High-speed spinning of collagen microfibers comprising aligned fibrils for creating artificial tendons.

Bundles of engineered collagen microfibers are promising synthetic tendons as substitutes for autogenous grafts. The purpose of this study was to develop high-speed and continuous spinning of collagen microfibers that involves stretching of collagen stream. Our study revealed the 'critical fibrillogenesis concentration (CFC)' of neutralized collagen solutions, which is defined as the upper limit of the collagen concentration at which neutralized collagen molecules remain stable as long as they are cooled (⩽10 °C). Neutralized collagen solutions at collagen concentrations slightly below the CFC formed cord-like collagen gels comprising longitudinally aligned fibrils when extruded from nozzles into an ethanol bath. Dry collagen microfibers with a controlled diameter ranging from 122 ± 2-31.2 ± 1.7 µm can be spun from the cord-like gels using nozzles of various sizes. The spinning process was improved by including stretching of collagen stream to further reduce diameter and increase linear velocity. We extruded a collagen solution through a 182 µm diameter nozzle while simultaneously stretching it in an ethanol bath during gelation and fiber formation. This process resembles the stretching of a melted thermoplastic resin because it solidifies during melt spinning. The mechanical properties of the stretched collagen microfibers were comparable to the highest literature values obtained using microfluidic wet spinning, as they exhibited longitudinally aligned fibrils both on their surface and in their core. Previous wet spinning methods were unable to generate collagen microfibers with a consistent tendon-like fibrillar arrangement throughout the samples. Although the tangent modulus (137 ± 7 MPa) and stress at break of the swollen bundles of stretched microfibers (13.8 ± 1.9 MPa) were lower than those of human anterior cruciate ligament, they were within the same order of magnitude. We developed a spinning technique that produces narrow collagen microfibers with a tendon-like arrangement that can serve as artificial fiber units for collagen-based synthetic tendons.

Plasticizer-gelatin mixed solutions as skin protection materials with flexible-film-forming capability.

To demonstrate the feasibility of plasticizer-gelatin solutions as novel skin protection materials from a physical aspect, we evaluated the rheological properties of the solutions and the mechanical properties and textures of their dried sheets and films. Three types of sugars and polyols were employed as organic plasticizers and mixed with gelatin in solutions at plasticizer/gelatin weight ratios of 0.13-1.67. The plasticizers minimally affected the viscosities and gelation temperatures of the gelatin solutions, but they remarkably softened dried gelatin sheets, except for propylene glycol. Glycerol exhibited the best plasticizing effects, but the sheets obtained using glycerol showed tacky textures. Preliminary investigations on the film-forming properties of the solutions on the human skin showed that the fructose-gelatin solution at a weight ratio of 1.0 formed a flexible thin film with a texture and mechanical properties similar to those of a commercially available polyurethane-based flexible film dressing. In terms of physical properties, we conclude that the fructose-gelatin solution has potential as a skin protection material that transforms from a solution to a film on the skin.

Effect of prophylactic dressings to reduce pressure injuries: a polymer-based skin model.

OBJECTIVE: This study evaluated the effect of pressure injury (PI) prophylactic dressings used for patients at high risk of PI development to reduce friction, shear force and pressure, and their combined force, in an original polymer-based skin model. METHOD: A low-friction outer-layer hydrocolloid (LFH) dressing and a multilayered silicone foam (MSF) dressing were used. Before application, compression and friction properties were measured. Our original experimental model-the 'simulated skin-shearing test'-consisted of: a weight; a polyurethane-based skin model containing a three-axis tactile sensor; dressings; a table covered with bedsheets; and a mechanical tester, by which the interface friction force, internal shear force and pressure were measured continuously during skin model movements. An estimated combined force generated by internal shear and pressure was represented as a vector. A model with no dressing was used as a control. RESULTS: The LFH dressing had significantly higher compression strength versus the MSF dressing. In contrast, the dynamic coefficient of friction was lower for the LFH dressing versus the MSF dressing (p<0.05). In simulated skin-shearing test results, shear forces were 0.45N and 0.42N for LFH and MSF dressings, respectively, with no significant difference. The estimated combined force was lower for the MSF dressing compared with that of the LFH dressing and control. CONCLUSION: The shear force-reducing effect in the skin model was equivalent between the LFH and MSF dressings. However, the MSF dressing significantly reduced the force generated by a combination of internal shear force and pressure compared with the LFH dressing.

Comparison of decellularization protocols for cultured cell-derived extracellular matrix-Effects on decellularization efficacy, extracellular matrix retention, and cell functions.

The in vitro reconstruction of the extracellular matrix (ECM) is required in tissue engineering and regenerative medicine because the ECM can regulate cell functions in vivo. For ECM reconstruction, a decellularization technique is used. ECM reconstructed by decellularization (dECM) is prepared from tissues/organs and cultured cells. Although decellularization methods have been optimized for tissue-/organ-derived dECM, the methods for cultured cell-derived dECM have not yet been optimized. Here, two physical (osmotic shocks) and five chemical decellularization methods are compared. The decellularization efficacies were changed according to the decellularization methods used. Among them, only the Triton X-100 and Tween 20 treatments could not decellularize completely. Additionally, when the efficacies were compared among different types of cells (monolayered cells with/without strong cell adhesion, multilayered cells), the efficacies were decreased for multilayered cells or cells with strong cell adhesion. Retained ECM contents tended to be greater in the dECM prepared by osmotic shocks than in those prepared by chemical methods. The contents impacted cell adhesion, shapes, growth and intracellular signal activation on the dECM. The comparison would be helpful for the optimization of decellularization methods for cultured cells, and it could also provide new insights into developing milder decellularization methods for tissues and organs.

Potential of temperature-response collagen-genipin sol as a novel submucosal injection agent for endoscopic resection: Acute and chronic phase study using living animals.

OBJECTIVES: We proposed a novel temperature-response collagen sol as a submucosal injection agent for endoscopic resection (ER) using pepsin-solubilized collagen (PSC) and genipin (Ge) in a prior study. This study aimed to evaluate the usefulness and safety of the sol (PSC/Ge) in acute and chronic phase experiments using living animals. METHODS: In experiment 1, we performed endoscopic submucosal dissection (ESD) for six pigs using normal saline (NS), sodium hyaluronate (SH), and PSC/Ge. We compared the required amount of each agent per unit area and procedure time. In experiment 2, we created artificial ulcers with endoscopic mucosal resection (EMR) for five pigs using NS and PSC sol. We compared the artificial ulcer residual rate at 7 and 14 days after EMR, and the scarring rate at 14 days after EMR. RESULTS: The required amount of agents per unit area for PSC/Ge (0.8 ± 0.8 mL/cm2 ) and SH (1.1 ± 0.8 mL/cm2 ) were significantly smaller than that for NS (1.8 ± 0.7 mL/cm2 ). The total procedure time did not have a statistical difference. The artificial ulcer residual rates were 47.3 ± 0.7% for NS and 40.3 ± 0.7% for PSC/Ge on day 7 (P = 0.51), and 15.0 ± 0.1% for NS and 10.2 ± 0.1% for PSC/Ge sol on day 14 (P = 0.35). The scarring rate on day 14 was 10% for NS and 20% for PSC/Ge. CONCLUSION: We demonstrated the feasibility of a novel temperature-response collagen gel as a submucosal injection agent for ER in the acute and chronic phase animal experiment.

Characterization and preliminary in vivo evaluation of a self-expandable hydrogel stent with anisotropic swelling behavior and endoscopic deliverability for use in biliary drainage.

We demonstrate the potential of a novel self-expandable biliary stent comprised of poly(vinyl alcohol) (PVA) hydrogel with anisotropic swelling behavior and endoscopic deliverability in vivo, using a porcine stent model. The mechanism underlying the anisotropic swelling behavior and endoscopic deliverability (i.e., flexibility) was investigated by scanning electron microscopy (SEM), small-angle X-ray scattering (SAXS), evaluation of the water content and swelling ratio, and three-point bending tests. The in vivo experiment using a porcine stent model indicated that the tube-shaped PVA hydrogel could effectively expand the biliary tract, without disturbing bile flow. SEM and SAXS showed that PVA hydrogels prepared by drying under extension showed structural orientation along the extension axis, leading to anisotropic swelling. The water content of the PVA hydrogel was found to be crucial for maintaining flexibility as well as endoscopic deliverability. In conclusion, this study demonstrated the novel concept of using a hydrogel stent as a self-expandable biliary stent.

Fibril matrices created with collagen from the marine fish barramundi for use in conventional three-dimensional cell culture.

Collagen obtained from fish offal (skin, scales, and bones) is required from some religious and ethnic groups, thus indirectly increasing demands for fish collagen for biomedical applications. The limitation of fish collagen is its lower thermal stability compared to mammalian collagen. In this study, we focused on collagen extracted from scales of the marine fish barramundi (Lates calcarifer) and demonstrated the suitability for the collagen to be utilized in collagen fibril matrices (CFM). Collagen was extracted from the scales through pepsin-digestion and purified (designated as "BC"). The denaturation temperature (Td) for BC was determined to be 36.4 °C, one of the highest among fish collagens. BC formed CFM which was thermally stable at 37 °C, while Td was lower than 37 °C. This could be explained by the fast fibril formation, initiating at temperatures near 20 °C in a temperature-elevated process. As a result, the NIH3T3 cells were successfully encapsulated in the CFM of BC and cultured three-dimensionally for 7 d. The cells spread and exhibited well-developed pseudopodia in the CFM of BC as observed in the CFM of pig collagen matrices. This is the first report on fish CFM used for conventional 3-D cell culture.

Closure of gastric perforations during endoluminal resection using a novel biodegradable collagen sol: A feasibility survival study on porcine model (with video).

OBJECTIVE: A prior study using porcine colon demonstrated the feasibility of a novel injectable, temperature?responsive, and biodegradable collagen sol (ICS) that transforms from a liquid to a gel state in response to body temperature for endoscopic closure of perforation during endoscopic resection (ER). This study aims to report the acute and survival outcomes of ICS for gastric perforations during ER. METHODS: In two experiments using nine live pigs under general anesthesia, four and six perforations (3-5\xA0mm) were created using an electrosurgical knife in acute and survival experiments, respectively. ICS was delivered to the perforations using an endoscopic catheter. In Experiment 1, a leak test and histopathology were performed on all explanted stomachs after euthanization. In Experiment 2, perforation sites were assessed by gastroscopy and histopathology 7, 14 and 28\xA0days post? RESULTS: In Experiment 1, gastroscopy confirmed complete closure of the perforations with ICS and no evidence of leak. Subsequent histopathology revealed a fixation of collagen gel (CG) as a sealant agent at the perforation sites. There were no adverse effects related with ESD or the use of ICS. In Experiment 2, histopathology revealed a fixation of CG as a sealant agent, replacement with granulation tissue and no CG; and fibrotic tissue at 7, 14 and 28\xA0days, respectively. CONCLUSIONS: This study presents a novel method using ICS, demonstrating promising efficacy and safety profile for endoscopic closure of perforations during ER. Further studies are necessary before translating to clinical use.

Evaluation of the Physiological Corneal Intrastromal Riboflavin Concentration and the Corneal Elastic Modulus After Violet Light Irradiation.

Purpose: KeraVio is a corneal crosslinking treatment modality that utilizes violet light (VL)-emitting glasses and topical epithelium-on riboflavin administration. We focus on the new KeraVio protocol without riboflavin. This study aims to quantify the physiological intrastromal concentrations of riboflavin in corneas without riboflavin decreases and evaluate the biomechanics of corneas after VL irradiation. Methods: Twelve human donor corneas were included in this study and randomly categorized into four groups. The corneas underwent four imbibition techniques (physiological riboflavin without drops, epithelial [epi]-on with 0.05% flavin adenine dinucleotide [FAD], epi-off with FAD, and 0.1% riboflavin epi-off). Corneas in the FAD epi-on, FAD epi-off, and riboflavin epi-off groups were instilled with the respective solution every 2 minutes for 30 minutes. An ex vivo experiment was conducted with 24 porcine corneas arranged into three treatment groups and one control group. Corneas in the KeraVio with FAD epi-on group were treated with VL irradiation at 0.31 mW/cm2 for 4.8 hours (5.4 J/cm2) and simultaneously received FAD drops every 30 minutes during the VL irradiation. Corneas in the group with KeraVio without FAD epi-on were only treated with VL irradiation (5.4 J/cm2). Results: We identified the original physiological riboflavin of human corneal stroma at a concentration of 0.31 ± 0.03 µg/g, but its value was approximately 39-fold smaller than that in the 0.1% riboflavin epi-off group. The group with KeraVio without FAD and the standard corneal crosslinking group showed a significant increase in biomechanical stability compared with the controls, whereas the elastic modulus in the treated groups was equivalent. Conclusions: We preliminarily identified physiological riboflavin in human corneas without adding riboflavin drops. The VL exposure may strengthen the corneal biomechanics without requiring the use of additional riboflavin drops. Translational Relevance: We preliminarily identified physiological riboflavin in the human cornea without adding riboflavin drops. VL irradiation without riboflavin drops may increase the corneal stiffness using physiological riboflavin.

Crosslinking Efficacy and Cytotoxicity of Genipin and Its Activated Form Prepared by Warming It in a Phosphate Buffer: A Comparative Study.

The aim of the present study was to compare the acute and cumulative cytotoxicity of intact (n-GE) and warmed genipin (w-GE), while investigating the differences in crosslinking capabilities of these two genipins by rheological and mechanical tests. The n-GE solution was prepared by dissolving genipin powder in a sodium phosphate buffer solution. The w-GE solution was prepared by warming the n-GE solution at 37 °C for 24 h. The mechanical tests for chitosan (CH)/genipin gels showed the crosslinking rate of w-GE was much greater than that of n-GE up until 6 h after preparation, whereas the degree of crosslinking of CH/n-GE gels became higher at 12 h. The ISO 10993-5 standard method, which is established specifically for evaluating cumulative cytotoxicity, determined equivalent IC50 for w-GE (0.173 mM) and n-GE (0.166 mM). On the other hand, custom-made cytotoxicity tests using a WST-8 assay after 1 h of cultivation showed that the acute cytotoxicity of w-GE was significantly higher than that of n-GE at concentrations between 0.1-5 mM. The acute cytotoxicity of w-GE should be taken into consideration in its practical uses, despite the fact that the much faster crosslinking of w-GE is useful as an effective cross linker for in-situ forming gels.

Novel temperature-responsive, biodegradable and injectable collagen sol for the endoscopic closure of colonic perforation holes: Animal study (with videos).

OBJECTIVES: Endoscopic submucosal dissection (ESD) poses a risk of intraprocedural perforation. We have developed a biodegradable injectable collagen sol that undergoes a liquid-to-gel formation in response to body temperature. Here, we investigated the feasibility of this novel collagen sol for the endoscopic closure of iatrogenic perforation holes. METHODS: In two experiments, 12 and 5 colonic perforation holes (3-5 mm) were made using an ESD knife in four and three live pigs under general anesthesia, respectively. In Experiment 1, collagen sol was delivered to the perforation holes using an endoscopic catheter. When the colon was expanded by CO2 insufflation, endo-clips were applied to the perforation holes. For Experiment 2, Collagen sol adjusted based on the Experiment 1 results was delivered to the perforation holes in the same manner. A leak test was performed for every colon after the pigs were killed, and the histology of the perforation sites was evaluated. RESULTS: In both experiments, collagen sol was smoothly delivered to the target area and fixed as a gel on the perforation holes. Experiment 1, 83% (10/12) of the perforation holes were completely closed, and all endo-clips were placed with composure. Experiment 2, all perforation holes were completely closed with collagen gel. There was no leak from the perforation holes. Histology revealed a fixation of the collagen gel as an embolus agent in the perforation holes. CONCLUSIONS: This novel collagen sol may be used for the endoscopic closure of intraprocedural perforation. Further studies will determine this collagen sol's clinical feasibility and safety.

Potential of temperature-response collagen-genipin sols as a novel submucosal injection material for endoscopic resection.

Background and study aims We developed a novel submucosal (SM) injection material that contained pepsin-solubilized collagen (PSC), genipin (Ge) and phosphate buffer (PB). The aim of this study was to validate safety and usability of it for endoscopic resection (ER). Materials and methods In preliminary studies, 1) appropriate warming time and concentration of Ge, and concentration of NaCl in PB, 2) storage modulus of PSC, Ge, and PB mixture (PSC/Ge), and PSC as a mechanical property, 3) histological finding after injection, and histological toxicity of PSC/Ge was evaluated. We injected PSC/Ge, PSC, sodium hyaluronate (SH), dextrose (DW), and normal saline (NS) into SM of resected porcine stomach. We compared mean height of mucosal elevation after immediate injection (MH) and mean retaining rate at 60 minutes (MR) as ex vivo study. Results Optimal condition of PSC/Ge was Ge 5.5 mMol with 24 hours worming time and NaCl 280 mMol. PSC/Ge had better mechanical property than PSC. It was efficiently integrated and confined to the SM with acceptable toxicity. MH of PSC/Ge (5.1 ±â€Š0.74 mm) and PSC (4.8 ±â€Š0.84 mm) were significantly higher than NS (3.2 ±â€Š0.84 mm). MR of PSC/Ge (100 ±â€Š0.0%) was significantly higher than NS (61.7 ±â€Š11.2%), DW (58.3 ±â€Š11.8%) and SH (61.8 ±â€Š8.6%). Conclusion PSC/Ge and PSC has potential to be safe and usable for ER. PSC/Ge was better than PSC because of better mechanical property than PSC.

The effect of alkaline pretreatment on the biochemical characteristics and fibril-forming abilities of types I and II collagen extracted from bester sturgeon by-products.

Non-mammalian collagens have attracted increasing attention for industrial and biomedical use. We have therefore evaluated extraction conditions and the biochemical properties of collagens from aquacultured sturgeon. Pepsin-soluble type I and type II collagen were respectively extracted from the skin and notochord of bester sturgeon by-products, with yields of 63.9 ±â€¯0.19% and 35.5 ±â€¯0.68%. Collagen extraction efficiency was improved by an alkaline pretreatment of the skin and notochord (fewer extraction cycles were required), but the final yields decreased to 56.2 ±â€¯0.84% for type I and 31.8 ±â€¯1.13% for type II. Alkaline pretreatment did not affect the thermal stability or triple-helical structure of both types of collagen. Types I and II collagen formed re-assembled fibril structures in vitro, under different conditions. Alkaline pretreatment slowed down the formation of type I collagen fibrils and specifically inhibited the formation of thick fibril-bundle structures. In contrast, alkaline pretreatment did not change type II collagen fibril formation. In conclusion, alkaline pretreatment of sturgeon skin and notochord is an effective method to accelerate collagen extraction process of types I and II collagen without changing their biochemical properties. However, it decreases the yield of both collagens and specifically changes the fibril-forming ability of type I collagen.

Ex vivo assessment of anchoring force of covered biflanged metal stent and covered self-expandable metal stent for interventional endoscopic ultrasound.

BACKGROUND AND AIMS: Endoscopic ultrasound (EUS)-guided transmural drainage using a covered biflanged metal stent (CBFMS) and a conventional tubular biliary covered self-expandable metal stent (CSEMS) has recently been performed by EUS experts. However, appropriate traction force of the sheath to prevent the migration during stent deployment is well unknown. Herein, we assessed the anchoring force (AF) of the distal flange in CBFMSs and CSEMSs. METHODS: The AFs of four CBFMSs (Stents AX, NG, PL, and SX) and six CSEMSs (Stents BF, BP, EG, HN, SP, and WF) were compared in an ex vivo setting. We assessed the AF produced by each stent using an EUS-guided transmural drainage model and an EUS-guided hepaticogastrostomy model consisting of sheet-shaped specimens of the stomach, gelatin gel, and gelatin tubes. RESULTS: For CBFMSs, the maximum AF of Stent AX was significantly higher than those of Stents PL and SX (P < 0.05) in the porcine model. In the gelatin series, all stents except Stent NG showed a nearly similar AF. For CSEMSs, Stents HN, EG, BF, and WF showed gradual AF elevation in the porcine stomach. Stents SP and BP showed a lower AF than the other four stents. For the gelatin setting, the maximum AF of Stents HN, EG, and WF was higher than those of the other stents regardless of the type of specimens. CONCLUSIONS: The significance of the AF and traction distance according to the property of various CBFMSs and CSEMSs could be elucidated using ex vivo models.

A novel method for continuous formation of cord-like collagen gels to fabricate durable fibers in which collagen fibrils are longitudinally aligned.

We developed a continuous formation method of cord-like collagen gels comprising fibrils preferentially aligned along the geometrical axes (CCGs). Collagen (2.5%) dissolved in a sodium phosphate buffer containing 280 mM of sodium chloride was introduced into a stainless cylinder (length 52 mm, diameter 2.0 mm) warmed at 38°C at a linear velocity of 2.5 mm/s. This process caused collagen fibril alignments under acute fibril formation in the cylinder, resulting in continuous formation of CCGs. Fibril formation rate, shear rate, and shear duration were substantial factors for successful CCG formation. Outstanding advantages of this method over conventional wet spinning include the capacity of this technique to form aligned fibrils in the entire gels and to control the diameter of cord-like gels over 1 mm. The air-drying of CCGs which were crosslinked with 1-ethyl-3-(3-dimethylaminopropyl)-carbodiimide and N-hydroxy-succinimide produced dry collagen fibers with cross-sectional areas of 0.0123-0.135 mm2 . Upon the rewetting of the fibers, they failed at a stress of 54.5 ± 7.8 MPa, which is higher than the mean failure stress of anterior cruciate ligament tissue (13.3-37.8 MPa). These findings indicate that the CCG formation method enables the fabrication of collagen fibers which are potential components of collagen-based artificial tendons. © 2018 Wiley Periodicals, Inc. J Biomed Mater Res Part B: Appl Biomater 107B: 1011-1023, 2019.

In situ gelation properties of a collagen-genipin sol with a potential for the treatment of gastrointestinal ulcers.

We investigated the potential of collagen-genipin sols as biomaterials for treating artificial ulcers following endoscopic submucosal dissection. Collagen sol viscosity increased with condensation, allowing retention on tilted ulcers before gelation and resulting in collagen gel deposition on whole ulcers. The 1.44% collagen sols containing genipin as a crosslinker retained sol fluidity at 23°C for >20 min, facilitating endoscopic use. Collagen sols formed gel depositions on artificial ulcers in response to body temperature, and high temperature responsiveness of gelation because of increased neutral phosphate buffer concentration allowed for thick gel deposition on tilted ulcers. Finally, histological observations showed infiltration of gels into submucosal layers. Taken together, the present data show that genipin-induced crosslinking significantly improves the mechanical properties of collagen gels even at low genipin concentrations of 0.2-1 mM, warranting the use of in situ gelling collagen-genipin sols for endoscopic treatments of gastrointestinal ulcers.

In Vitro Parallel Evaluation of Antibacterial Activity and Cytotoxicity of Commercially Available Silver-Containing Wound Dressings.

PURPOSE: This study evaluated the in vitro antibacterial activity and cytotoxicity of various commercially available silver-containing dressings (Ag dressing). METHODS: Biohesive Ag (hydrocolloid, silver sulfadiazine), Aquacel® Ag (nonwoven fabric, ionic silver [Ag]), Algisite™ Ag (nonwoven fabric, Ag), Mepilex® Ag (foam, silver sulfate), and PolyMem® Ag (foam, nanocrystalline silver) were tested for characteristics of Ag release, antibacterial activity, and cytotoxicity. The release of Ag was investigated in cell culture medium at immersion periods of 6, 24, and 48 hours. The antibacterial activity against Staphylococcus aureus and Pseudomonas aeruginosa were accessed by a disc diffusion test. The cytotoxicity was evaluated using V79 cells, by an extraction method. RESULTS: The cytotoxicity was not a monotonic function of the antibacterial activity among the Ag dressings and could not be simply explained by Ag-release properties. Biohesive Ag was regarded as a slow-release Ag dressing, showing the lowest cytotoxicity, while the antibacterial activity was classified as "strong" or "significant" against the two species of bacteria. Aquacel Ag and Algisite Ag showed higher antibacterial activity and cytotoxic effects, which were supported by the higher Ag release. Mepilex Ag showed the highest release of Ag, and the cytotoxicity was the highest among the Ag dressings. However, the antibacterial activity was classified as "significant" or "no activity" for P. aeruginosa and S. aureus, respectively. PolyMem Ag showed the lowest Ag release, and the antibacterial activity classified as "significant" or "no activity" for S. aureus and P. aeruginosa, respectively, whereas the cytotoxicity was similar to those of Aquacel Ag and Algisite Ag. CONCLUSION: The efficacy and adverse effects of the Ag dressings revealed differences that should be considered by clinicians during wound management.

A novel fabrication method to create a thick collagen bundle composed of uniaxially aligned fibrils: an essential technology for the development of artificial tendon/ligament matrices.

In this study, we developed a fabrication method for thick collagen gel bundles comprising uniaxially aligned fibrils of sufficient size for filling defects in ligament tissues. The fabrication involved rotary shearing to dense collagen sols using a rheometer and then warming them from 23°C to 37°C to trigger gelation upon rotation. Gelation due to collagen fibril formation was accelerated by increased concentrations of neutral phosphate buffer, and fibril alignment occurred within 20 s during the early stage of rapid gelation. Fabrication of gels was completed with slippage between gels and the movable upper plate, and well-aligned fibrils along the rotation direction were observed in the marginal regions of disc-shaped gels. Gel thickness could be increased from 1 to 3 mm with homogeneous alignment of fibrils in the entire sample. The alignment of fibrils improved mechanical properties against tensile loads that were placed parallel to the alignment axis. Elongation of cultured fibroblast along the alignment was observed on the gels. The present method will enable the bottom-up fabrication of an artificial tendon for ligament reconstruction and repair.

Evaluation of gelatin hydrogel as a potential carrier for cell transportation.

We prepared uncleaved gelatin composed mainly of collagen α-, ß-, and γ-chains. Gelation and melting of uncleaved gelatin occurred rapidly with moderate decrease and increase in temperature (23°C-37°C). The viability of cells encapsulated in the gelatin gel was greater than 96% after 7 d at 23°C.

Temperature-responsive gelation of type I collagen solutions involving fibril formation and genipin crosslinking as a potential injectable hydrogel.

We investigated the temperature-responsive gelation of collagen/genipin solutions using pepsin-solubilized collagen (PSC) and acid-solubilized collagen (ASC) as substrates. Gelation occurred in the PSC/genipin solutions at genipin concentrations 0-2 mM under moderate change in temperature from 25 to 37°C. The PSC/genipin solutions exhibited fluidity at room temperature for at least 30 min, whereas the ASC/genipin solutions rapidly reached gel points. In specific cases PSC would be preferred over ASC as an injectable gel system. The temperature-responsive gelation of PSC/genipin solutions was due to temperature responses to genipin crosslinking and collagen fibril formation. The elastic modulus of the 0.5% PSC/genipin gel system could be adjusted in a range of 2.5 to 50 kPa by the PSC and genipin concentrations, suggesting that a PSC/genipin solution is a potential injectable gel system for drug and cell carriers, with mechanical properties matching those of living tissues.