Therefore, the masses were adjusted to pH 5, concentrated by centrifugation on a tube centrifuge (special design) up to a dry matter content of 10% and acidified again by addition of low volumes of 1 M hydrochloric acid. This mass had to be aged for 24 h minimum by storage at 4 °C. The collagen dispersions were processed to threads by a cylinder spinning system (Biedermann & Wolschendorf OHG; Saalfeld, Germany) with a volume of 6 cm3 and a maximum force of 10 kN. The spinneret design depended on the experimental conditions. Cylindrical and conical nozzles with diameters between 250 and 500 μm were used. The spinning temperature was fixed in all experiments to 26 °C. The spun primary thread was coagulated in a
The fibre parameters such as tensile strength and elongation at break of the fibres were determined with the automatic testing system Fafegraph M (Textechno, Mönchengladbach, Germany) according to DIN EN ISO 5079 with 25 replicates for each sample. The analyses were performed at 20 °C and 60% relative humidity, with a feed of 10 mm/min. The fineness was investigated according to DIN EN ISO 1973. During SEM-measurements the fibres were fixed on sample holders and were subsequently coated with gold using a magnetron sputter coating device Polaron SC7620 (Quorum Technologies Ltd., Ringmer, UK). The measurements were carried out with a Leica S440i (Leica Microsystems GmbH, Wetzlar, Germany) at 20 kV. X-ray diffraction was measured on a Bruker D8 Advance diffractometer using CuKα radiation (λ = 0.154 nm) in transmission. The generator system operated at 40 kV. The manufactured collagen fibres were prepared for measurement by taking some pieces together in a parallel orientation and fixing them with cellulose nitrate. The aqueous dispersion was brought as film on a glass plate and dried on air.
Fig. 1. SEM-measurements of fibres manufactured with a conical spinneret from dispersions of porcine collagen (a) and bovine hide collagen (b) at the same magnification.
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lyophilised and weighed. Insolubility was calculated according to Insolubility [%] = m (residue dry, after enzymatic treatment) [μg] / m (dry, before enzymatic treatment) [μg] × 100. 2.8. Cytotoxicity Cytotoxicity was measured by a screening method, testing the inhibition of proliferation of extracts of the threads according to EN ISO 10993-5. The samples were washed two times with cell culture medium (DMEM-FBS) and further extracted with DMEM-FBS for 7 days at 37 +/− 2 °C. An “eluate” without sample acted as negative control and as positive control a solution of 7.5% DMSO in cell culture medium was used. All eluates were diluted with DMEM-FBS to 55.1, 36.7, 24.5, 16.3, 10.9 and 7.3 vol.%. The diluted eluates were plated out in 96-well plates, seeded with 50 μl of a freshly prepared cell suspension (73.5 × 104 cells of L929 mouse fibroblasts). The cells were incubated for 72 h at 37 +/− 2 °C (5% CO2; 95% air). Finally the protein content was measured photometric using BCA protein quantification test (Absorption A at 550 nm). The eluates showed cytotoxic behaviour, when proliferation inhibition (PI) was N30% calculated according to PI [%] = 100 − ((A550 sample)−(A550 blank))/ (A550 negative control)−(A550 blank)) × 100. 3. Results and discussion Two different collagen materials, namely thermoplastic collagen and collagen dispersion were processed to threads using wet and melt spinning techniques, respectively. 3.1. Collagen dispersion Fig. 2. WAXS reflexes (vertical — black; horizontal — grey) of a film prepared from dispersion before fibre forming (a) and the manufactured fibres (b). Peak 1 reflects the intermolecular lateral packing. Peak 2 results from amorphous scatter. Peak 3 shows the periodic axial rise per residue.
2.5. Cross linking Cross linking was performed by soaking the samples in aqueous solutions of glutaraldehyde and EDC (1-ethyl-3-(3dimethylaminopropyl)-carbodiimide), respectively. With formaldehyde the fibres were cross linked in gas phase by storing them overnight at room temperature in a tightly closed exsiccator which contained some milliliters of fresh formaldehyde solution (30%). This cross linking in gas phase allows to avoid wetting in aqueous solutions during cross linking. 2.6. Differential scanning calorimetrie (DSC) DSC is a common method to get hints about denatured and native parts of collagen as well as to measure cross linking. Samples of 1– 5 mg were put in aluminium pans, an excess of an aqueous solution of 0.9% NaCl was added and the pans were sealed tightly. The thermograms were recorded in a Perkin Elmer DSC 7 using a heating rate of 5 °C/min. 2.7. Enzymatic digestion Fibre samples were digested enzymatically to get hints about their degradation behaviour. For this, 100 μg trypsin was dissolved in 1000 μl H2O deion. Up to 1 mg of each sample was soaked in a 800 μl freshly prepared solution of 0.2 M NH4HCO3 in H2O deion and 200 μl of the trypsin solution was added. The vials were incubated at 37 °C for 2 h and 200 μl of the trypsin solution was added again and incubated for 2 h at 37 °C. Then, the samples were spun down, the supernatant was discarded and washed three times with H2O deion. Finally the centrifugate was
The collagen dispersion was prepared and formed to threads by a wet spinning technique as described above. The resulting wet strands were precipitated in a coagulation bath, dried and wound up on a bobbin. Various parameters influenced the stability of the fibres: the tenacity depended on the collagen source, the isolation method, the swelling behaviour, the solid content of the mass, the composition of the coagulation bath and the spinneret design. Fig. 1 presents SEM-pictures of fibres spun from porcine and bovine dispersions with a conical shaped spinneret. The porcine fibres (Fig. 1a) show a filamentous surface structure and the structural elements are oriented in parallel. On the contrary the bovine hide fibres (Fig. 1b) show structures which are irregular containing separated clew-like forms. The latter fibres were very brittle and broke easily when slightly touched. The native collagen fibrils of bovine skin are thicker and longer than the porcine ones [17]. Furthermore, because of the older age of the cattle compared to the pigs when slaughtered the bovine skin collagen shows a higher natural cross linking degree [18]. Presumably, these structural differences cause the difference in fibre appearance. Therefore, all further wet spinning trials were made with porcine material. Both, the manufactured collagen fibres as well as the film prepared from the dispersion showed the same WAXS reflexes (Fig. 2a, b). As described by Maxwell et al. [19] the reflexes correspond to the different structural units of collagen. Peak 1 (0.86 nm−1) represents
Table 1 Textile physical properties of threads made from collagen dispersions depending on the mixture of the precipitation agents ethanol and acetone (3.8% dry matter content; pH 4.0; cylindrical spinneret). Sample EtOH: Thread properties acetone Thickness Fineness [μm] [tex]
Tenacity [cN/tex]
sd [%]
P1 P2 P3
8.48 8.30 5.75
12.5 20.3 16.5 18.8 25.5 13.1
9:1 8:2 7:3
89 118 140
9.8 11.8 19.7
Elongation sd [%] [%] 19.47 24.3 36.6
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Table 2 Textile physical properties of threads made from collagen dispersions depending on spinneret design and dry matter content (pH 4.0; precipitation agent ethanol:acetone 9:1). Sample
P1 P4 P5
Dry matter content
Spinneret design
Thread properties Thickness [μm]
Fineness [tex]
Tenacity [cN/tex]
sd [%]
Elongation [%]
sd [%]
3.8 11.1 3.8
cylindrical cylindrical conical
89 179 170
9.8 28.1 20.0
8.48 7.12 4.65
12.5 12.5 22.5
20.3 28.5 14.6
19.5 15.9 35.0
scattering related to the intermolecular lateral packing of the collagen molecules. The broad reflex (peak 2) reflects primarily the amorphous scattering of the sample and peak 3 (0.29 nm) corresponds to the periodicity of the axial rise per residue or the distances between the Gly-X-Y repeats, respectively. The intensity of the scattering reflexes of the film manufactured from the collagen dispersion did not vary significantly with different scattering angles. However, the scattering signal of the processed collagen fibre showed different intensities depending on the angle. While peak 1 of the vertical measurement is much higher than that at the horizontal angle peak 3 disappears completely in a horizontal direction. Purslow et al. [24] described the WAXS signals of a single collagen fibril bundle compared with the reflections of a disperse population of collagen fibril bundles. They showed that the angle dependent scattering intensity depends on their orientation. Therefore, especially peak 1 reflecting the lateral packing of the collagen molecules clearly indicates an orientation of the collagen fibril bundles of the dispersion by the spinning procedure in axial direction. The tenacity of the fibres made from collagen dispersions depended on the composition of the coagulation bath (Table 1). By the use of ethanol (technical grade, 96%) no stable fibres could be prepared, whereas respectable stability was found after the addition of moderate amounts of acetone. In our experiments, adding of 10% (P1) and 20% (P2) acetone respectively, resulted in the best fibre stability (8.5 cN/tex or 8.3 cN/tex). However, if the acetone concentration was further increased the stability decreased again to 5.8 cN/tex (P3). In contrast, the elongation at break decreased with increasing acetone content during precipitation. It stood out that the standard deviations showed high values of 12 to 25% for tenacity and elongation reflecting still a low accuracy during manufacturing process. The mechanical properties of the wet spun fibres also depended on the spinneret design (Table 2). A cylindrical shape led to a tenacity which was twice (P1, 8.48 cN/tex) as much as that of a conical design (P5, 4.65 cN/tex), using the same spinning conditions. In addition, elongation at break decreased from 20.3% to 14.6% by using the conical spinneret. In our trials we adjusted fibre thickness and fineness, respectively by varying the dry matter content of the collagen dispersion to be spun.
Expectedly, increasing dry matter content led to higher thickness of the fibres and the tenacity decreased slightly with increasing thickness (P1 and P4, Table 2). These tenacities found in our experiments are in the same range found by other authors (4 to 12 cN/tex) [14,15,20]. Merely Vasilev et al. achieved higher values (20–23 cN/tex; 0,5 tex) by coagulation of acid soluble collagen in acetone/ethanol mixtures for 60 s at a spinning speed of around 1 m/min [13]. For comparison, synthetic fibres, which are used as degradable suture material show tenacities 3 to 6 times higher (30 to 57 cN/tex [21]) than those, which were found for the fibres prepared from collagen dispersions. 3.2. Thermoplastic collagen To obtain threads from thermoplastic collagen the prepared granules were fed into the single-screw extruder, melt on and pressed through the cylindrical spinneret. The fibres were directed over two wind up systems. By using different speeds of the rolls it was possible to achieve a draft of spun threads, being a common technique to improve the stability of polymer threads. In most cases this process was stable enough to wind up an “endless” thread. The monofils from thermoplastic collagen were not as stable as those prepared from the collagen suspensions. Anyhow, high yarded monofils could be manufactured and also used for further processing e.g. textile structures (Fig. 3). In the first trials the threads were embroidered on a polyvinyl alcohol carrier, which is one possible technique to manufacture textile structures from these threads. Dry threads of thermoplastic collagen behaved in a brittle way, but breaking was prevented by the addition of glycerol as plasticizer. With increasing glycerol concentration the tenacity of the threads decreased and elongation increased (Fig. 4). At concentrations of glycerol lower than 15% winding up and drafting of the threads were not possible. The most stable spinning conditions were achieved using glycerol contents between 25% and 35%. Without any draft only low tenacities (0.99–1.12 cN/tex) were obtained. Drafting of the fibres containing 25% glycerol up to a ratio of 1:2 led to an improved tenacity (1.58 cN/tex) and a slightly reduced elongation at break. Furthermore, glycerol is known as a humidifying agent for proteins [22,23]. In general, in our investigation the mechanical properties of
Fig. 3. TC fibres wound up on rolls (left) and embroidered on a polyvinyl alcohol carrier (right).
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M. Meyer et al. / Materials Science and Engineering C 30 (2010) 1266–1271 Table 4 Onset temperatures of DSC thermograms of wet spun and thermoplastically spun threads before and after cross linking (CL) with 1% glutaraldehyde.
P1 TC 1.0
Non CL
CL
38 ± 0.5 °C 32 ± 0.1 °C
56 ± 2 °C 40 ± 1 °C
3.3. Comparative measurements
Fig. 4. Tenacity (●) and elongation at break (▲) of TC fibres depending on the glycerol content.
Fig. 5. Tenacity (●) and elongation at break (▲) of TC fibres depending on the relative humidity of the surrounding atmosphere.
the threads were measured at 60% relative humidity (RH). To measure the dependency of the mechanical parameters on the humidity, the draught TC fibres containing 25% of glycerol were stored at different RH and measured again. Increasing humidity from 33% RH up to 78% RH led to a decrease of a quarter of the stability at low humidity (Fig. 5), the corresponding elongation at break increased from 55% to 93%, showing high standard deviations, however. Cross linking is often used to improve the collagen's mechanical stability and stability against enzymatic digestion and resorption [8]. Therefore, it was tried to increase the mechanical stability of the threads by an additional treatment with various cross linking agents like formaldehyde (FA) and 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide (EDC), respectively (Table 3). The low stability of the fibres could be improved by 3.5 to 4.5 times by cross linking and the corresponding elongation at break was reduced from 125% without cross linking to 39% by cross linking with EDC.
As a degradable biomaterial collagen is resorbed after being implanted or during its use in cell culture experiments. The resorption time depends on the several criteria amongst others nativity and cross link degree of the collagen but also parameters of the environment as pH, temperature, buffering concentration. Samples of both fibre types were investigated by DSC and by enzymatic digestion to correlate the nativity of the collagen, cross link degree and the susceptibility to enzymatic degradation. The nativity of the collagen of the wet spun threads could be confirmed (Tonset = 38 °C) in contrast to TC which showed a very small melting peak at low Tonset = 32 °C reflecting the situation of a highly concentrated gelatin gel. By cross linking with 1% glutaraldehyde the denaturation temperature of the wet spun fibres increased up to Tonset = 56 °C in contrast to the TC fibres reaching just Tonset = 40 °C (Table 4). To learn more about the susceptibility to enzymatic cleavage TC fibres with and without cross linking were digested in a trypsin solution as well as wet spun threads before and after denaturation. The TC samples cross linked by FA and by 1% GA showed only slight degradation over 8 h (Fig. 6), whereas lower concentration of GA led to a higher susceptibility against trypsin. Threads prepared from TC which were not treated by cross linking agents were degraded completely within 2 h. In contrast, one half of the fibres prepared from collagen dispersion could be solved by tryptic digest when the collagen of the fibres was held in native state. If the collagen was denatured by heat before digestion the samples were destroyed within less than 1 h as it was found for TC without cross linking (Fig. 7). Therefore, both, cross linking in the case of TC and preservation of the native structure in the case of the collagen dispersion improve stability against tryptic degradation of the samples. Finally, the threads made of TC and post treated with different cross linking agents as well as those made from collagen dispersion were investigated regarding their cytotoxicity against mouse fibroblasts. Both fibre types (dispersion; TC) which had been cross linked by formaldehyde behaved highly cytotoxic (Inhibition N30% at highest eluate concentration). The fibres which had been cross linked with 1% GA
Table 3 Textile physical properties of TC fibres cross linked by different cross linking agents. EDC — 1-ethyl-3-(3-dimethylaminopropyl)-carbodiimide. Sample Nr.
Properties of threads Post treatment
Thickness Fineness Tenacity [μm] [tex] [cN/tex]
TC1.0 – 315 TC1.1 EDC 401 TC1.2 formaldehyde 333
152 131 127
0.68 2.85 2.52
sd [%]
Elongation sd [%] [%]
17.8 125 51.7 39 41.3 54
17.8 32.7 56.1
Fig. 6. Enzymatic digests of TC fibres depending on the cross linking agent and cross linking degree. ● — formaldehyde; Δ — 1% glutaraldehyde (GA); ▼ — 0.1% GA; ○ — 0.01% GA; ■ — without cross linking.
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Acknowledgements We thank Oliver Kotlarski for the preparation of thermoplastic collagen and his important share in melt extrusion. The German Ministry of Economy is gratefully acknowledged for financial support.
References
Fig. 7. Enzymatic digests of fibres made from collagen dispersion. ○ — native; ● — denatured.
showed cytotoxicity as well (Table 5). However, materials which were treated with lower concentration of GA showed considerably lower inhibition. The fibres treated with a solution of 0.1% GA and lower concentration as well as those without cross linking were not evaluated as cytotoxic.
4. Conclusion Both of the two different technologies wet and melt spinning allowed to form threads of several dozen meters in length. Nevertheless, both techniques show pros and cons. Wet spinning of collagen dispersions is much more laborious compared to dry spinning of thermoplastic collagen because dispersions show low dry matter contents and the collagen needs to be precipitated by an organic solvent. However, by wet spinning fibres were achieved with higher tenacity than those prepared from TC. In contrast processing of thermoplastic collagen is much easier than wet spinning of dispersion. To enhance the fibre properties of the TC threads different strategies were successful as cross linking and stretching of the filaments. The physical parameters to be achieved depend on the raw material to be used as well as the spinning conditions. The quality of the fibres prepared from collagen dispersion is up to the composition of the coagulation bath and the spinneret design, whereas that of TC depends on the glycerol concentration and the draft of the fibres. In addition, by cross linking the stability against trypsin digestion was increased several times. Using a maximum of 0.1% glutaraldehyde in a solution for cross linking acceptable low proliferation inhibition was observed interpreted as low cytotoxic effect. Despite the relatively low mechanical stability, it was possible to embroider the collagen fibres on polymeric carriers. Therefore, the different techniques of fibre forming led to collagen threads suitable to be further processed by textile technologies.
Table 5 Cytotoxicity on mouse fibroblasts of diluted sample eluates. TC — thermoplastic collagen; FA — formaldehyde treatment (gas phase); GA — glutaraldehyde treatment. Sample
Inhibition [%] at eluate concentration
Material
Cross linked
44.4%
29.6%
19.8%
13.2%
8.8%
5.9%
Collagen dispersion Collagen dispersion TC TC TC TC TC
– FA – FA 0.01% GA 0.1% GA 1% GA
3 100 14 100 22 23 66
1 100 9 100 17 18 27
0 92 6 100 14 10 18
0 76 0 100 9 8 12
0 54 0 100 6 4 8
0 17 0 82 1 2 4
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Michael Meyer studied biology at Albert Ludwigs Universität Freiburg, Germany, process engineering at Bergakademie Freiberg, Germany and he finished his doctorate in 2001 in macromolecular chemistry at the Technical University of Dresden, Germany. Since 2002 he is the head of the Department Leather/Biopolymers of the Research Institute of Leather and Plastic Sheeting, Freiberg, Germany. The main focus of his scientific work is R&D of collagen materials and their characterisation especially for the medical device industry.
Hagen Baltzer studied forestry at the Technical University of Dresden and process engineering at Bergakademie Freiberg. To date he works at the Department Leather/ Biopolymers of the Research Institute of Leather and Plastic Sheeting, Freiberg, Germany. He is specialised in process development of collagen raw materials and in manufacturing processes of medical devices.
Katrin Schwikal studied chemistry at the Friedrich Schiller University Jena, where she received her diploma in 2004 and her PhD in 2007 in the group of Prof. Thomas Heinze. Since 2007 she joined the Thuringian Institute of Textile and Plastics Research, Rudolstadt. Her research activities are focused on the characterisation, shaping and functionalization of biopolymers, especially polysaccharides and proteins.