Toshia Fujisato

Control of myotube contraction using electrical pulse stimulation for bio-actuator

The contractility of tissue-engineered muscle on the application of electrical signals is required for the development of bio-actuators and for muscle tissue regeneration. Investigations have already reported on the contraction of myotubes differentiated from myoblasts and the construction of tissue-engineered skeletal muscle using electrical pulses. However, the relationship between myotube contraction and electrical pulses has not been quantitatively evaluated. We quantitatively investigated the effect of electrical pulse frequency on the excitability of myotubes and developed bio-actuators made of tissue-engineered skeletal muscle. C2C12 cells were seeded on a collagen-coated dish and in collagen gel and were cultured in Dulbecco’s modified Eagle’s medium (DMEM) containing 10% fetal bovine serum and antibiotics. When the cells reached confluence or after 2 days in culture, the medium was shifted to DMEM containing 7% horse serum to allow them to differentiate to C2C12 myotubes. We electrically stimulated the myotubes and tissue-engineered skeletal muscle, and contractions were observed under a microscope. The myotubes contracted synchronously with electrical pulses between 0.5 and 5 Hz and unfused tetanus was generated at 10 Hz. The contractile performance of tissue-engineered skeletal muscle made of collagen gel and C2C12 was similar to that of the myotubes. Both the rheobase and chronaxie of the myotubes were lowest when the electric field was applied parallel to the myotube axis, and the values were 8.33 ± 2.78 mA and 1.19 ± 0.38 ms, respectively. The motion of C2C12 myotube contraction depended on the pulse frequency and showed anisotropy in the electric field. These results suggest that a tissue-engineered bio-actuator may be controlled using electrical signals.

Preparation and Recellularization of Tissue Engineered Bioscaffold for Heart Valve Replacement

Tissue engineered grafts based on polymeric or acellular xenogeneric matrices have been widely studied, and found to have greater durability and functionality with growth potential and less immunogenicity than current bioprostheses. On the other hand, there are still several problems to be solved such as degradation control of biodegradable polymeric scaffolds and unwanted transfer of unknown animal related infectious diseases. In this chapter, our novel tissue processing of decellularization named PowerGraft by ultrahigh pressure treatment for safe tissue transplantation is reported. Porcine heart valves were isolated under sterile conditions and treated by cold isostatic pressing (CIP) at 4°C for disruption of donor cells. The cell debris was then washed out in PBS under microwave irradiation at 4°C. The tissues were completely cell free when they were treated by a CIP of 980 MPa (10,000 atm) for 10 min. There was no porcine endogeneous retrovirus (PERV) detected in the treated tissue. There were no significant changes in biomechanical properties of breaking strength and elastic modulus. From the in vitro incubation test, the tissues were disinfected when CIP was applied to the tissues contaminated by normal bacteria floras. The endothelial cells were well seeded on the acellular bioscaffold by the roller and circulation culture systems sequentially. This PowerGraft processing may provide a more durable and safe bioscaffold for tissue transplantation.

Peripheral nerve regeneration and electrophysiological recovery with CIP-treated allogeneic acellular nerves.

Acellular nerve grafts are a desirable alternative to autografts, both because the source of acellular nerves is potentially unlimited and because they have the same matrix structure as natural nerves, which would facilitate axon growth from the defective nerve stump. Although some acellular nerves have been developed, most of them were studied in isogenic transplantation models and evaluated only by histological observation. In the present study, novel allogeneic acellular nerves prepared using the cold isostatic pressuring (CIP) method were developed and assessed as a potential substitute for autografts. The host immune response to acellular nerves and fresh nerves was analyzed using Lewis rats as donors and SD rats as recipients, which is the allogeneic transplantation model, by subcutaneous implantation for one month. In addition, sciatic nerve transplantation into a 10-mm nerve gap was carried out using the same model, and the axonal growth in acellular nerve transplantation was evaluated histologically and electrophysiologically, and compared with that of axons in the autograft transplant area. The subcutaneously implanted acellular nerves contained more macrophages and less vasculature than the allogeneic fresh nerves. In spite of these results of the subcutaneous implantation, Schwann cell infiltration in the graft transplanted into the sciatic nerve gap was observed after the short-term transplantation. The myogenic potential, which was measured as an index of electrophysiological function in acellular nerve transplantation, was also recovered in the long-term transplantation. Our results indicate that the acellular nerves developed herein have the potential to support nerve regeneration and might be useful as an alternative to autografts.

The Rapid Inactivation of Porcine Skin by Applying High Hydrostatic Pressure without Damaging the Extracellular Matrix

We previously reported that high hydrostatic pressure (HHP) of 200 MPa for 10 minutes could induce cell killing. In this study, we explored whether HHP at 200 MPa or HHP at lower pressure, in combination with hyposmotic distilled water (DW), could inactivate the skin, as well as cultured cells. We investigated the inactivation of porcine skin samples 4 mm in diameter. They were immersed in either a normal saline solution (NSS) or DW, and then were pressurized at 100 and 200 MPa for 5, 10, 30, or 60 min. Next, we explored the inactivation of specimens punched out from the pressurized skin 10 × 2 cm in size. The viability was evaluated using a WST-8 assay and an outgrowth culture. The histology of specimens was analyzed histologically. The mitochondrial activity was inactivated after the pressurization at 200 MPa in both experiments, and no outgrowth was observed after the pressurization at 200 MPa. The arrangement and proportion of the dermal collagen fibers or the elastin fibers were not adversely affected after the pressurization at 200 MPa for up to 60 minutes. This study showed that a HHP at 200 MPa for 10 min could inactivate the skin without damaging the dermal matrix.

Preparation of inactivated human skin using high hydrostatic pressurization for full-thickness skin reconstruction

We have reported that high-hydrostatic-pressure (HHP) technology is safe and useful for producing various kinds of decellularized tissue. However, the preparation of decellularized or inactivated skin using HHP has not been reported. The objective of this study was thus to prepare inactivated skin from human skin using HHP, and to explore the appropriate conditions of pressurization to inactivate skin that can be used for skin reconstruction. Human skin samples of 8 mm in diameter were packed in bags filled with normal saline solution (NSS) or distilled water (DW), and then pressurized at 0, 100, 150, 200 and 1000 MPa for 10 minutes. The viability of skin after HHP was evaluated using WST-8 assay. Outgrowth cells from pressurized skin and the viability of pressurized skin after cultivation for 14 days were also evaluated. The pressurized skin was subjected to histological evaluation using hematoxylin and eosin staining, scanning electron microscopy (SEM), immunohistochemical staining of type IV collagen for the basement membrane of epidermis and capillaries, and immunohistochemical staining of von Willebrand factor (vWF) for capillaries. Then, human cultured epidermis (CE) was applied on the pressurized skin and implanted into the subcutis of nude mice; specimens were subsequently obtained 14 days after implantation. Skin samples pressurized at more than 200 MPa were inactivated in both NSS and DW. The basement membrane and capillaries remained intact in all groups according to histological and immunohistological evaluations, and collagen fibers showed no apparent damage by SEM. CE took on skin pressurized at 150 and 200 MPa after implantation, whereas it did not take on skin pressurized at 1000 MPa. These results indicate that human skin could be inactivated after pressurization at more than 200 MPa, but skin pressurized at 1000 MPa had some damage to the dermis that prevented the taking of CE. Therefore, pressurization at 200 MPa is optimal for preparing inactivated skin that can be used for skin reconstruction.

Decellularization of porcine carotid by the recipient’s serum and evaluation of its biocompatibility using a rat autograft model

Recently, decellularized tissues for organ transplantation and regeneration have been actively studied in the field of tissue engineering. In the decellularization process, surfactants such as sodium dodecyl sulfate (SDS) have been most commonly used to remove cellular components from the tissue. However, the residual surfactant may be cytotoxic in vivo and has been reported to hinder remodeling after implantation. In addition, treatment with surfactants may destroy the important extracellular matrix (ECM) structure that allows the decellularized tissue to function as a scaffold for cells. In this study, decellularized tissues with high biocompatibility were created using the recipient’s serum. By immersing a heterogeneous tissue in serum conditioned to activate the complement system and DNase I, its cellular components could be removed. Compared to an SDS-treated graft, the serum-treated graft preserved the native structure of its ECM. When subcutaneously implanted into an isogenic inbred rat, the graft treated with the recipient’s serum resulted in less immunorejection than did the SDS-treated graft.

Verification of the Inactivation of Melanocytic Nevus in vitro Using a Newly Developed Portable High Hydrostatic Pressure Device

High hydrostatic pressure (HHP) technology is a physical method for inactivating tissue. We reported that nevus specimens were inactivated after HHP at 200 MPa and that the inactivated nevus could be used as autologous dermis for covering skin defects. In this study, we verified the inactivation of nevus specimens using a newly developed portable HHP device which will be used in a clinical trial. Nevus tissue specimens were obtained from 5 patients (mean age 7.2 years, range 1-19). We cultured fibroblasts and nevus cells from the tissue specimens and then evaluated their inactivation after HHP at 200 MPa by confirming the attachment of the suspensions and by the live/dead staining of the suspensions, through the dissociation of the cells on chamber slides and by the live/dead staining of the remaining cells. The cells were also quantitatively evaluated by WST-8 assay. We then confirmed the inactivation of the nevus specimens after HHP using explant culture. Our results indicated that fibroblasts and nevus cells were inactivated after HHP at 200 MPa, with the exception of a small percentage of green-colored cells, which reflected the remaining activity of the cellular esterases after HHP. No cells migrated from the nevus specimens after HHP at 200 MPa. We verified the inactivation of fibroblasts and nevus cells cultured from nevus specimens, and in the nevus samples themselves after pressurization at 200 MPa using this device. This device could be used in clinical trials for giant congenital melanocytic nevi and may thus become useful in various medical fields.

Inactivation of Human Nevus Tissue Using High Hydrostatic Pressure for Autologous Skin Reconstruction: A Novel Treatment for Giant Congenital Melanocytic Nevi.

Giant congenital melanocytic nevi are intractable lesions associated with a risk of melanoma. High hydrostatic pressure (HHP) technology is a safe physical method for producing decellularized tissues without chemicals. We have reported that HHP can inactivate cells present in various tissues without damaging the native extracellular matrix (ECM). The objectives of this study were to inactivate human nevus tissue using HHP and to explore the possibility of reconstructing skin using inactivated nevus in combination with cultured epidermis (CE). Human nevus specimens 8 mm in diameter were pressurized by HHP at 100, 200, 500, and 1000 MPa for 10 min. The viability of specimens just after HHP, outgrowth of cells, and viability after cultivation were evaluated to confirm the inactivation by HHP. Histological evaluation using hematoxylin-eosin staining and immunohistochemical staining for type IV collagen was performed to detect damage to the ECM of the nevus. The pressurized nevus was implanted into the subcutis of nude mice for 6 months to evaluate the retention of human cells. Then, human CE was applied on the pressurized nevus and implanted into the subcutis of nude mice. The viability of pressurized nevus was not detected just after HHP and after cultivation, and outgrowth of fibroblasts was not observed in the 200, 500, and 1000 MPa groups. Human cells were not observed after 6 months of implantation in these groups. No apparent damage to the ECM was detected in all groups; however, CE took on nevus in the 200 and 500 MPa groups, but not in the 1000 MPa group. These results indicate that human nevus tissue was inactivated by HHP at more than 200 MPa; however, HHP at 1000 MPa might cause damage that prevents the take of CE. In conclusion, all cells in nevus specimens were inactivated after HHP at more than 200 MPa and this inactivated nevus could be used as autologous dermis for covering full-thickness skin defects after nevus removal. HHP between 200 and 500 MPa will be optimal to reconstruct skin in combination with cultured epidermal autograft without damage to the ECM.

Reconstruction of small diameter arteries using decellularized vascular scaffolds

Although artificial vessels are available for large diameter arteries, there are no artificial vessels for small diameter arteries of < 4 mm. We created a decellularized vascular scaffold (length, 10 mm; outer diameter, 1.5 mm; inner diameter, 1.3 mm) from rat abdominal arteries. We measured the biomechanical characteristics of the scaffolds, implanted them to defects made in rat carotid arteries, and evaluated their patency and the endothelial cell linings. Silastic grafts were implanted as controls. The decellularized scaffolds demonstrated similar mechanical characteristics to normal arteries. All of the control grafts were occluded. Fibroblast-like cells were discovered in the thrombus, and fibrous organization was apparent. In contrast, patency of the grafts in 10 of 12 animals was observed 4 weeks after implantation. The internal cavity of the patent scaffold was completely lined by endotheliallike cells. Thus, the possibility of small artery reconstruction using decellularized scaffolds was demonstrated.

An evaluation of the engraftment and the blood flow of porcine skin autografts inactivated by high hydrostatic pressure.

We previously reported that exposure to a high hydrostatic pressure (HHP) of 200 MPa could completely inactivate porcine skin without damaging the extracellular matrix. In this study, we used an autologous porcine skin graft model and explored whether the skin inactivated by HHP could be engrafted without inflammation to the residual cellular components. Twenty-one full-thickness skin grafts of 1.5 × 1.5 cm in size were prepared from a minipig (n = 2). Grafts were either nonpressurized or pressurized to 100, 150, 200, 300, 500, or 1000 MPa (n = 3) and randomly implanted on the fascia and removed at 1 and 4 weeks after grafting. All grafts showed complete engraftment at the macroscopic level and microcirculation was detected by a full-field laser speckle perfusion imager. The epidermis was removed and skin appendages were not observed in the grafts pressurized to more than 200 MPa. Azan and Elastica van Gieson staining showed no sign of dermal collagen fiber degeneration, while elastin fibers were observed. The fibroblasts and capillaries were observed to have infiltrated to dermis in all groups without severe inflammation. In conclusion, we showed that skin inactivated by HHP up to 1000 MPa could be engrafted successfully without removing cellular remnants.