Yuko Fujihara

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.

Immunological response to tissue-engineered cartilage derived from auricular chondrocytes and a PLLA scaffold in transgenic mice.

The immune response against biomaterials in tissue-engineered constructs could potentially worsen the outcome of tissue regeneration, but immunological reactions between host and donor in tissue-engineered constructs remain to be clarified. In the present study, we syngenically transplanted tissue-engineered cartilage constructs consisting of C57BL/6 mice auricular chondrocytes and poly-l-lactic acid scaffolds (MW:200,000) into EGFP transgenic mice of C57BL/6 background, and evaluated the response by the localization of donor-derived and host-derived cells, the latter of which were distinguished by the presence of EGFP. While donor-derived cells constituted the areas of regenerated cartilage, host-derived cells were increased in number for the initial two weeks, and then decreased and excluded to non-cartilage areas thereafter. Furthermore, EGFP positivity was mostly co-localized with that of F4/80, suggesting most of the host-derived cells in the tissue-engineered constructs could be macrophages. Immunohistochemical staining of the tissue-engineered cartilage constructs revealed expression of factors related to immune privilege in chondrocytes, such as macrophage migration inhibitory factor (MIF), fas ligand (FasL) and others. Co-culture of chondrocytes and macrophages in vitro increased the expression of MIF and FasL in the chondrocytes, suggesting that chondrocytes in tissue-engineered cartilage constructs could regulate the actions of host-derived macrophages by expressing factors related to immune privilege.

Tissue Reactions to Engineered Cartilage Based on Poly-L-Lactic Acid Scaffolds

Tissue reactions against poly-L-lactic acid (PLLA) in engineered cartilage may influence the size or maturity of regenerative tissue. To understand the biological events in these reactions, we subcutaneously transplanted engineered constructs of PLLA scaffolds with or without human chondrocytes or atelocollagen in nude mice and evaluated neovascularization and macrophage activation, which can be assessed even in nude mice. Although not showing cartilage regeneration, PLLA alone demonstrated dense localization of macrophages and blood vessels, as well as a high level of interleukin-1 beta and tissue hemoglobin at 2 and 8 weeks. Otherwise, constructs with PLLA and chondrocytes with or without atelocollagen (PLLA/cell/gel or PLLA/cell) formed mature cartilage by 8 weeks, which was more prominent in PLLA/cell/gel. Although accumulation of macrophages and blood vessels in PLLA/cell/gel and PLLA/cell was comparable with that in PLLA at 2 weeks, that in PLLA/cell/gel markedly decreased by 8 weeks, with blood vessels and macrophages excluded into non-cartilage areas. Macrophage migration inhibitory factor could be involved in these suppressed tissue reactions, because it was expressed in chondrocytes of engineered cartilage. Intense tissue reactions inevitably occurred in biopolymers alone, but it is possible that maturation of engineered cartilage suppressed these reactions, which may contribute to circumventing deformity or malformation of engineered tissues.

Macrophage-inducing FasL on chondrocytes forms immune privilege in cartilage tissue engineering, enhancing in vivo regeneration.

To obtain stable outcomes in regenerative medicine, controlling inflammatory reactions is a requirement. Previously, auricular chondrocytes in tissue‐engineered cartilage have been shown to express factors related to immune privilege including Fas ligand (FasL) in mice. Since elucidation of mechanism on immune privilege formed in cartilage regeneration may contribute to suppression of excessive inflammation, in this study, we investigated the function of FasL and induction of immune privilege in tissue‐engineered cartilage using a mouse subcutaneous model. When cocultured, auricular chondrocytes of FasL‐dysfunctional mice, C57BL/6JSlc‐gld/gld (gld), induced less cell death and apoptosis of macrophage‐like cells, RAW264, compared with chondrocytes of C57BL/6 mice (wild), suggesting that FasL on chondrocytes could induce the apoptosis of macrophages. Meanwhile, the viability of chondrocytes was hardly affected by cocultured RAW264, although the expression of type II collagen was decreased, indicating that macrophages could hamper the maturation of chondrocytes. Tissue‐engineered cartilage containing gld chondrocytes exhibited greater infiltration of macrophages, with less accumulation of proteoglycan than did wild constructs. Analysis of the coculture medium identified G‐CSF as an inducer of FasL on chondrocytes, and G‐CSF‐treated tissue‐engineered cartilage showed less infiltration of macrophages, with increased formation of cartilage after transplantation. The interactions between chondrocytes and macrophages may increase G‐CSF secretion in macrophages and induce FasL on chondrocytes, which in turn induce the apoptosis of macrophages and suppress tissue reactions, promoting the maturation of tissue‐engineered cartilage. These findings provide scientific insight into the mechanism of autologous chondrocyte transplantation, which could be applied as a novel strategy for cartilage tissue engineering. Stem Cells 2014;32:1208–1219

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.

Controlled delivery of bFGF to recipient bed enhances the vascularization and viability of an ischemic skin flap

Therapeutic angiogenesis is a promising approach to treat ischemic skin flaps. We delivered basic fibroblast growth factor (bFGF) to the recipient bed of a rat dorsal skin flap by a drug delivery system with acidic gelatin hydrogel microspheres (AGHMs), and assessed augmentation of neovascularization and flap viability. An axial skin flap was elevated on the back of male Sprague–Dawley rats, and bFGF solution or bFGF‐impregnated AGHMs were injected into the recipient bed. The dose of bFGF in the bFGF solution was set to 15 (Sol‐15 group), 50 (Sol‐50 group), or 150 μg (Sol‐150 group). Correspondingly, 2 mg AGHMs were impregnated with 15 (AGHM‐15 group), 50 (AGHM‐50 group), or 150 μg (AGHM‐150 group) bFGF. Other groups of animals received phosphate‐buffered saline (Sol‐Cont group) or phosphate‐buffered saline‐impregnated AGHMs (AGHM‐Cont group) as controls. Seven days later, analyses of the area of necrosis, microangiographic findings, and histological findings in the flap were carried out. The area of necrosis in the AGHM‐150 group was significantly smaller than that in the other groups. Microangiographic and histological analyses showed that neovascularization of the ischemic skin flap significantly increased in the AGHM‐150 group as compared with the Sol‐150 group and the AGHM‐Cont group. These findings suggest that continuous delivery of bFGF to the recipient bed by bFGF‐impregnated AGHMs enhances the viability of an ischemic skin flap.

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.

Selection of highly osteogenic and chondrogenic cells from bone marrow stromal cells in biocompatible polymer-coated plates.

To enrich the subpopulation that preserves self-renewal and multipotentiality from conventionally prepared bone marrow stromal cells (MSCs), we attempted to use 2-methacryloyloxyethyl phosphorylcholine (MPC) polymer-coated plates that selected the MSCs with strong adhesion ability and evaluated the proliferation ability or osteogenic/chondrogenic potential of the MPC polymer-selected MSCs. The number of MSCs that were attached to the MPC polymer-coated plates decreased with an increase in the density of MPC unit (0-10%), whereas no significant difference in the proliferation ability was seen among these cells. The surface epitopes of CD29, CD44, CD105, and CD166, and not CD34 or CD45, were detectable in the cells of all MPC polymer-coated plates, implying that they belong to the MSC category. In the osteogenic and chondrogenic induction, the MSCs selected by the 2-5% MPC unit composition showed higher expression levels of osteoblastic and chondrocytic markers (COL1A1/ALP, or COL2A1/COL10A1/Sox9) at passage 2, compared with those of 0-1% or even 10% MPC unit composition, while the enhanced effects continued by passage 5. The selection based on the adequate cell adhesiveness by the MPC polymer-coated plates could improve the osteogenic and chondrogenic potential of MSCs, which would provide cell sources that can be used to treat the more severe and various bone/cartilage diseases.

Controlled Delivery of bFGF Remodeled Vascular Network in Muscle Flap and Increased Perfusion Capacity Via Minor Pedicle

BACKGROUND The vascular pedicles of superficial skeletal muscles are classified as the dominant pedicle and the minor pedicle. The dominant pedicle can reliably provide adequate circulation to the entire muscle, but the minor pedicle cannot. Therefore, it is impossible to elevate an entire muscle flap based solely on the minor pedicle. We evaluated whether the delivery of basic fibroblast growth factor (bFGF) would remodel the vascular network in the muscle flap and increase the perfusion capacity via the minor pedicle. METHODS Using a controlled delivery system with acidic gelatin hydrogel microspheres, bFGF (50 microg, FGF group) or phosphate-buffered saline (PBS group) was delivered to the rabbit gracilis muscle, which contained a single dominant pedicle and a minor pedicle. Seven days later, the gracilis muscle was elevated as a muscle flap on the minor pedicle, and the flap was wrapped with a silicone sheet to avoid vessel in-growth from surrounding tissue. At 7 d after operation, flap viability, microangiograms, and regional blood flow were evaluated. RESULTS Flap viability in the FGF group (73.6 +/- 20.5%) was significantly higher than that in the PBS group (19.7 +/- 23.5%). Postmortem angiograms of the flap showed obvious communications between the distal vascular networks of the minor and dominant pedicles. Angiographic score and regional blood flow of the flap were significantly higher in the FGF group than in the PBS group. CONCLUSIONS Controlled delivery of bFGF to rabbit gracilis muscle increased the perfusion capacity via the minor pedicle, and the minor pedicle became able to solely provide sufficient circulation to the entire muscle.

Tissue responses against tissue-engineered cartilage consisting of chondrocytes encapsulated within non-absorbable hydrogel

To disclose the influence of foreign body responses raised against a non‐absorbable hydrogel consisting of tissue‐engineered cartilage, we embedded human/canine chondrocytes within agarose and transplanted them into subcutaneous pockets in nude mice and donor beagles. One month after transplantation, cartilage formation was observed in the experiments using human chondrocytes in nude mice. No significant invasion of blood cells was noted in the areas where the cartilage was newly formed. Around the tissue‐engineered cartilage, agarose fragments, a dense fibrous connective tissue and many macrophages were observed. On the other hand, no cartilage tissue was detected in the autologous transplantation of canine chondrocytes. Few surviving chondrocytes were observed in the agarose and no accumulation of blood cells was observed in the inner parts of the transplants. Localizations of IgG and complements were noted in areas of agarose, and also in the devitalized cells embedded within the agarose. Even if we had inhibited the proximity of the blood cells to the transplanted cells, the survival of the cells could not be secured. We suggest that these cytotoxic mechanisms seem to be associated not only with macrophages but also with soluble factors, including antibodies and complements. Copyright