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Cell immobilization in gelatin–hydroxyphenylpropionic acid hydrogel fibers
Biomaterials 30 (2009) 3523–3531
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Biomaterials journal homepage: www.elsevier.com/locate/biomaterials
Cell immobilization in gelatin–hydroxyphenylpropionic acid hydrogel fibers Min Hu, Motoichi Kurisawa, Rensheng Deng, Choon-Meng Teo, Annegret Schumacher, Ya-Xuan Thong, Lishan Wang, Karl M. Schumacher, Jackie Y. Ying* Institute of Bioengineering and Nanotechnology, 31 Biopolis Way, The Nanos, Singapore 138669, Singapore
a r t i c l e i n f o
a b s t r a c t
Article history: Received 14 December 2008 Accepted 7 March 2009 Available online 27 March 2009
Gelatin–hydroxyphenylpropionic acid (Gtn–HPA) hydrogels are highly porous and biodegradable materials. Herein we report a fiber spinning method that can produce cell-seeded solid and hollow hydrogel fibers by enzymatically cross-linking Gtn–HPA in solutions flowing within a capillary tube. The cell-immobilized hydrogel fibers, with feature sizes down to 20 mm, are formed as a result of continuous cross-linking of cell-mixed hydrogel precursors in a multiphase laminar flow. This fiber formation process is mild enough to retain the cell viability. The continuous fiber formation, simultaneous cell encapsulation, as well as versatile combination of fiber structures provided by this approach make it a promising and effective technique for the preparation of cell-seeded hydrogel scaffolds and carriers for tissue engineering. Ó 2009 Elsevier Ltd. All rights reserved.
Keywords: Cell encapsulation Cross-linking Gelatin Hydrogel Scaffold
1. Introduction Gelatin is a protein derived by partial hydrolysis of natural collagen, which is typically extracted from the bone, skin, tendon, cartilage, intestine and other mammalian tissues or organs [1,2]. Gelatin-based hydrogels contain arginine–glycine–aspartic acid (RGD) peptide sequences that mimic many features of the extracellular matrix (ECM). Due to their excellent biocompatibility [3], biodegradability [4] and mechanical strength [5], gelatin-based hydrogels are regarded as ideal biomaterials for applications in tissue engineering [6–9] and cell transplantation [10–14]. In particular, gelatin-based hydrogel scaffolds in filament- and tubulelike constructs are potentially useful for tissue engineering of damaged or lost small anatomical structures, such as peripheral nerves [15–18], muscle fibers, small blood vessels [19–23], and kidney tubules. However, fabricating these ultrafine gelatin scaffolds and homogeneously immobilizing cells in the hydrogel matrices remain a major challenge [24,25]. Conventionally, cells are immobilized in the hydrogels following the fabrication of the hydrogel scaffolds [24]. Small hydrogel scaffolds such as fibers and tubules can be prefabricated by electrostatic spinning [26,27], extrusion [28,29] or centrifugal molding [30]. Hydrogels that are synthesized via harsh chemical reaction conditions or with cytotoxic chemical solvents may be used as scaffolds only after thorough removal of the hazardous or toxic chemicals
* Corresponding author. Tel.: þ65 6824 7100; fax: þ65 6478 9020. E-mail address: [email protected] (J.Y. Ying). 0142-9612/$ – see front matter Ó 2009 Elsevier Ltd. All rights reserved. doi:10.1016/j.biomaterials.2009.03.004
and solvents. This greatly complicates the fabrication process, and dictates the need to conduct cell seeding in a separate process. In addition, such sequential processing would not allow for the uniform distribution and/or controlled positioning of cells within the hydrogel matrices. Since most of the cells are immobilized on the surface of the hydrogel, this two-step process may not be applicable to cell encapsulation whereby the immobilized cells should be completely surrounded by hydrogel matrices to exclude immunocytes and antibodies from the immune system of the body [12–14]. Recently, a spinning technology has been reported to be capable of continuous formation of cell-loaded alginate fibers in a coaxial flow [31,32]. As the coaxial flow is a two-phase laminar flow, only solid alginate fibers (single-layered with or without cells embedded) can be processed by this approach. In this study, we developed Gtn–HPA hydrogel fiber through the oxidative coupling of HPA moiety, which was catalyzed by hydrogen peroxide (H2O2) and horseradish peroxidase (HRP). By enzymatically cross-linking Gtn–HPA hydrogel in solutions flowing laminarly within a triple-orifice extruder, we presented a novel spinning system that could continuously produce cell-seeded hydrogel fibers in both solid (single- or dual-layered) and hollow structures. We chose enzyme-mediated cross-linking system for the fabrication of hydrogel fibers because the gelation rate could be tuned to be very high by increasing the HRP concentration. This was vital for the continuous production of fibers in a multiphase coaxial flow. Secondly, the cell proliferation rate could be controlled by the mechanical properties of the hydrogel fibers, which were tailored through the concentrations of Gtn–HPA and H2O2. Herein, we reported the formation of cell-immobilized hydrogel fibers with
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diameters as small as 20 mm. This was achieved via the transverse diffusion of cross-linkers to the cell-mixed hydrogel precursors in multiphase laminar flow. We further demonstrated that the fiber spinning process was conducted under mild conditions, such that the viability of live cells was retained after immobilization in the Gtn–HPA hydrogel fibers. 2. Materials and methods 2.1. Materials Gelatin (8–14 kDa), HRP and H2O2 (31 wt%) were purchased from Wako (Osaka, Japan), Tokyo Kasei Kogyo Co. (Tokyo, Japan) and MGC Pure Chemicals (Singapore), respectively. 40 -6-Diamidino-2-phenylindole (DAPI) was obtained from Invitrogen Corp. (CA, USA). ArtisanÔ Hematoxylin and Eosin (H&E) stain kit was purchased from Dako (CA, USA). PKH2 Green Fluorescent Cell Linker Kit for General Cell Labeling (PKH2-GL) and PKH26 Red Fluorescent Cell Linker Kit for General Cell Labeling (PKH26-GL) were obtained from Sigma–Aldrich (Singapore). Live/dead viability/cytotoxicity assay kit for mammalian cells (containing calcein and EthD-1) was purchased from Invitrogen Corp. (CA, USA). Phosphate buffered saline (PBS) (150 mM, pH 7.3) was supplied by media preparation facility at Biopolis, Singapore. All chemicals were used without further purification. 2.2. Synthesis of Gtn–HPA conjugates Gtn–HPA conjugates were synthesized by a general carbodiimide/active ester-mediated coupling reaction (see Fig. 1A). 3,4-HPA (3.32 g, 20 mmol), N-hydroxysuccinimide (NHS; 3.2 g, 27.8 mmol) and 1-ethyl-3-(3-dimethylaminopropyl)-carbodiimide (EDC; 3.82 g, 20 mmol) were dissolved in 250 ml of water and dimethylformamide (volume ratio ¼ 3:2). The reaction was conducted at room temperature and pH 4.7 for 5 h. Upon completion, 10 g of gelatin in 150 ml of water
A
were added to the reaction mixture, and stirred overnight at room temperature and pH 4.7. The mixture was then extensively dialyzed against 100 mM of NaCl solution, 25% ethanol and water in sequence for 1 day each, and lyophilized. The percentage of amine groups of gelatin introduced with HPA (i.e. degree of conjugation) was determined by the conventional 2,4,6-trinitrobenzene sulfonic acid (TNBS) method [33] to be 90%. The yield was 7.0 g. The Gtn–HPA conjugates could be cross-linked by the enzymatic oxidative reaction of HPA moieties using HRP and H2O2 (see Fig. 1B). This was similar to the cross-linking of hyaluronic acid–tyramine (HA–Tyr) conjugates via the oxidative coupling of Tyr moieties [34,35] using HRP and H2O2. HRP has been frequently used as a catalyst for the oxidative coupling of phenol derivatives under mild reaction conditions. In this case, the oxidative coupling of phenol proceeded at the C–C and C–O positions between phenols. 2.3. Multiphase laminar flow from a triple-orifice extruder As shown in Fig. 2A, our fiber spinning system consisted of a triple-orifice extrusion head, three syringe pumps and a liquid bath for fiber collection. The jiglike extrusion head was fashioned in such a way that there were five holes as inlets at the top (one in the center and four at the sides), and a single orifice as outlet at the bottom (where the solutions would merge). Two small stainless steel tubes (18G and 25G) were inserted into the extrusion head and protruded from the outlet orifice (1.7 mm inner diameter). A coaxial structure with concentric liquid annuli/orifices was achieved at the outlet of the extrusion head by precisely machining the head and assembling the tubes. The extrusion head not only held the tubes in position, but also connected the three concentric annuli/orifices individually to programmable syringe pumps. Coaxially attached to the orifices of the extrusion head was a connection funnel through which the liquids from the three concentric orifices were first merged and then discharged to the liquid bath. The straight part of the funnel was a glass capillary with an inner diameter of 0.78 mm and a length of 15 mm. When solutions including hydrogel precursors and cross-linkers were pumped through the three (inner, middle and outer) annuli/orifices, the fluids at low
OH
OH EDC/NHS
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NH CO
OH NH CO
HRP/H2O2 PBS
NH CO
O
H
O
CO NH
OH
OH Gelatin-HPA conjugate
CO NH
Gelatin-HPA hydrogel Fig. 1. Synthesis of the Gtn–HPA conjugate and hydrogel. A) Gtn–HPA conjugate is synthesized by a general carbodiimide/active ester-mediated coupling reaction. B) The Gtn–HPA conjugates are cross-linked by the enzymatic oxidative reaction of HPA moieties using H2O2 and HRP.
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Extrusion head
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Fig. 2. Multiphase laminar flow and its application in forming cell-seeded Gtn–HPA hydrogel fibers. A) Multiphase laminar flow from a triple-orifice extruder. Three orifices are coaxially nested in the extruder and individually connected to three syringe pumps. Due to the concentric geometry, the three solutions (inner, middle and outer) would merge at the exit of the orifices and form a triple-layered coaxial flow at low Reynolds numbers in the connection funnel. The outlet of the funnel (capillary tube) is immersed in, rather than hung above, a liquid bath to eliminate the influence of liquid surface tension on the laminar flow. B) Cell-seeded single-layered Gtn–HPA solid fiber can be formed by pumping cellmixed hydrogel precursor (Gtn–HPA þ HRP þ cells) through the inner orifice and H2O2 solution through the middle orifice. C) Cell-seeded Gtn–HPA hollow fiber can be obtained by switching the solutions in the inner and middle orifices of B. D) Cell-seeded dual-layered Gtn–HPA solid fiber can be synthesized by mixing Gtn–HPA and another type of cells with H2O2, and pumping the mixture through the inner orifice of C. In B–D, PBS is pumped through the outer orifice as a sheath fluid.
Reynolds numbers would merge in the connection funnel and form a triple-layered coaxial laminar flow. Meanwhile, small reactive species in the solutions might diffuse transversely through the interfaces of the laminar flow [36]. As a result, chemical reactions would occur progressively, and a hydrogel fiber was derived in the coaxial laminar flow. If cells were dispersed in the hydrogel precursors before pumping into the extrusion head, they would be encapsulated in the hydrogel matrices simultaneously during the hydrogel cross-linking process. 2.4. Fabrication of cell-seeded Gtn–HPA hydrogel fibers To effectively spin fine Gtn–HPA fibers, a rapid rate of cross-linking of the hydrogel was necessary given the swift transverse diffusion of reactive species in the coaxial laminar flow. Therefore, Gtn–HPA conjugates should be gelled rapidly via
enzymatic oxidative reaction. The gelation rate of Gtn–HPA hydrogel could be controlled by varying the amount of HRP. We could also control the mechanical strength of the fiber by changing the concentrations of Gtn–HPA conjugates and H2O2. The mechanical strength of the hydrogel matrices would affect the quality of cell growth and rate of cell proliferation. Besides a fast cross-linking rate, it was also essential that the fibers were not too fragile to handle, were flexible, and yet would not compromise the quality of cell growth. To meet these criteria, we have chosen the concentrations of Gtn–HPA, HRP and H2O2 in PBS solutions to be 25 mg/ml, 6.25 units/ml and 0.5 wt%, respectively. Depending on the orifices through which Gtn–HPA þ HRP þ cells, H2O2, or Gtn– HPA þ H2O2 þ cells were pumped, three types of cell-seeded Gtn–HPA hydrogel fibers (single-layered, dual-layered and hollow) could be formed from the same triple-orifice extruder (see Fig. 2B–D). Moreover, other combinations and variations
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of cell-hydrogel patterns could also be achieved. Dual-layered fibers, for example, might consist of only one type of cells, encapsulated either in the core or in the outer layer of the hydrogel fibers. A sheath flow was created by pumping PBS solution through the outer orifice of the extrusion head. It served as a lubricant between the solidified fibers and the capillary inner surface to convey the solidified fibers to the liquid bath without clogging the outlet of the extrusion head. To maintain an isotonic condition for cell immobilization, PBS solution was also used for the bath liquid. 2.5. Cell culture Madin-Darby canine kidney (MDCK) cells and NIH/3T3 fibroblasts immobilized in Gtn–HPA hydrogel solid fibers were cultured in Dulbecco’s Modified Eagle’s Medium (DMEM) supplemented with 5% (v/v) fetal calf serum (FCS) and 0.5% (v/v) antibiotic–antimycotic mix at 37 C and 5% CO2. Human microvascular endothelial (HME) cells and human proximal tubular (HPT) cells immobilized in Gtn–HPA hydrogel hollow fibers were respectively cultured in endothelial cell growth medium (EGM) and renal epithelial cell growth medium (REGM) supplemented with 0.5% (v/v) of penicillin/streptomycin (100 units/ml) at 37 C and 5% CO2. Cell culture medium was refreshed every two days, but only half of the medium in the well plate was replaced each time. 2.6. Characterization of Gtn–HPA hydrogel and fibers 2.6.1. Mechanical test Rheological measurements of the hydrogel formation were performed with a HAAKE Rheoscope 1 rheometer (Karlsruhe, Germany) using a cone and plate geometry to determine the storage modulus. In a similar fashion, Gtn–HPA mixture was prepared and quickly transferred to the rheometer. Measurements were taken in the dynamic oscillatory mode with a constant deformation of 1% and a frequency of 1 Hz. For this study, concentrations of Gtn–HPA and HRP in PBS solutions were fixed at 25 mg/ml and 0.01247 units/ml. H2O2 concentrations in PBS were varied. 2.6.2. Optical microscopy An inverted Olympus IX71 optical microscope equipped with a DP70 digital color camera was employed to study the surface morphology and cryosections of the hydrogel fibers incorporated with or without cells. Fluorescent imaging of duallayered solid fibers and live/dead assay of the immobilized cells were also performed on this inverted microscope. 2.6.3. Scanning electron microscopy (SEM) Morphology of pure Gtn–HPA hydrogel fibers was also characterized with a field emission SEM (JEOL JSM-7400F) at an acceleration voltage of 8.0 kV and a working distance of 8.0 mm. Before the fibers were loaded into the SEM chamber for imaging, samples of pure hydrogel fibers were first rinsed in sufficient deionized (DI) water to remove the residues of PBS, followed by freezing in a 35 mm petri dish at 80 C for 12 h. Next, the frozen samples were dehydrated in a freeze dryer (Labconco Freezone 12L) for 24 h. To reveal the porous structures within the hydrogel fibers, the dried samples were broken into short segments in liquid nitrogen. The segments were then fixed on a stage using a conductive tape, and sputter-coated with platinum for 80 s. 2.6.4. Cryosectioning and DAPI staining The distribution of cells in the hydrogel fibers were checked by cryosectioning and DAPI staining. DAPI is known to form fluorescent complexes with natural double-stranded DNA in both living and fixed cells. Hydrogel fibers were cut into short segments, and fixed with tissue embedding medium Tissue-TekÒ (Sakura Finetek, Japan) at 80 C for 12 h. The frozen fiber segments were then sectioned by a cryostat (LEICA CM3050S) to a thickness of 10 mm each, and transferred to microscopy glass slides for DAPI staining. The sectioned samples on microscopy glass slides were washed once with PBS solution, and the cells were fixed with 70% ethanol for 20 min at room temperature. After applying PBS-buffered DAPI solution (10 mg/ml), the samples were left at room temperature for w15 min, and washed with PBS solution. The cell morphology was observed using the inverted fluorescence microscope at an excitation wavelength of 350 nm. 2.6.5. Live/dead assay The viability of cells after encapsulation in hydrogel fibers was checked with a live/dead viability/cytotoxicity assay kit for mammalian cells. The staining solution was prepared by adding 1 ml of Calcein stock solution (Component A, 4 mM solution in dimethyl sulfoxide (DMSO)) and 1 ml of ethidium homodimer-1 (EthD1) stock solution (Component B, 2 mM solution in 1:4 DMSO/H2O) to 2 ml of PBS solution in a dark environment. After applying the staining solution to the cellencapsulated hydrogel fibers in a 35 mm petri dish, the petri dish was wrapped with aluminum foil and incubated at 37 C in a 5% CO2 incubator for 45 min. The stained cells were visualized using the inverted fluorescence microscope. Pictures were taken with the appropriate filter block swb (lex ¼ 420 nm, lem ¼ 515 nm, dichroic filter: 500 nm) for Calcein and swg (lex ¼ 480–550 nm, lem ¼ 590 nm, dichroic filter: 570 nm) for EthD-1.
3. Results 3.1. Gtn–HPA hydrogel stiffness vs. H2O2 concentration Fig. 3 shows the effects of H2O2 concentration on the gel stiffness. Keeping the HRP concentration and Gtn–HPA concentration constant at 0.01247 units/ml and 25 mg/ml, respectively, the storage modulus (G0 ) increased as the H2O2 concentration increased from 0.005 to 0.01 wt%. Further increase in H2O2 concentration resulted in the decline of G0 . 3.2. Gtn–HPA fibers without cells Fig. 4A shows pure Gtn–HPA solid fibers spun through the triple-orifice extruder. The fibers were extruded with an inner hydrogel precursor (Gtn–HPA þ HRP) flow rate of 5 ml/min, a middle H2O2 flow rate of 20 ml/min, and an outer PBS flow rate of 75 ml/min. The Gtn–HPA solid fibers were transparent with fairly uniform diameters in DI water (left image). The porous structures were revealed by SEM after freeze drying and cutting (right image). Gtn–HPA hydrogel fibers were found to be mechanically strong and elastic; even the smallest fibers obtained (20 mm in diameter) could be handled without breakage. Fig. 4B shows the pure Gtn–HPA hollow fibers formed by the same triple-orifice extruder. These fibers were extruded with an inner H2O2 flow rate of 15 ml/min, a middle hydrogel precursor (Gtn–HPA þ HRP) flow rate of 35 ml/ min, and an outer PBS flow rate of 50 ml/min. Both the lumen (hollow part) and the wall (solid part) of the hollow fibers were clearly visible under optical microscope and through histological cryosectioning. 3.3. Cell-seeded Gtn–HPA fibers Fig. 5A shows a single-layered cell-seeded Gtn–HPA hydrogel solid fiber. MDCK cells were used for this immobilization study. The fiber was extruded by an inner hydrogel precursor (Gtn– HPA þ HRP þ MDCK cells) flow rate of 7.5 ml/min, a middle H2O2 flow rate of 17.5 ml/min, and an outer PBS flow rate of 75 ml/min. MDCK cell suspension was added to and uniformly mixed with the Gtn–HPA þ HRP solution prior to the loading of this precursor solution into the syringe pump. The optical micrograph and cryosection image showed that a uniform distribution of MDCK cells was achieved within the hydrogel fibers. Fig. 5B illustrates the cell-seeded Gtn–HPA hydrogel hollow fibers. These fibers were obtained by switching the inner and middle flows in single-layered fiber formation. Specifically, the
Fig. 3. Effect of H2O2 concentration on the stiffness of bulk Gtn–HPA hydrogel. The concentrations of HRP and Gtn–HPA were kept at 0.0125 units/ml and 25 mg/ml, respectively.
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Fig. 4. A) Solid fibers of pure Gtn–HPA hydrogel without cells. (Left) Optical micrograph of fibers in DI water. (Right) SEM image of a fiber after freeze drying and cutting, illustrating its porous structure. B) Hollow fibers of pure Gtn–HPA hydrogel without cells. (Left) Optical micrograph of fibers in DI water. (Right) Cryosectional image of a hollow fiber stained with hematoxylin and eosin. Concentrations of species in PBS solutions before cross-linking were Gtn–HPA: 25 mg/ml, HRP: 6.25 units/ml, and H2O2: 0.5 wt%.
hollow fibers were formed with an inner H2O2 flow rate of 2.5 ml/ min, a middle hydrogel precursor (Gtn–HPA þ HRP þ MDCK cells) flow rate of 7.5 ml/min, and an outer PBS flow rate of 90 ml/min. The lumina of the fibers were discernible through histology. Fig. 5C shows the cell-seeded, dual-layered Gtn–HPA hydrogel fibers. Both MDCK cells and HME cells were immobilized in the same hydrogel fibers, with HME cells lining the periphery and MDCK cells occupying the core. The fibers were extruded by an inner hydrogel precursor (Gtn–HPA þ H2O2 þ MDCK cells) flow rate of 15 ml/min, a middle hydrogel precursor (Gtn–HPA þ HRP þ HME cells) flow rate of 12.5 ml/min, and an outer PBS sheath flow rate of 72.5 ml/min. In this case, the solution with Gtn–HPA þ H2O2 þ MDCK cells replaced the function of PBS-diluted H2O2 solution in the previous hollow fiber extrusion. When merged with the solution containing Gtn– HPA þ HRP þ HME cells from the middle orifice, cross-linking of the double-layered Gtn–HPA hydrogel fibers would occur at the flow interface via the transverse diffusion of both H2O2 and HRP. To distinguish between the different types of cells, the HME cells and MDCK cells were labeled with fluorescent cell linkers, PKH2-GL and PKH26-GL, respectively, which incorporated reporter molecules onto the cell membrane. These labeled cells retained their biological and proliferation activities, enabling cell tracking to be conducted with ease.
the matrices of the Gtn–HPA hollow fibers showed that more than 95% of the cells survived the in situ cross-linking process. This confirmed that the enzymatic cross-linking of Gtn–HPA hydrogel was a mild chemical reaction that was not cytotoxic. Cell viability was further verified by the results of cell culture within the Gtn–HPA hydrogel fibers. MDCK cells were found to proliferate well within the single-layered fibers, forming a monolayer on the hydrogel surface after 4 days of incubation (Fig. 6C). Similar results were observed in the NIH/3T3-seeded single-layered fibers after 10 days of incubation in DMEM (Fig. 6D). The proliferation of cells and formation of monolayer were also observed for the hollow fibers seeded with HME cells (Fig. 6E) and HPT cells (Fig. 6F) after 8 days of incubation in EGM and REGM supplemented with 0.5% (v/v) penicillin/streptomycin (100 units/ml), respectively. Although the morphology of the cells in (or on the surface of) the hydrogel fibers depended on the type of cells immobilized, the presence of the RGD sequences in gelatin and the porosity in the hydrogel have promoted the attachment and proliferation of mammalian cells in these systems. In soft tissue engineering, the formation of a monolayer of epithelial, proximal tubular or similar type of cells on the inner surfaces of a tubular scaffold is a key step towards restoring the function of damaged/lost native tissues. 4. Discussion
3.4. Cell viability after seeding 4.1. Stiffness of Gtn–HPA hydrogel To evaluate the influence of this one-step immobilization process on cell viability, we collected the cell-seeded hydrogel fibers from the liquid bath. Parts of the fibers were analyzed using the live/dead assay, while the rest were incubated in the cell culture medium. The live/dead assay (Fig. 6A and B) of cells embedded in
The stiffness of hydrogel has been reported to affect the degradability of proteolysis-sensitive hydrogels and the cell proliferation rate [37]. Thus, it would be important to control the stiffness of hydrogel for three-dimensional (3D) cell culture. As an
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Fig. 5. Fibers of cell-seeded Gtn–HPA hydrogel. A) (Left) Optical micrograph and (right) cryosectional image of single-layered solid fiber seeded with MDCK cells. B) (Left) Optical micrograph and (right) cryosectional image of hollow fibers seeded with MDCK cells. C) (Left) Optical micrograph and (right) fluorescent image of dual-layered solid fiber with HME cells lining the periphery and MDCK cells located at the center. The HME cells were labeled with PKH2 green fluorescence, while the MDCK cells were labeled with PKH26 red fluorescence. Concentrations of species in PBS solutions before cross-linking were Gtn–HPA: 25 mg/ml, HRP: 6.25 units/ml, H2O2: 0.5 wt% (in A and B) or 0.01 wt% (in C), MDCK cells: 106 cells/ml, and HME cells: 106 cells/ml (in C only). Cryosections in A and B were stained with DAPI, which was known to form fluorescent complexes when bond to DNA at the cell nuclei.
oxidant, H2O2 played an important role in the enzymatic oxidative reaction of HPA moieties. In this reaction, the H2O2 concentration affected the stiffness of the cross-linked hydrogel (Fig. 3). At the lower H2O2 concentration range, G0 increased with the increase of H2O2 concentration, suggesting that HRP was continuously oxidized by H2O2. However, G0 would decrease with further increase in H2O2 concentration. It has been reported previously that the activity of HRP decreased with excess H2O2 [38,39], which could explain the decline in G0 when H2O2 concentration was increased beyond 0.01 wt%. 4.2. Effect of H2O2 concentration As the stiffness of hydrogel directly influenced the quality of cell growth in the matrices, a concentration of 0.5 wt% of H2O2 was used for the production of Gtn–HPA hydrogel fibers for optimal mechanical strength and cell growth. Although the enzymatic
oxidative reaction did not involve or affect the cells, our toxicity studies indicated that the MDCK cells could only withstand a H2O2 concentration of <0.25 wt% in the absence of HRP and Gtn–HPA. Even at a low H2O2 concentration of 0.025 wt%, only 50% of the cells survived after 12 h, implying that H2O2 was highly toxic to the cells. However, when 0.5 wt% of H2O2 was used in the spinning of hydrogel fibers, the cells were found to proliferate well regardless of whether they were seeded after extrusion or were co-extruded. Although this might seem contradictory at first, the reaction between H2O2 and Gtn–HPA and the diffusion of H2O2 out of the hydrogel were not taken into account in the toxicity studies. In other words, the tolerance limit of 0.25 wt% of H2O2 only applied to MDCK cells that were in direct contact with H2O2 and for a relatively long period of time (12 h). The H2O2 concentration was reduced to 0.01 wt% when it was mixed with the solutions containing Gtn–HPA and HME cells for the dual-layered fiber extrusion.
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Fig. 6. Cell viability after seeding within the hydrogel matrices. A) Live assay of the immobilized MDCK cells. Live MDCK cells were labeled with calcein that fluoresced green. B) Dead assay of the immobilized MDCK cells. The nuclei of dead MDCK cells were labeled with EthD-1 that fluoresced red. C) A solid fiber embedded with MDCK cells after 4 days of incubation in DMEM. The cells proliferated and formed a monolayer on the surface of the fiber. D) Growth of NIH/3T3 cells in a solid fiber after 10 days of culture in DMEM. E) A hollow fiber loaded with HME cells after 8 days of incubation in EGM. A monolayer of cells was formed on both the inner and outer surfaces of the hollow fiber. F) A hollow fiber loaded with HPT cells after incubation for 8 days in REGM. A monolayer of cells was formed on both the inner and outer surfaces of the hollow fiber.
4.3. Control of the fiber spinning process During the fiber spinning process, clogging of the extruder orifices could occur if the flow rates and cross-linking kinetics were not properly matched in the connection funnel. If the diffusioncontrolled ionic cross-linking was too fast compared to the flow rate in the funnel, then the hydrogel would be cross-linked prematurely and blocking the orifice of the extruder. On the other hand, if the cross-linking speed was too slow, the hydrogel fibers might not be completely cross-linked in the funnel before being discharged from the glass capillary. These issues were tackled by controlling the cross-linking kinetics (i.e. by varying the HRP concentration) and/or by changing the flow rates of the solutions. 4.4. Advantages of the fiber spinning technique Cell-seeded filament- and tubule-like hydrogel grafts are potentially applicable in engineering similar structures observed in
various organs. For example, cell-seeded fibers might be useful for cell delivery to impaired parenchymal organs, such as liver and kidney. Cell-seeded tubular structures could be employed as substitution for diseased small vessels e.g. in coronary heart disease or peripheral vascular diseases. These tubular structures could also be employed in renal tissue engineering. Significant efforts have been devoted towards implementing these cell-seeded grafts, especially in tubular structures. Reported approaches include cell loading [24], mold casting [30,40,41], cellsheet rolling [42], and 3D bio-printing [43]. In cell loading, a porous tubular scaffold is prefabricated with synthetic or modified biological materials for structural support. The live cells are then loaded into the networks of the tubular structure by soaking, perfusion, centrifugal force or vacuum application. As the tubule fabrication process is separate from the cell seeding procedure, even the biocompatible polymers synthesized under harsh chemical reactions can be used as the scaffolds. On the other hand, the cell loading may not be efficient in such two-step processes. Also, it
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may not be very easy to disperse the cells uniformly or to attain a controlled seeding density in the polymers. Hydrogel mold casting overcomes the above issues by premixing the cells with the hydrogel precursors before pouring them into a tubule mold for gelation. Due to the difficulty of de-molding, however, this method is not effective for preparing long tubular scaffolds with a diameter of <1 mm. Also, uniformity of cell distribution in the hydrogel matrices may not be easily achieved. Cell-sheet rolling is used to prepare cell-seeded tubular grafts by wrapping cultured cellular sheets into tubular structures. Multilayered hydrogel scaffolds can be embedded with different types of cells to mimic the structure of small native arteries. 3D bio-printing uses inkjet technology to fabricate cell-seeded tubular structures. As a bottom-up approach, different types of cells could be uniformly immobilized and deployed in the hydrogel matrices. Although promising results have been achieved with these two approaches, challenges still remain in the efficient fabrication of small tubular scaffolds (with <1 mm diameter). As a continuous spinning process, our method provided for the efficient production of cell-seeded fiber- and tubule-like hydrogel grafts. Simultaneous scaffold fabrication and cell seeding allowed for the controlled distribution of multiple types of cells within the scaffolds. Our efficient process could facilitate the treatment of damaged or lost anatomical structures. Gelatin and HPA were selected as the fiber materials for this study. However, the method of in situ cross-linking of Gtn–HPA hydrogel precursor(s) in a multiphase laminar flow could also be applied to other hydrogels or gels that were chemically cross-linked (by radical polymerization, chemical reaction, irradiation, enzymes, etc.) or physically cross-linked (by ionic interactions, crystallization, amphiphilic block and graft copolymers, hydrogen bonds, protein interactions, temperature/pH changes, etc.) [44]. By shining visible or ultraviolet light on the multiphase laminar flow, for instance, a (di)acrylate or methacrylate end-capped water-soluble polymer could be cross-linked into a tubular structure in coaxial flow. 5. Conclusion We have demonstrated the homogeneous immobilization of cells in novel gelatin-based hydrogel fibers. By enzymatically crosslinking cell-mixed, fast-gelling Gtn–HPA conjugates in solutions flowing laminarly through a triple-orifice extrusion head, cellseeded solid (single-layered or dual-layered) and hollow hydrogel structures could be obtained in a single step, with uniform cell distribution and high cell viability. As live cells (and growth factors) could be simultaneously seeded in the hydrogel matrix during the fiber formation process, this technology provided us with a convenient platform to prepare cell/drug-containing hydrogel carriers/ scaffolds for 3D cell culture/co-culture, cell transplantation, as well as soft tissue engineering. Acknowledgments This work is supported by the Institute of Bioengineering and Nanotechnology (Biomedical Research Council, Agency for Science, Technology and Research, Singapore). Appendix Figures with essential colour discrimination. Certain figures in this article, in particular parts of Figure 2 are difficult to interpret in black and white. The full colour images can be found in the on-line version, at doi:10.1016/j.biomaterials.2009.03.004.
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Cell immobilization in gelatin–hydroxyphenylpropionic acid hydrogel fibers Min Hu, Motoichi Kurisawa, Rensheng Deng, Choon-Meng Teo, Annegret Schumacher, Ya-Xuan Thong, Lishan Wang, Karl M. Schumacher, Jackie Y. Ying* Institute of Bioengineering and Nanotechnology, 31 Biopolis Way, The Nanos, Singapore 138669, Singapore
a r t i c l e i n f o
a b s t r a c t
Article history: Received 14 December 2008 Accepted 7 March 2009 Available online 27 March 2009
Gelatin–hydroxyphenylpropionic acid (Gtn–HPA) hydrogels are highly porous and biodegradable materials. Herein we report a fiber spinning method that can produce cell-seeded solid and hollow hydrogel fibers by enzymatically cross-linking Gtn–HPA in solutions flowing within a capillary tube. The cell-immobilized hydrogel fibers, with feature sizes down to 20 mm, are formed as a result of continuous cross-linking of cell-mixed hydrogel precursors in a multiphase laminar flow. This fiber formation process is mild enough to retain the cell viability. The continuous fiber formation, simultaneous cell encapsulation, as well as versatile combination of fiber structures provided by this approach make it a promising and effective technique for the preparation of cell-seeded hydrogel scaffolds and carriers for tissue engineering. Ó 2009 Elsevier Ltd. All rights reserved.
Keywords: Cell encapsulation Cross-linking Gelatin Hydrogel Scaffold
1. Introduction Gelatin is a protein derived by partial hydrolysis of natural collagen, which is typically extracted from the bone, skin, tendon, cartilage, intestine and other mammalian tissues or organs [1,2]. Gelatin-based hydrogels contain arginine–glycine–aspartic acid (RGD) peptide sequences that mimic many features of the extracellular matrix (ECM). Due to their excellent biocompatibility [3], biodegradability [4] and mechanical strength [5], gelatin-based hydrogels are regarded as ideal biomaterials for applications in tissue engineering [6–9] and cell transplantation [10–14]. In particular, gelatin-based hydrogel scaffolds in filament- and tubulelike constructs are potentially useful for tissue engineering of damaged or lost small anatomical structures, such as peripheral nerves [15–18], muscle fibers, small blood vessels [19–23], and kidney tubules. However, fabricating these ultrafine gelatin scaffolds and homogeneously immobilizing cells in the hydrogel matrices remain a major challenge [24,25]. Conventionally, cells are immobilized in the hydrogels following the fabrication of the hydrogel scaffolds [24]. Small hydrogel scaffolds such as fibers and tubules can be prefabricated by electrostatic spinning [26,27], extrusion [28,29] or centrifugal molding [30]. Hydrogels that are synthesized via harsh chemical reaction conditions or with cytotoxic chemical solvents may be used as scaffolds only after thorough removal of the hazardous or toxic chemicals
* Corresponding author. Tel.: þ65 6824 7100; fax: þ65 6478 9020. E-mail address: [email protected] (J.Y. Ying). 0142-9612/$ – see front matter Ó 2009 Elsevier Ltd. All rights reserved. doi:10.1016/j.biomaterials.2009.03.004
and solvents. This greatly complicates the fabrication process, and dictates the need to conduct cell seeding in a separate process. In addition, such sequential processing would not allow for the uniform distribution and/or controlled positioning of cells within the hydrogel matrices. Since most of the cells are immobilized on the surface of the hydrogel, this two-step process may not be applicable to cell encapsulation whereby the immobilized cells should be completely surrounded by hydrogel matrices to exclude immunocytes and antibodies from the immune system of the body [12–14]. Recently, a spinning technology has been reported to be capable of continuous formation of cell-loaded alginate fibers in a coaxial flow [31,32]. As the coaxial flow is a two-phase laminar flow, only solid alginate fibers (single-layered with or without cells embedded) can be processed by this approach. In this study, we developed Gtn–HPA hydrogel fiber through the oxidative coupling of HPA moiety, which was catalyzed by hydrogen peroxide (H2O2) and horseradish peroxidase (HRP). By enzymatically cross-linking Gtn–HPA hydrogel in solutions flowing laminarly within a triple-orifice extruder, we presented a novel spinning system that could continuously produce cell-seeded hydrogel fibers in both solid (single- or dual-layered) and hollow structures. We chose enzyme-mediated cross-linking system for the fabrication of hydrogel fibers because the gelation rate could be tuned to be very high by increasing the HRP concentration. This was vital for the continuous production of fibers in a multiphase coaxial flow. Secondly, the cell proliferation rate could be controlled by the mechanical properties of the hydrogel fibers, which were tailored through the concentrations of Gtn–HPA and H2O2. Herein, we reported the formation of cell-immobilized hydrogel fibers with
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diameters as small as 20 mm. This was achieved via the transverse diffusion of cross-linkers to the cell-mixed hydrogel precursors in multiphase laminar flow. We further demonstrated that the fiber spinning process was conducted under mild conditions, such that the viability of live cells was retained after immobilization in the Gtn–HPA hydrogel fibers. 2. Materials and methods 2.1. Materials Gelatin (8–14 kDa), HRP and H2O2 (31 wt%) were purchased from Wako (Osaka, Japan), Tokyo Kasei Kogyo Co. (Tokyo, Japan) and MGC Pure Chemicals (Singapore), respectively. 40 -6-Diamidino-2-phenylindole (DAPI) was obtained from Invitrogen Corp. (CA, USA). ArtisanÔ Hematoxylin and Eosin (H&E) stain kit was purchased from Dako (CA, USA). PKH2 Green Fluorescent Cell Linker Kit for General Cell Labeling (PKH2-GL) and PKH26 Red Fluorescent Cell Linker Kit for General Cell Labeling (PKH26-GL) were obtained from Sigma–Aldrich (Singapore). Live/dead viability/cytotoxicity assay kit for mammalian cells (containing calcein and EthD-1) was purchased from Invitrogen Corp. (CA, USA). Phosphate buffered saline (PBS) (150 mM, pH 7.3) was supplied by media preparation facility at Biopolis, Singapore. All chemicals were used without further purification. 2.2. Synthesis of Gtn–HPA conjugates Gtn–HPA conjugates were synthesized by a general carbodiimide/active ester-mediated coupling reaction (see Fig. 1A). 3,4-HPA (3.32 g, 20 mmol), N-hydroxysuccinimide (NHS; 3.2 g, 27.8 mmol) and 1-ethyl-3-(3-dimethylaminopropyl)-carbodiimide (EDC; 3.82 g, 20 mmol) were dissolved in 250 ml of water and dimethylformamide (volume ratio ¼ 3:2). The reaction was conducted at room temperature and pH 4.7 for 5 h. Upon completion, 10 g of gelatin in 150 ml of water
A
were added to the reaction mixture, and stirred overnight at room temperature and pH 4.7. The mixture was then extensively dialyzed against 100 mM of NaCl solution, 25% ethanol and water in sequence for 1 day each, and lyophilized. The percentage of amine groups of gelatin introduced with HPA (i.e. degree of conjugation) was determined by the conventional 2,4,6-trinitrobenzene sulfonic acid (TNBS) method [33] to be 90%. The yield was 7.0 g. The Gtn–HPA conjugates could be cross-linked by the enzymatic oxidative reaction of HPA moieties using HRP and H2O2 (see Fig. 1B). This was similar to the cross-linking of hyaluronic acid–tyramine (HA–Tyr) conjugates via the oxidative coupling of Tyr moieties [34,35] using HRP and H2O2. HRP has been frequently used as a catalyst for the oxidative coupling of phenol derivatives under mild reaction conditions. In this case, the oxidative coupling of phenol proceeded at the C–C and C–O positions between phenols. 2.3. Multiphase laminar flow from a triple-orifice extruder As shown in Fig. 2A, our fiber spinning system consisted of a triple-orifice extrusion head, three syringe pumps and a liquid bath for fiber collection. The jiglike extrusion head was fashioned in such a way that there were five holes as inlets at the top (one in the center and four at the sides), and a single orifice as outlet at the bottom (where the solutions would merge). Two small stainless steel tubes (18G and 25G) were inserted into the extrusion head and protruded from the outlet orifice (1.7 mm inner diameter). A coaxial structure with concentric liquid annuli/orifices was achieved at the outlet of the extrusion head by precisely machining the head and assembling the tubes. The extrusion head not only held the tubes in position, but also connected the three concentric annuli/orifices individually to programmable syringe pumps. Coaxially attached to the orifices of the extrusion head was a connection funnel through which the liquids from the three concentric orifices were first merged and then discharged to the liquid bath. The straight part of the funnel was a glass capillary with an inner diameter of 0.78 mm and a length of 15 mm. When solutions including hydrogel precursors and cross-linkers were pumped through the three (inner, middle and outer) annuli/orifices, the fluids at low
OH
OH EDC/NHS
NH CO
O COO N
COOH
3-(4-Hydroxyphenyl)propionic acid (HPA)
Gelatin
O OH Gelatin-HPA conjugate
B
NH CO
OH NH CO
HRP/H2O2 PBS
NH CO
O
H
O
CO NH
OH
OH Gelatin-HPA conjugate
CO NH
Gelatin-HPA hydrogel Fig. 1. Synthesis of the Gtn–HPA conjugate and hydrogel. A) Gtn–HPA conjugate is synthesized by a general carbodiimide/active ester-mediated coupling reaction. B) The Gtn–HPA conjugates are cross-linked by the enzymatic oxidative reaction of HPA moieties using H2O2 and HRP.
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Extrusion head
Programmable syringe pumps Feeding zone
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c
c
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Fig. 2. Multiphase laminar flow and its application in forming cell-seeded Gtn–HPA hydrogel fibers. A) Multiphase laminar flow from a triple-orifice extruder. Three orifices are coaxially nested in the extruder and individually connected to three syringe pumps. Due to the concentric geometry, the three solutions (inner, middle and outer) would merge at the exit of the orifices and form a triple-layered coaxial flow at low Reynolds numbers in the connection funnel. The outlet of the funnel (capillary tube) is immersed in, rather than hung above, a liquid bath to eliminate the influence of liquid surface tension on the laminar flow. B) Cell-seeded single-layered Gtn–HPA solid fiber can be formed by pumping cellmixed hydrogel precursor (Gtn–HPA þ HRP þ cells) through the inner orifice and H2O2 solution through the middle orifice. C) Cell-seeded Gtn–HPA hollow fiber can be obtained by switching the solutions in the inner and middle orifices of B. D) Cell-seeded dual-layered Gtn–HPA solid fiber can be synthesized by mixing Gtn–HPA and another type of cells with H2O2, and pumping the mixture through the inner orifice of C. In B–D, PBS is pumped through the outer orifice as a sheath fluid.
Reynolds numbers would merge in the connection funnel and form a triple-layered coaxial laminar flow. Meanwhile, small reactive species in the solutions might diffuse transversely through the interfaces of the laminar flow [36]. As a result, chemical reactions would occur progressively, and a hydrogel fiber was derived in the coaxial laminar flow. If cells were dispersed in the hydrogel precursors before pumping into the extrusion head, they would be encapsulated in the hydrogel matrices simultaneously during the hydrogel cross-linking process. 2.4. Fabrication of cell-seeded Gtn–HPA hydrogel fibers To effectively spin fine Gtn–HPA fibers, a rapid rate of cross-linking of the hydrogel was necessary given the swift transverse diffusion of reactive species in the coaxial laminar flow. Therefore, Gtn–HPA conjugates should be gelled rapidly via
enzymatic oxidative reaction. The gelation rate of Gtn–HPA hydrogel could be controlled by varying the amount of HRP. We could also control the mechanical strength of the fiber by changing the concentrations of Gtn–HPA conjugates and H2O2. The mechanical strength of the hydrogel matrices would affect the quality of cell growth and rate of cell proliferation. Besides a fast cross-linking rate, it was also essential that the fibers were not too fragile to handle, were flexible, and yet would not compromise the quality of cell growth. To meet these criteria, we have chosen the concentrations of Gtn–HPA, HRP and H2O2 in PBS solutions to be 25 mg/ml, 6.25 units/ml and 0.5 wt%, respectively. Depending on the orifices through which Gtn–HPA þ HRP þ cells, H2O2, or Gtn– HPA þ H2O2 þ cells were pumped, three types of cell-seeded Gtn–HPA hydrogel fibers (single-layered, dual-layered and hollow) could be formed from the same triple-orifice extruder (see Fig. 2B–D). Moreover, other combinations and variations
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of cell-hydrogel patterns could also be achieved. Dual-layered fibers, for example, might consist of only one type of cells, encapsulated either in the core or in the outer layer of the hydrogel fibers. A sheath flow was created by pumping PBS solution through the outer orifice of the extrusion head. It served as a lubricant between the solidified fibers and the capillary inner surface to convey the solidified fibers to the liquid bath without clogging the outlet of the extrusion head. To maintain an isotonic condition for cell immobilization, PBS solution was also used for the bath liquid. 2.5. Cell culture Madin-Darby canine kidney (MDCK) cells and NIH/3T3 fibroblasts immobilized in Gtn–HPA hydrogel solid fibers were cultured in Dulbecco’s Modified Eagle’s Medium (DMEM) supplemented with 5% (v/v) fetal calf serum (FCS) and 0.5% (v/v) antibiotic–antimycotic mix at 37 C and 5% CO2. Human microvascular endothelial (HME) cells and human proximal tubular (HPT) cells immobilized in Gtn–HPA hydrogel hollow fibers were respectively cultured in endothelial cell growth medium (EGM) and renal epithelial cell growth medium (REGM) supplemented with 0.5% (v/v) of penicillin/streptomycin (100 units/ml) at 37 C and 5% CO2. Cell culture medium was refreshed every two days, but only half of the medium in the well plate was replaced each time. 2.6. Characterization of Gtn–HPA hydrogel and fibers 2.6.1. Mechanical test Rheological measurements of the hydrogel formation were performed with a HAAKE Rheoscope 1 rheometer (Karlsruhe, Germany) using a cone and plate geometry to determine the storage modulus. In a similar fashion, Gtn–HPA mixture was prepared and quickly transferred to the rheometer. Measurements were taken in the dynamic oscillatory mode with a constant deformation of 1% and a frequency of 1 Hz. For this study, concentrations of Gtn–HPA and HRP in PBS solutions were fixed at 25 mg/ml and 0.01247 units/ml. H2O2 concentrations in PBS were varied. 2.6.2. Optical microscopy An inverted Olympus IX71 optical microscope equipped with a DP70 digital color camera was employed to study the surface morphology and cryosections of the hydrogel fibers incorporated with or without cells. Fluorescent imaging of duallayered solid fibers and live/dead assay of the immobilized cells were also performed on this inverted microscope. 2.6.3. Scanning electron microscopy (SEM) Morphology of pure Gtn–HPA hydrogel fibers was also characterized with a field emission SEM (JEOL JSM-7400F) at an acceleration voltage of 8.0 kV and a working distance of 8.0 mm. Before the fibers were loaded into the SEM chamber for imaging, samples of pure hydrogel fibers were first rinsed in sufficient deionized (DI) water to remove the residues of PBS, followed by freezing in a 35 mm petri dish at 80 C for 12 h. Next, the frozen samples were dehydrated in a freeze dryer (Labconco Freezone 12L) for 24 h. To reveal the porous structures within the hydrogel fibers, the dried samples were broken into short segments in liquid nitrogen. The segments were then fixed on a stage using a conductive tape, and sputter-coated with platinum for 80 s. 2.6.4. Cryosectioning and DAPI staining The distribution of cells in the hydrogel fibers were checked by cryosectioning and DAPI staining. DAPI is known to form fluorescent complexes with natural double-stranded DNA in both living and fixed cells. Hydrogel fibers were cut into short segments, and fixed with tissue embedding medium Tissue-TekÒ (Sakura Finetek, Japan) at 80 C for 12 h. The frozen fiber segments were then sectioned by a cryostat (LEICA CM3050S) to a thickness of 10 mm each, and transferred to microscopy glass slides for DAPI staining. The sectioned samples on microscopy glass slides were washed once with PBS solution, and the cells were fixed with 70% ethanol for 20 min at room temperature. After applying PBS-buffered DAPI solution (10 mg/ml), the samples were left at room temperature for w15 min, and washed with PBS solution. The cell morphology was observed using the inverted fluorescence microscope at an excitation wavelength of 350 nm. 2.6.5. Live/dead assay The viability of cells after encapsulation in hydrogel fibers was checked with a live/dead viability/cytotoxicity assay kit for mammalian cells. The staining solution was prepared by adding 1 ml of Calcein stock solution (Component A, 4 mM solution in dimethyl sulfoxide (DMSO)) and 1 ml of ethidium homodimer-1 (EthD1) stock solution (Component B, 2 mM solution in 1:4 DMSO/H2O) to 2 ml of PBS solution in a dark environment. After applying the staining solution to the cellencapsulated hydrogel fibers in a 35 mm petri dish, the petri dish was wrapped with aluminum foil and incubated at 37 C in a 5% CO2 incubator for 45 min. The stained cells were visualized using the inverted fluorescence microscope. Pictures were taken with the appropriate filter block swb (lex ¼ 420 nm, lem ¼ 515 nm, dichroic filter: 500 nm) for Calcein and swg (lex ¼ 480–550 nm, lem ¼ 590 nm, dichroic filter: 570 nm) for EthD-1.
3. Results 3.1. Gtn–HPA hydrogel stiffness vs. H2O2 concentration Fig. 3 shows the effects of H2O2 concentration on the gel stiffness. Keeping the HRP concentration and Gtn–HPA concentration constant at 0.01247 units/ml and 25 mg/ml, respectively, the storage modulus (G0 ) increased as the H2O2 concentration increased from 0.005 to 0.01 wt%. Further increase in H2O2 concentration resulted in the decline of G0 . 3.2. Gtn–HPA fibers without cells Fig. 4A shows pure Gtn–HPA solid fibers spun through the triple-orifice extruder. The fibers were extruded with an inner hydrogel precursor (Gtn–HPA þ HRP) flow rate of 5 ml/min, a middle H2O2 flow rate of 20 ml/min, and an outer PBS flow rate of 75 ml/min. The Gtn–HPA solid fibers were transparent with fairly uniform diameters in DI water (left image). The porous structures were revealed by SEM after freeze drying and cutting (right image). Gtn–HPA hydrogel fibers were found to be mechanically strong and elastic; even the smallest fibers obtained (20 mm in diameter) could be handled without breakage. Fig. 4B shows the pure Gtn–HPA hollow fibers formed by the same triple-orifice extruder. These fibers were extruded with an inner H2O2 flow rate of 15 ml/min, a middle hydrogel precursor (Gtn–HPA þ HRP) flow rate of 35 ml/ min, and an outer PBS flow rate of 50 ml/min. Both the lumen (hollow part) and the wall (solid part) of the hollow fibers were clearly visible under optical microscope and through histological cryosectioning. 3.3. Cell-seeded Gtn–HPA fibers Fig. 5A shows a single-layered cell-seeded Gtn–HPA hydrogel solid fiber. MDCK cells were used for this immobilization study. The fiber was extruded by an inner hydrogel precursor (Gtn– HPA þ HRP þ MDCK cells) flow rate of 7.5 ml/min, a middle H2O2 flow rate of 17.5 ml/min, and an outer PBS flow rate of 75 ml/min. MDCK cell suspension was added to and uniformly mixed with the Gtn–HPA þ HRP solution prior to the loading of this precursor solution into the syringe pump. The optical micrograph and cryosection image showed that a uniform distribution of MDCK cells was achieved within the hydrogel fibers. Fig. 5B illustrates the cell-seeded Gtn–HPA hydrogel hollow fibers. These fibers were obtained by switching the inner and middle flows in single-layered fiber formation. Specifically, the
Fig. 3. Effect of H2O2 concentration on the stiffness of bulk Gtn–HPA hydrogel. The concentrations of HRP and Gtn–HPA were kept at 0.0125 units/ml and 25 mg/ml, respectively.
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Fig. 4. A) Solid fibers of pure Gtn–HPA hydrogel without cells. (Left) Optical micrograph of fibers in DI water. (Right) SEM image of a fiber after freeze drying and cutting, illustrating its porous structure. B) Hollow fibers of pure Gtn–HPA hydrogel without cells. (Left) Optical micrograph of fibers in DI water. (Right) Cryosectional image of a hollow fiber stained with hematoxylin and eosin. Concentrations of species in PBS solutions before cross-linking were Gtn–HPA: 25 mg/ml, HRP: 6.25 units/ml, and H2O2: 0.5 wt%.
hollow fibers were formed with an inner H2O2 flow rate of 2.5 ml/ min, a middle hydrogel precursor (Gtn–HPA þ HRP þ MDCK cells) flow rate of 7.5 ml/min, and an outer PBS flow rate of 90 ml/min. The lumina of the fibers were discernible through histology. Fig. 5C shows the cell-seeded, dual-layered Gtn–HPA hydrogel fibers. Both MDCK cells and HME cells were immobilized in the same hydrogel fibers, with HME cells lining the periphery and MDCK cells occupying the core. The fibers were extruded by an inner hydrogel precursor (Gtn–HPA þ H2O2 þ MDCK cells) flow rate of 15 ml/min, a middle hydrogel precursor (Gtn–HPA þ HRP þ HME cells) flow rate of 12.5 ml/min, and an outer PBS sheath flow rate of 72.5 ml/min. In this case, the solution with Gtn–HPA þ H2O2 þ MDCK cells replaced the function of PBS-diluted H2O2 solution in the previous hollow fiber extrusion. When merged with the solution containing Gtn– HPA þ HRP þ HME cells from the middle orifice, cross-linking of the double-layered Gtn–HPA hydrogel fibers would occur at the flow interface via the transverse diffusion of both H2O2 and HRP. To distinguish between the different types of cells, the HME cells and MDCK cells were labeled with fluorescent cell linkers, PKH2-GL and PKH26-GL, respectively, which incorporated reporter molecules onto the cell membrane. These labeled cells retained their biological and proliferation activities, enabling cell tracking to be conducted with ease.
the matrices of the Gtn–HPA hollow fibers showed that more than 95% of the cells survived the in situ cross-linking process. This confirmed that the enzymatic cross-linking of Gtn–HPA hydrogel was a mild chemical reaction that was not cytotoxic. Cell viability was further verified by the results of cell culture within the Gtn–HPA hydrogel fibers. MDCK cells were found to proliferate well within the single-layered fibers, forming a monolayer on the hydrogel surface after 4 days of incubation (Fig. 6C). Similar results were observed in the NIH/3T3-seeded single-layered fibers after 10 days of incubation in DMEM (Fig. 6D). The proliferation of cells and formation of monolayer were also observed for the hollow fibers seeded with HME cells (Fig. 6E) and HPT cells (Fig. 6F) after 8 days of incubation in EGM and REGM supplemented with 0.5% (v/v) penicillin/streptomycin (100 units/ml), respectively. Although the morphology of the cells in (or on the surface of) the hydrogel fibers depended on the type of cells immobilized, the presence of the RGD sequences in gelatin and the porosity in the hydrogel have promoted the attachment and proliferation of mammalian cells in these systems. In soft tissue engineering, the formation of a monolayer of epithelial, proximal tubular or similar type of cells on the inner surfaces of a tubular scaffold is a key step towards restoring the function of damaged/lost native tissues. 4. Discussion
3.4. Cell viability after seeding 4.1. Stiffness of Gtn–HPA hydrogel To evaluate the influence of this one-step immobilization process on cell viability, we collected the cell-seeded hydrogel fibers from the liquid bath. Parts of the fibers were analyzed using the live/dead assay, while the rest were incubated in the cell culture medium. The live/dead assay (Fig. 6A and B) of cells embedded in
The stiffness of hydrogel has been reported to affect the degradability of proteolysis-sensitive hydrogels and the cell proliferation rate [37]. Thus, it would be important to control the stiffness of hydrogel for three-dimensional (3D) cell culture. As an
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Fig. 5. Fibers of cell-seeded Gtn–HPA hydrogel. A) (Left) Optical micrograph and (right) cryosectional image of single-layered solid fiber seeded with MDCK cells. B) (Left) Optical micrograph and (right) cryosectional image of hollow fibers seeded with MDCK cells. C) (Left) Optical micrograph and (right) fluorescent image of dual-layered solid fiber with HME cells lining the periphery and MDCK cells located at the center. The HME cells were labeled with PKH2 green fluorescence, while the MDCK cells were labeled with PKH26 red fluorescence. Concentrations of species in PBS solutions before cross-linking were Gtn–HPA: 25 mg/ml, HRP: 6.25 units/ml, H2O2: 0.5 wt% (in A and B) or 0.01 wt% (in C), MDCK cells: 106 cells/ml, and HME cells: 106 cells/ml (in C only). Cryosections in A and B were stained with DAPI, which was known to form fluorescent complexes when bond to DNA at the cell nuclei.
oxidant, H2O2 played an important role in the enzymatic oxidative reaction of HPA moieties. In this reaction, the H2O2 concentration affected the stiffness of the cross-linked hydrogel (Fig. 3). At the lower H2O2 concentration range, G0 increased with the increase of H2O2 concentration, suggesting that HRP was continuously oxidized by H2O2. However, G0 would decrease with further increase in H2O2 concentration. It has been reported previously that the activity of HRP decreased with excess H2O2 [38,39], which could explain the decline in G0 when H2O2 concentration was increased beyond 0.01 wt%. 4.2. Effect of H2O2 concentration As the stiffness of hydrogel directly influenced the quality of cell growth in the matrices, a concentration of 0.5 wt% of H2O2 was used for the production of Gtn–HPA hydrogel fibers for optimal mechanical strength and cell growth. Although the enzymatic
oxidative reaction did not involve or affect the cells, our toxicity studies indicated that the MDCK cells could only withstand a H2O2 concentration of <0.25 wt% in the absence of HRP and Gtn–HPA. Even at a low H2O2 concentration of 0.025 wt%, only 50% of the cells survived after 12 h, implying that H2O2 was highly toxic to the cells. However, when 0.5 wt% of H2O2 was used in the spinning of hydrogel fibers, the cells were found to proliferate well regardless of whether they were seeded after extrusion or were co-extruded. Although this might seem contradictory at first, the reaction between H2O2 and Gtn–HPA and the diffusion of H2O2 out of the hydrogel were not taken into account in the toxicity studies. In other words, the tolerance limit of 0.25 wt% of H2O2 only applied to MDCK cells that were in direct contact with H2O2 and for a relatively long period of time (12 h). The H2O2 concentration was reduced to 0.01 wt% when it was mixed with the solutions containing Gtn–HPA and HME cells for the dual-layered fiber extrusion.
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Fig. 6. Cell viability after seeding within the hydrogel matrices. A) Live assay of the immobilized MDCK cells. Live MDCK cells were labeled with calcein that fluoresced green. B) Dead assay of the immobilized MDCK cells. The nuclei of dead MDCK cells were labeled with EthD-1 that fluoresced red. C) A solid fiber embedded with MDCK cells after 4 days of incubation in DMEM. The cells proliferated and formed a monolayer on the surface of the fiber. D) Growth of NIH/3T3 cells in a solid fiber after 10 days of culture in DMEM. E) A hollow fiber loaded with HME cells after 8 days of incubation in EGM. A monolayer of cells was formed on both the inner and outer surfaces of the hollow fiber. F) A hollow fiber loaded with HPT cells after incubation for 8 days in REGM. A monolayer of cells was formed on both the inner and outer surfaces of the hollow fiber.
4.3. Control of the fiber spinning process During the fiber spinning process, clogging of the extruder orifices could occur if the flow rates and cross-linking kinetics were not properly matched in the connection funnel. If the diffusioncontrolled ionic cross-linking was too fast compared to the flow rate in the funnel, then the hydrogel would be cross-linked prematurely and blocking the orifice of the extruder. On the other hand, if the cross-linking speed was too slow, the hydrogel fibers might not be completely cross-linked in the funnel before being discharged from the glass capillary. These issues were tackled by controlling the cross-linking kinetics (i.e. by varying the HRP concentration) and/or by changing the flow rates of the solutions. 4.4. Advantages of the fiber spinning technique Cell-seeded filament- and tubule-like hydrogel grafts are potentially applicable in engineering similar structures observed in
various organs. For example, cell-seeded fibers might be useful for cell delivery to impaired parenchymal organs, such as liver and kidney. Cell-seeded tubular structures could be employed as substitution for diseased small vessels e.g. in coronary heart disease or peripheral vascular diseases. These tubular structures could also be employed in renal tissue engineering. Significant efforts have been devoted towards implementing these cell-seeded grafts, especially in tubular structures. Reported approaches include cell loading [24], mold casting [30,40,41], cellsheet rolling [42], and 3D bio-printing [43]. In cell loading, a porous tubular scaffold is prefabricated with synthetic or modified biological materials for structural support. The live cells are then loaded into the networks of the tubular structure by soaking, perfusion, centrifugal force or vacuum application. As the tubule fabrication process is separate from the cell seeding procedure, even the biocompatible polymers synthesized under harsh chemical reactions can be used as the scaffolds. On the other hand, the cell loading may not be efficient in such two-step processes. Also, it
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may not be very easy to disperse the cells uniformly or to attain a controlled seeding density in the polymers. Hydrogel mold casting overcomes the above issues by premixing the cells with the hydrogel precursors before pouring them into a tubule mold for gelation. Due to the difficulty of de-molding, however, this method is not effective for preparing long tubular scaffolds with a diameter of <1 mm. Also, uniformity of cell distribution in the hydrogel matrices may not be easily achieved. Cell-sheet rolling is used to prepare cell-seeded tubular grafts by wrapping cultured cellular sheets into tubular structures. Multilayered hydrogel scaffolds can be embedded with different types of cells to mimic the structure of small native arteries. 3D bio-printing uses inkjet technology to fabricate cell-seeded tubular structures. As a bottom-up approach, different types of cells could be uniformly immobilized and deployed in the hydrogel matrices. Although promising results have been achieved with these two approaches, challenges still remain in the efficient fabrication of small tubular scaffolds (with <1 mm diameter). As a continuous spinning process, our method provided for the efficient production of cell-seeded fiber- and tubule-like hydrogel grafts. Simultaneous scaffold fabrication and cell seeding allowed for the controlled distribution of multiple types of cells within the scaffolds. Our efficient process could facilitate the treatment of damaged or lost anatomical structures. Gelatin and HPA were selected as the fiber materials for this study. However, the method of in situ cross-linking of Gtn–HPA hydrogel precursor(s) in a multiphase laminar flow could also be applied to other hydrogels or gels that were chemically cross-linked (by radical polymerization, chemical reaction, irradiation, enzymes, etc.) or physically cross-linked (by ionic interactions, crystallization, amphiphilic block and graft copolymers, hydrogen bonds, protein interactions, temperature/pH changes, etc.) [44]. By shining visible or ultraviolet light on the multiphase laminar flow, for instance, a (di)acrylate or methacrylate end-capped water-soluble polymer could be cross-linked into a tubular structure in coaxial flow. 5. Conclusion We have demonstrated the homogeneous immobilization of cells in novel gelatin-based hydrogel fibers. By enzymatically crosslinking cell-mixed, fast-gelling Gtn–HPA conjugates in solutions flowing laminarly through a triple-orifice extrusion head, cellseeded solid (single-layered or dual-layered) and hollow hydrogel structures could be obtained in a single step, with uniform cell distribution and high cell viability. As live cells (and growth factors) could be simultaneously seeded in the hydrogel matrix during the fiber formation process, this technology provided us with a convenient platform to prepare cell/drug-containing hydrogel carriers/ scaffolds for 3D cell culture/co-culture, cell transplantation, as well as soft tissue engineering. Acknowledgments This work is supported by the Institute of Bioengineering and Nanotechnology (Biomedical Research Council, Agency for Science, Technology and Research, Singapore). Appendix Figures with essential colour discrimination. Certain figures in this article, in particular parts of Figure 2 are difficult to interpret in black and white. The full colour images can be found in the on-line version, at doi:10.1016/j.biomaterials.2009.03.004.
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