Satoru Nishizawa

The optimization of porous polymeric scaffolds for chondrocyte/atelocollagen based tissue-engineered cartilage

To broaden the clinical application of cartilage regenerative medicine, we should develop an implant-type tissue-engineered cartilage with firmness and 3-D structure. For that, we attempted to use a porous biodegradable polymer scaffold in the combination with atelocollagen hydrogel, and optimized the structure and composition of porous scaffold. We administered chondrocytes/atelocollagen mixture into the scaffolds with various kinds of porosities (80-95%) and pore sizes (0.3-2.0 mm), consisting of PLLA or related polymers (PDLA, PLA/CL and PLGA), and transplanted the constructs in the subcutaneous areas of nude mice. The constructs using scaffolds of excessively large pore sizes (>1 mm) broke out on the skin and impaired the host tissue. The scaffold with the porosity of 95% and pore size of 0.3 mm could effectively retain the cells/gel mixture and indicated a fair cartilage regeneration. Regarding the composition, the tissue-engineered cartilage was superior in PLGA and PLLA to that in PLA/CA and PDLA. The latter two showed the dense accumulation of macrophages, which may deteriorate the cartilage regeneration. Although PLGA or PLLA has been currently recommended for the scaffold of cartilage, the polymer for which biodegradation was exactly synchronized to the cartilage regeneration would improve the quality of the tissue-engineered cartilage.

Aptitude of Auricular and Nasoseptal Chondrocytes Cultured Under a Monolayer or Three-Dimensional Condition for Cartilage Tissue Engineering

To elucidate the characterizations of chondrocytes originating from auricular cartilage (donors: 10-15 years) and nasoseptal one (20-23 years), we evaluated proliferation or matrix synthesis of both cells cultured under monolayer and collagen type I (COL1) three-dimensional (3D) conditions. Three passages were needed until cell numbers of auricular chondrocytes in the 3D culture increased 1000-fold, although those in monolayer culture or nasoseptal monolayer and 3D cells reached a 1000-fold increase at four passages. When we cultured the tissue-engineered cartilage pellets made of the chondrocytes proliferated at 1000-fold increase, the pellets of monolayer cells maintained their sizes during the culture period. However, those of nasoseptal 3D cells began to shrink at day 1 and became approximately one-tenth in size at day 21. The downsizing of pellets may result from the upregulation of tumor necrosis factor (TNF)-alpha or the related proteinases, including matrix metalloproteinases (MMPs)-1, -2, and -3, and cathepsin B, suggesting that the nasoseptal chondrocytes, which are physiologically separated from COL1, may be hardly adapted for the COL1 3D proliferation condition. Ideally, these characteristics would have been compared between the chondrocytes from donors that are completely matched in ages. However, according to our data using closely matched ones, the auricular chondrocytes seemed to more rapidly proliferate and produce less proteinases during this 3D culture than the nasoseptal ones.

Early stage foreign body reaction against biodegradable polymer scaffolds affects tissue regeneration during the autologous transplantation of tissue-engineered cartilage in the canine model.

To overcome the weak points of the present cartilage regenerative medicine, we applied a porous scaffold for the production of tissue-engineered cartilage with a greater firmness and a 3D structure. We combined the porous scaffolds with atelocollagen to retain the cells within the porous body. We conducted canine autologous chondrocyte transplants using biodegradable poly-l-lactic acid (PLLA) or poly-dl-lactic-co-glycolic acid (PLGA) polymer scaffolds, and morphologically and biochemically evaluated the time course changes of the transplants. The histological findings showed that the tissue-engineered constructs using PLLA contained abundant cartilage 1, 2, and 6 months after transplantation. However, the PLGA constructs did not possess cartilage and could not maintain their shapes. Biochemical measurement of the proteoglycan and type II collagen also supported the superiority of PLLA. The biodegradation of PLGA progressed much faster than that of PLLA, and the PLGA had almost disappeared by 2 months. The degraded products of PLGA may evoke a more severe tissue reaction at this early stage of transplantation than PLLA. The PLLA scaffolds were suitable for cartilage tissue engineering under immunocompetent conditions, because of the retarded degradation properties and the decrease in the severe tissue reactions during the early stage of transplantation.

Growth factor contents of autologous human sera prepared by different production methods and their biological effects on chondrocytes

To discuss the autologous serum production for cartilage tissue engineering, we compared three kinds of sera: whole blood‐derived serum (WBS), platelet‐containing plasma‐derived serum (PCS), and plasma‐derived serum (PDS), on the growth factor contents and their biological effects on human auricular chondrocytes. EGF, VEGF and PDGF levels were highest in WBS, while PCS and PDS followed WBS. The proliferation effects of WBS were the most pronounced, followed by that of PCS, both of which realized a 1000‐fold‐increase in chondrocyte numbers at the third passage, whereas PDS reached it after passage 4. No significant differences were observed in histology or cartilaginous matrix measurements of tissue‐engineered cartilage produced from chondrocytes cultured under different serum conditions. WBS would be clinically useful because of its potent proliferation effects, while PCS, which possibly saves the red cell concentrate, may be an option in cases where there are elevated risks of blood loss.

The application of atelocollagen gel in combination with porous scaffolds for cartilage tissue engineering and its suitable conditions.

For improving the quality of tissue-engineered cartilage, we examined the in vivo usefulness of porous bodies as scaffolds combined with an atelocollagen hydrogel, and investigated the suitable conditions for atelocollagen and seeding cells within the engineered tissues. We made tissue-engineered constructs using a collagen sponge (CS) or porous poly(L-lactide) (PLLA) with human chondrocytes and 1% hydrogel, the concentration of which maximized the accumulation of cartilage matrices. The CS was soft with a Youngs modulus of less than 1 MPa, whereas the porous PLLA was very rigid with a Youngs modulus of 10 MPa. Although the constructs with the CS shrank to 50% in size after a 2-month subcutaneous transplantation in nude mice, the PLLA constructs maintained their original sizes. Both of the porous scaffolds contained some cartilage regeneration in the presence of the chondrocytes and hydrogel, but the PLLA counterpart significantly accumulated abundant matrices in vivo. Regarding the conditions of the chondrocytes, the cartilage regeneration was improved in inverse proportion to the passage numbers among passages 3-8, and was linear with the cell densities (10(6) to 10(8) cells/mL). Thus, the rigid porous scaffold can maintain the size of the tissue-engineered cartilage and realize fair cartilage regeneration in vivo when combined with 1% atelocollagen and some conditioned chondrocytes.

Involvement of fibroblast growth factor 18 in dedifferentiation of cultured human chondrocytes

Objective:  Chondrocytes inevitably decrease production of cartilaginous matrices during long‐term cultures with repeated passaging; this is termed dedifferentiation. To learn more concerning prevention of dedifferentiation, we have focused here on the fibroblast growth factor (FGF) family that influences chondrocyte proliferation or differentiation.

Evaluation of the implant type tissue-engineered cartilage by scanning acoustic microscopy

The tissue-engineered cartilages after implantation were nonuniform tissues which were mingling with biodegradable polymers, regeneration cartilage and others. It is a hard task to evaluate the biodegradation of polymers or the maturation of regenerated tissues in the transplants by the conventional examination. Otherwise, scanning acoustic microscopy (SAM) system specially developed to measure the tissue acoustic properties at a microscopic level. In this study, we examined acoustic properties of the tissue-engineered cartilage using SAM, and discuss the usefulness of this devise in the field of tissue engineering. We administered chondrocytes/atelocollagen mixture into the scaffolds of various polymers, and transplanted the constructs in the subcutaneous areas of nude mice for 2 months. We harvested them and examined the sound speed and the attenuation in the section of each construct by the SAM. As the results, images mapping the sound speed exhibited homogenous patterns mainly colored in blue, in all the tissue-engineered cartilage constructs. Contrarily, the images of the attenuation by SAM showed the variation of color ranged between blue and red. The low attenuation area colored in red, which meant hard materials, were corresponding to the polymer remnant in the toluidine blue images. The localizations of blue were almost similar with the metachromatic areas in the histology. In conclusion, the SAM is regarded as a useful tool to provide the information on acoustic properties and their localizations in the transplants that consist of heterogeneous tissues with various components.

Administration of the insulin into the scaffold atelocollagen for tissue‐engineered cartilage

Three-dimensional culture of the tissue-engineered cartilage constructs may increase the matrix production, but central necrosis must occur if the construct becomes large. To increase the cell viability in the middle part of constructs and to enhance the in vivo cartilage regeneration, we attempted to administer the insulin into the scaffold. Insulin is known to strongly enhance the matrix production in the chondrocytes. The pellets of human auricular chondrocytes with atelocollagen hydrogel were 3D-cultured in the medium. The comparison among three groups (insulin mixed in the atelocollagen, insulin added to the medium, and control group, i.e.; insulin in neither atelocollagen nor medium) revealed that both insulin mixed in the atelocollagen and that in the medium could effectively promoted the cell viability and matrix synthesis of the chondrocytes. The daily assay also showed the gradual release of insulin from the atelocollagen hydrogel, suggesting that this material may work as a control release of insulin. We actually transplanted the poly-L-lactide porous scaffolds carrying the chondrocytes and the atelocollagen mixed with or without insulin, into the nude mice, showing that glycosaminoglycan accumulation was evident in the group with insulin although less without insulin. We thus showed the possibility to enhance the in vivo cartilage regeneration, when administered insulin into the atelocollagen hydrogel.

The effects of rapid- or intermediate-acting insulin on the proliferation and differentiation of cultured chondrocytes.

In cartilage regenerative medicine, which is highly expected in the face of our aging society, insulin is the potent factor for culture media. To secure the safety of culture media, we attempted to use medical insulin formulations, and compared their effects on human articular or auricular chondrocytes between regular human insulin (R) and neutral protamine hagedorn insulin (N). In monolayer culture with the media containing either R or N, the cell growth reached approximately 15-fold-increase in 6 days, which showed no significant difference between them. These cells showed the equivalent ability to produce cartilage matrices, both in vitro and in vivo. Also, in the 3D culture of the dedifferentiated chondrocytes, either R or N increased gene expression of type II collagen at 3-4 folds in the combination with other growth factors, compared with basal medium, while insulin could similarly enhance both the redifferentiation and cartilage maturation. The in vitro half-life of each insulin in the presence of chondrocytes neither decreased within 3 days, suggesting little degradation in the culture media, unlike in the body. Although both R and N showed similar biological effects on cultured chondrocytes, we may choose the R for clinical practice because of its pure composition.

Preclinical and clinical research on bone and cartilage regenerative medicine in oral and maxillofacial region

Abstract Recently, there have been remarkable advances in regenerative medicine, and almost all disorders of the oral and maxillofacial region could be research targets of regenerative medicine. Meanwhile, treatment in this region has been well established using biomaterials, prostheses, and microsurgery. Therefore, to surpass such a conventional approach as an alternative, regenerative medicine should take an approach of being less invasive and/or more effective. In this report, we present our preclinical and clinical research on bone and cartilage regenerative medicine in the oral and maxillofacial region. Regarding bone regenerative medicine, we have tried to develop artificial bone that would maximize bone formation at the transplanted site, but would subsequently be replaced by autologous bone. We have made custom-made artificial bone (CT-Bone) using alpha-tricalcium phosphate (α-TCP) particles and an ink-jet printer, and have conducted clinical research and trials on 30 patients. To develop tissue-engineered cartilage with proper three-dimensional (3D) morphological form and mechanical strength, we have optimized the culture medium of chondrocytes and the scaffold. Following a preclinical study confirming efficacy and safety, we have conducted clinical research in three patients with nasal deformity associated with cleft lip and palate, and are now starting multicenter clinical research.