One-Pot Preparation of Collagen Tubes Using Diffusing Gelation.

One-pot gelation in capillary glass tubes with carbonate-based buffer solution allows the formation of hollow collagen gels (collagen tubes) with an outer diameter of 1 mm or less. The preparation conditions of collagen concentration, buffer concentration, and capillary diameter impacted the ratio and size of the hollow gel and allowed for morphological control of the cavity. The morphology of the hollows suggests that their vacancies are the result of macroscopic phase separation and pinning due to gelation. Mechanical strength measurements of the dried collagen gel tubes demonstrated that the collagen concentration determines their Young's modulus and maximum stress and that the material is strong enough for practical use. In vitro seeding studies of vascular endothelial cells demonstrated the possible formation of endothelial cells in layers in the gel lumen.

Introduction

Collagen, which accounts for one-third of all proteins in the animal body, is present in all animals’ intercellular connective tissues[1] and promotes cell adhesion,[1] proliferation,[2] and differentiation.[3] Based on these functions, collagen is used as a highly biocompatible and bioresorbable medical material in various forms. Sheets of collagen have been commercialized, especially as medical burn dressings[4] and skin wound healing materials.[5] Bundles of collagen fibers and threads have been extensively studied because of their biochemical and structural homology to natural tendons and ligaments.[6] For example, xerogel threads, prepared by vitrifaction and drying of collagen gel, have been reported to have a remarkable potential to prevent tissue inflammation-induced fibrosis and tissue adhesion.[7] Collagen rods with a macroscopic hollow cavity are being studied and applied as bioabsorbable nerve conduit materials for peripheral nerve repair[8] and as dressings for digestive organs.[9] These hollow cavities inside the collagen can be used for artificial blood vessels.[10] Since the cavities can be filled with drugs, researchers have studied drug carriers’ applications. For example, a collagen tube filled with platelet-enriched fibrin in the hole can reduce neuropathic pain.[11] Nerve growth factor-incorporated collagen carriers have been reported to improve the repair of spinal cord defects.[12] This broad applicability potential raises the need for a fabrication method that allows control over desired physical properties for hollow collagen structures, such as size and mechanical strength.[11]

A common technique for collagen tube formation is to use a collagen film and then curl the film.[13] Although this procedure is simple to form a hollow formation, the potential for leakage of functional internal materials from the overlapped area is undeniable. In addition, glutaraldehyde, which is typically used to attach films, has cellular toxicity.[14] Against this background, other simple methods to form collagen tubes without films and controllable outer and inner diameters are desired. In this paper, we present a one-pot method for the tube formation of collagen gels from collagen solutions. The tube is formed when the buffer solution gelates the collagen solution in a capillary. The size of the capillary used can determine the outer diameter of the gel tube, which allows us to obtain thinner and controllable shapes compared to previous methods. Our approach to obtain collagen tubes is inspired by collagen shrinkings of its body during gelation. This gel shrinking results in multiple microscopic pores in the collagen gel obtained by the buffer solution diffusion from the interface.[15] Here, we discovered that the gelation with the buffer solution in capillaries forms a single hollow. The following describes the dependence of the collagen concentration and buffer solution on (1) the hollowing condition of the gel rod prepared, (2) the tube-tip diameter of gels, and (3) the starting position of a single hollow cavity in a gel tube and discusses the formation mechanism underlying these dependencies. Next, the mechanical properties of the assembled collagen tube threads will be presented. At last, we show the in vitro culturing results of vascular endothelial cells to evaluate the tissue-forming ability of the collagen tubes.

Materials and Methods

Materials

Collagen solution (type I porcine atelocollagen) was purchased from Nippon Meat Packers Inc., Japan. The carbonate buffer solution was purchased from DKK-TOA as a powder reagent for a pH 10.02 standard solution (powder). These were used without further purification.

Preparation of Collagen Hydrogel Rods

The collagen solution was freeze-dried, and the obtained powder was then dissolved in distilled water to prepare 0.33, 0.66, 1.5, and 6.0 wt % collagen solutions. Carbonate buffer solutions as the gelling agent were prepared by diluting the powdered reagent with distilled water to adjust 8, 13, 18, 25, 35, and 50 mM. The collagen solutions were filled into glass capillaries with an inner diameter of 1.89 mm and then sealed at one end with fluorine sealing tape. The capillary tube containing the collagen solution was immersed vertically into a plastic cell of 10 mm2 filled with 15 mL of carbonate buffer solution by placing the sealed end downward and covering the opposite end of the capillary with the buffer solution and placed in cold storage (5 °C) for 5 days. After leaving the gel to form in the capillaries, the gel formed in the capillaries was removed using tweezers and then rinsed with distilled water three times.

Microscopic Observation and Analysis

An optical microscope (Leica DMI3000 B, Leica Microsystems, Wetzlar, Germ) observed the resulting samples under bright-field and crossed-nicol conditions. Microscopy images of these gels were taken with a digital camera (QICAM Fast 1394; QIMAGING, Burnaby, Canada). The hollow cavity diameters (di) inside the gel and the outer diameter (do) of the gel rod were measured from the polarized light microscopy images, and the cavity area per cross-sectional area was calculated by ∑di(x)2/d02. The distance from the glass capillary end directly in contact with the carbonate buffer (diffusion end) to the unification distance of cavities (xuni) was measured from the overview images obtained using image analysis software (Image-Pro Plus 7.0.0, Media Cybernetics, Inc. Rockville). Relative unification distances (xRUD) for various samples are calculated by xRUD = xuni/x0, where x0 is the gel rod distance between the diffusion end and another end of the glass capillary. Confocal laser scanning microscopy (CLSM) was utilized to visualize internal fluorescein isothiocyanate (FITC)-labeled collagen and spatially determine collagen-rich areas. CLSM image stacks were acquired using a Nikon C1 (Nikon Corporation, Japan) equipped with an argon laser (488 nm, 15 mW) illuminator. FITC-labeled collagen was prepared as follows: a small amount of FITC (Dojindo Laboratory, Kumamoto, Japan) was added to 1.0 wt % collagen solution (20 mL) and FITC was reacted with collagen (room temperature, 1 h). The reaction mixture was dialyzed with the HCl solution (pH 3.0) by a cellulose semipermeable membrane (MWCO 8000–14,000 Da, Viskase, IL) for 24 h (three times) to remove unreacted FITC.

Viscosity Measurements

The collagen solutions’ viscosity with carbonate buffer was measured for collagen concentrations of 0.5, 1.0, and 1.5 wt % with carbonate buffer of 0.0, 0.6, and 1.2 mM using a steady-state stress Brookfield viscometer (Brookfield DV-II+ programmable Vescometer, Middleboro, MA). The spindle was used as a parallel plate, the measurement temperature was 5 °C, and the measurements were performed on approximately 1.4 mL of the sample at rotational speeds from 0.01 to 0.1 rpm, depending on the torque value.

Preparation and Mechanical Strength Measurement of Collagen Xerogel Threads

The gel rods were dried in a constant temperature and humidity chamber at 20 °C and 60% RH for 24 h under load with a 10 mg weight attached at the one end to prevent shrinking by drying, and we obtained collagen xerogel threads. Tensile stress–strain curves of collagen-dried threads were measured at 28 °C using an AND force tester MCT-2150. The initial gage length was set at 20 mm. The cross-head speed was set at 0.5 mm/s. Every measurement was performed on ten or more test threads. The tensile stress (σ) was calculated as σ = F/πrdry2, where F is the loading force and rdry is the initial radius of the thread, which was obtained from an optical microscope image before the tensile tests. The dried radius (rdry) was typically 0.24 mm. The strain at a stretch (ε) is defined as the change in the length (l) of the sample relative to the initial size of the gage (l0), ε = (l – l0) × 100/l0%. Three parameters characterized the mechanical strength of individual test threads: maximum stress (σmax), maximum strain (εmax), and elastic modulus (E, Young’s modulus). σmax is the point at which the specimen reached its highest stress value, εmax is the strain at which the strain began to break off the samples, and Young’s modulus is determined by fitting a linear trend line between two manually selected points in the linear portion of the curve from the initial toe to the yield point of the failure zone. Young’s modulus (E) was calculated using equation E = σ/ε.

Cell Culture and Observation

Mouse endothelial cell line MS-1 (CRL-2279) was obtained from the ATCC (Rockville, Md.). Cells were cultured in RPMI-1640 medium (Fujifilm, Tokyo, Japan) containing 10% fetal bovine serum (FBS), 100 μg/mL streptomycin, and 100 μg/mL penicillin. The culture medium was changed every 2 days. All cells were grown at 37 °C in 5% CO2. First, the collagen xerogel rod was placed in a 6 cm diameter culture dish. The collagen xerogel rod was then rehydrated in 3 mL of complete medium. After 2 h of the rehydration, a culture solution containing 2 × 105 cells was seeded over the sample. Samples were fixed with 10% formalin and embedded in paraffin after 7 days of culture. Histological examinations were performed in hematoxylin–eosin (H&E)-stained sections.

Results and Discussion

Phase Diagram

Figure shows the microscopy images of collagen gel rods prepared by diffusion of the carbonate buffer solution into a collagen solution enclosed in glass capillaries (A and B were prepared from 1 and 3 wt % collagen solutions, respectively). Images of panels A and B were obtained under bright-field observation, and those of panels A’ and B’ were obtained under crossed-nicol observation. These bright-field and polarized light microscopy images reveal a narrow, elongated region inside the gel with different color contrasts only in the gel prepared with 1 wt % aqueous collagen solution. The polarized light microscopy image indicated that the formed gel part exhibits an intense transmitted light, but the elongated zone has a lower transmitted light. The long and narrow region parallel to the capillary with little polarization appears at around 1% collagen concentration. We investigated this less polarized pathway using confocal laser microscopy, as shown in Figure . The optically sliced images in the direction of the Z-axis of the lying gel rod (parallel to the sample glass plate) suggested that the long path has no fluorescence. In contrast, fluorescence from labeled collagen appeared almost uniformly over the top and bottom of the images on the Z-axis. These combined results indicated that the elongated and unpolarized pathway lacks collagen. The path ejected water and was filled with continuous air when a syringe needle was inserted into the gel-less portion and air was pumped (Figure S1). A ring-shaped gel with an internal cavity was obtained when the resulting gel rod was physically sliced across a cross-sectional section using a cutter and the wetting water was wiped off (Figure S2). This finding implied a macroscopic water phase filling in the pathway.

Figure 1

Microscopy images of the collagen gel rod prepared using (A) 1 wt % and (B) 3 wt % collagen solutions. A and B show under bright-field images. A′ and B′ show crossed-nicol polarized light microscopy images. White bars are 1 mm.

Figure 2

Confocal laser microscopy images of the optically sliced gel rod lying on the cover glass in the Z-axis direction. The collagen gel was gelated with FITC-labeled 1 wt % collagen solution in 25 mM carbonate buffer. The gel rods were optically sliced about every 50 μm. The white bar is 1 mm.

Figure shows a phase diagram of the collagen concentration and carbonate buffer region where the hollow cavity is formed. The single elongated hollow appeared inside the collagen gel rod prepared by around 1 wt % collagen. No hollow cavity was observed in the 3–6 wt % concentrated collagen solution, although the gel forms. At 2 wt % collagen concentration, enough content of the buffer solution was required (carbonate buffer concentration >35 mM) to form the hollow cavity. In contrast, gels prepared with lower collagen concentrations (prepared under 0.5 wt % collagen concentration) had randomly shorter and multiple holes in the interior of the gel rod (Figure S3). Under conditions of high buffer concentrations (carbonate buffer solution >25 mM), the limiting collagen concentration for single hollow cavity formation was below 0.66 wt %. In contrast, the lower buffer concentration (carbonate buffer solution <25 mM) can form a hollow cavity even at 0.66 wt % collagen concentration. In a sufficiently low-concentration buffer solution (carbonate buffer solution concentration <8 mM), singlet cavities were observed in the gel under 0.66–1.5 wt % collagen solution. To summarize, a single hollow cavity is formed in the gel at collagen concentrations of 0.66–2.0 wt % and buffer concentrations of 8–50 mM, as shown in the phase diagram in Figure . The above results indicate that the formation of the hollow cavity requires lower buffer concentrations for low collagen concentrations and, in contrast, higher buffer concentrations for high collagen concentrations. More insufficient buffers below 2 mM and higher buffers above 60 mM failed to form any gel. The reason for the failure to form hollow cavities in the above regions is regarded as insufficient gelation, considering that collagen gelation results from charge neutralization. A highly concentrated buffer solution negatively charges the collagen molecules and would fail to form a gel. The internal size of the capillary in which gels form was independent of the condition of the single hollow formation.

Figure 3

Phase diagram of collagen concentration and carbonate buffer concentration showing the regions where intragel cavities are formed and the typical morphologies, long single hollow (○), short single hollow (Δ), and no cavities (×), are observed in the collagen gel rod. The images in the phase diagram are typical polarizing optical microscopy images under crossed Nicols observed in these areas. The bars inside the pictures are 1 mm.

Cavity Ratio and Unification Distance of the Hollow Cavities inside Collagen Gel Rods

Gel rods usually have multiple cavities at the diffusion end of the buffer, and a single hollow cavity appears away from this diffusing end, as shown in Figure . In the typical gel rod with a single cavity, most of the multiple cavities near the diffusing end of the buffer disappear and become a single cavity at a certain distance (unification distance xuni), as shown in the overall image of Figure A. To characterize the hollows inside gels, the cavity ratio (Scavity = ∑di(x)2/di(x)2) was determined (Figure B). This cavity ratio (Scavity) was plotted against the relative distance (x/x0) from the diffusion end, as shown in Figure C. This plot reveals that the cavity volume decreases from the diffusion edge and stays almost constant from the unification distance (xuni). In this buffer diffusing gelation, the buffer will be more diluted with the distance from the diffusion end due to its consumption. Thus, the gel tube morphology would be affected by the distance from the diffusion end. The effect of the distance from the diffusion end on the morphology implies that the initial concentrations of carbonate buffer and collagen affect the cavity formation. Figure shows the dependence of buffer and collagen concentration on (A) cavity ratio (Scavity) and (B) relative unification distance (xRUD). As expected, the Scavity increased with the gelling agent concentration; this fraction was a positive linear function of the buffer concentration at any collagen concentration (Figure A). This result agrees that Scavity is significant around the diffusion end (Figure A). The xRUD moved away from the diffusion end as the buffer concentration increased (Figure A). In contrast, the Scavity (Figure A′) and xRUD (Figure B′) were inversely proportional to the collagen concentration. The above results are not inconsistent because the higher collagen concentration leads to the higher consumption of the buffer solution when collagen is gelatinized. The tube thickness is given by in the single hollow area and is a positive linear function for collagen concentration and an inversely proportional function for buffer concentration. The length of a single cavity is given by x0(1 – xRUD). Thus, it is a positive proportional function for collagen concentration and an inversely proportional function for buffer concentration.

Figure 4

Characteristics of the cavities in a gel rod. (A) Overall image of a typical gel rod with a single-cavity pathway; 1.0 wt % collagen solution, 25 mM buffer solution, and 1.89 mm capillary diameter. (B) Schematic drawings to explain cavity ratio Scavity and relative unification distance xRUD. (C) Scavity for the collagen tube gel of (A) at relative distance x/x0.

Figure 5

Relationships of the preparation conditions to Scavity and xRUD. Dependencies of Scavity on (A) carbonate buffer and (A′) collagen concentration. Dependencies of xRUD on (B) carbonate buffer and (B′) collagen concentration.

Based on the information obtained from our results, we will briefly discuss the formation mechanism of the single hollow cavity. The collagen enclosed in the capillaries became cloudy when the buffer solution was introduced into the capillary. This white turbidity spreads to the lower part of the capillary with gelation (Figure S3). Because a cloudy gel is formed finally, resulting cavities are formed in the simultaneous phase separation and gelation system. Gelation can freeze a phase-separated system resulting from thickening caused by the increased molecular weight and network formation;[16−18] this thickening was confirmed by the viscosity increase of the aqueous collagen solution mixed with the diluted carbonate buffer solution as a function of collagen concentration and buffer concentration (Figure S4). The kinetics of the simultaneous system of phase separation and gelation[19] leads us to the following. (i) The water phase and collagen-rich phase formed by the phase separation coarsens with time, but the gelation pins this process (thickening). (ii) Highly concentrated gelling agents tend to pin the size of the phase separation in the initial process and form a small water phase. (iii) Lower viscosity in low collagen concentration solutions leads to quick coarsening to macroscopic phase separation. In this system, a glass capillary confined the collagen polymer solution. This glass interface would attract the aqueous phase resulting from the separation because the container glass interface has a higher affinity for aqueous phases than the polymer-rich gel phase. Considering the conditions confined by a glass capillary and the kinetics mentioned above, we can estimate the tube-forming steps of the collagen solution as follows. (1) The buffer solution’s diffusion cancels the collagen molecules’ charge at the diffusion port, causing simultaneous gelation and shrinking due to phase separation. This contraction forms a gap composed of an aqueous phase near the capillary wall. (2) The aqueous phase near the wall diffuses ions faster than the central part of the glass capillary (because of no consumption and no polymer interruption), resulting in a gelation and polymer concentration from the wall side direction. (3) The collagen condensation near the wall (phase separation) reduces the polymer concentration in the central region of the capillary; thus, an aqueous phase eventually appears in this region. The shape is then gelatinized (immobilized). A highly concentrated buffer near the diffuser inlet can pin multiple small domains in the early stages of coarsening, and numerous cavities are formed there. Conversely, the lower buffer far from the diffusion end, consumed and diluted by gelation, leads to coarsening before an increase in viscosity, and the aqueous phase unifies into a single hollow cavity by Ostwald ripening.[20] This scenario can explain the collagen and buffer concentration dependence on xRUD and Scavity presented above. If collagen condensation near the glass wall was symmetrical to the cylindrical axis, the cavities would appear at the center of the gel rod. However, the cavity appears offset more or less from the cylinder axis center, as shown in Figure . This cavity dislocation from the center demonstrates that the buffer diffusion plane is no longer parallel to the plane of the diffusion end as it progresses, and the collagen separates asymmetrically around the cylinder axis.

Mechanical Properties

To know the mechanical properties of the collagen tube, we investigate properties obtained from the tensile test. Figure shows the typical stress–strain curves of dried collagens tubes prepared with (A) various collagen concentrations and (B) various buffer solutions. The resulting stress–strain curve is classified into two phases with different tensile strains in the initial and later phases; the initial one below about 10% strain has a more significant slope, and the latter one after the strain has a smaller slope. In the case of the dried threads of collagen formed from 1.0 wt % collagen solution in 25 mM buffer solution, Young’s moduli were 33 and 15 MPa in the initial and second stages, respectively; the initial Young’s modulus was about 2 times larger than that of the second stage. The initial properties were reversible for the mechanical stretch, though they are irreversible for the sample elongated into the latter region, resulting in no return to the original length before the stretch. Next, we discuss the dependence of Young’s modulus, maximum stress, and maximum strain resulting from these stress–strain curves on the collagen and buffer concentrations. Young’s moduli in the early and late regions increased with the collagen concentration, while the buffer solution had no significant impact on the moduli, as shown in Figure A,C. The maximum tensile stress also increased with the collagen concentration (Figure D). The tensile stress of the collagen rod prepared at 1.5 wt % collagen concentration was about double that of the 0.66 wt % collagen concentration. The maximum strain decreased with the collagen concentration, contrary to the maximum stress. The buffer concentration mainly affects the maximum tensile stress and strain, especially in the 0.66 and 1.5 wt % collagen samples (see Figure D,E). The effects on maximum stress and Young’s modulus of collagen concentration may be due to the cross-link point density because Young’s modulus of polymeric elastic materials is inversely proportional to the distance between the inter-cross-link points.[21] The cloudiness during the gelation of aqueous collagen solutions is mainly the result of the formation of microscopic collagen fibrils. The fiber aggregates will be produced by polymer–molecule interactions associated with neutralizing the collagen molecule’s dissociated groups by the carbonate ions. Since the collagen concentration determines the number of network knots, these mechanical properties must depend on our gelatinization concentration. We observed little dependence on buffer concentration, possibly because 8 mM is a high enough concentration to neutralize most collagen molecules, and thus, the cross-link density was saturated. These resulting mechanical properties demonstrated that Young’s moduli and maximum strains of the dried collagen tubing threads were comparable to those of nylon,[22] and the maximum stress was more than twice more substantial. We believe that our collagen threads have sufficient mechanical properties to handle in the gas phase, whereas further research is required on their cellular affinity, mechanical properties in solution, and degradation stability if they are to be applied to medical materials such as sutures and regenerative medicine materials.

Figure 6

Mechanical strength characteristics of the dried collagen threads. Typical stress–strain curves of various collagen rods obtained by diffusing (A) 25 mM buffer concentration into collagen solutions with various concentrations and (B) various buffers in the concentration into 1.0 wt % collagen. (C) Young’s modulus E of the initial and later phase, (D) maximum stress σmax, and (E) maximum strain εmax dependencies on the collagen and the buffer concentrations.

Usefulness as a Cell Culture Scaffold

The usefulness as a cell culture scaffold is defined by the viability and migration of cultured cells. In addition, in a culture scaffold with a luminal structure, maintenance of the luminal structure under the culture condition is essential. As shown in Figure , the endothelial cells seeded on the surface of the collagen tube migrated, proliferated, and covered the lumen of the tube. The construction of a vascular network is an urgent issue in current regenerative medicine, but a technique for artificially creating a complicated capillary network has not been established. Our collagen tube has excellent patency and biocompatibility with cells, which can be a fundamental technology to solve the problem mentioned above.

Figure 7

Micrograph of vascular endothelial cells (cell line, MS-1, ATCC) observed in the cavity of a collagen rod. The collagen rod was placed in a culture solution seeded with 2 × 105 cells and incubated at 37° C, 5% CO2, for 7 days. H&E staining was used to detect the vascular endothelial cells. The white area surrounded by the colored area indicates the cavity of the collagen rod. Arrows indicate vascular endothelial cells. The white bar is 0.5 mm.

Conclusions

We have presented a one-step method to obtain less than 0.5 mm collagen tubular gels in diameter. The tubular gel can be fabricated by gelling a collagen solution packed in a glass capillary using a carbonate buffer solution from one end of the capillary. A single hole was found in gels formed at collagen concentrations of 0.66–2.0 wt % and buffer concentrations of 8–50 mM. The hole volume in the gels is large and multiple near the carbonate buffer diffusion end and then decreases with the distance, leading to a single hole. Downstream from that single hole-forming distance, the hole diameters become nearly constant. The hole volume is proportional to the carbonate buffer concentration. The dependence of the volume on the gelation buffer concentration is reduced for higher collagen concentrations. These hole-forming distances are farther from the end of the buffer diffusion, especially at low collagen concentrations. The mechanical property measurements demonstrated that the collagen concentration significantly increases Young’s modulus and maximum stress, resulting in almost similar mechanical properties to nylon threads. Since the microdiameter collagen tube has excellent patency and performance as a cell culture carrier, it is expected to contribute significantly to constructing the capillary network in regenerative medicine.