EunAh Lee

A new role of substance P as an injury-inducible messenger for mobilization of CD29 + stromal-like cells

Tissue injury may create a specific microenvironment for inducing the systemic participation of stromal-like cells in the repair process. Here we show that substance P is an injury-inducible factor that acts early in the wound healing process to induce CD29+ stromal-like cell mobilization. Likewise, mobilization of such cells also occurs in uninjured mice, rats and rabbits if substance P is intravenously injected. Upon further characterization these substance P–mobilized CD29+ cells were found to be similar to stromal cells from a number of connective tissues, including bone marrow (that is, bone marrow stromal cells, or BMSCs). Both substance P injection and transfusion of autologously derived substance P–mobilized CD29+ cells from uninjured rabbits accelerated wound healing in an alkali burn model. Also, epithelial engraftment of the transfused cells into the injured tissue occurred during the wound healing. Finally, using human BMSCs as a test population, we show that substance P stimulates transmigration, cell proliferation, activation of the extracellular signal–related kinases (Erk) 1 and 2 and nuclear translocation of β-catenin in vitro. This finding highlights a previously undescribed function of substance P as a systemically acting messenger of injury and a mobilizer of CD29+ stromal-like cells to participate in wound healing.

Substance P stimulates the recovery of bone marrow after the irradiation.

The therapeutic use of ionizing radiation (e.g., X‐rays and γ‐rays) needs to inflict minimal damage on non‐target tissue. Recent studies have shown that substance P (SP) mediates multiple activities in various cell types, including cell proliferation, anti‐apoptotic responses, and inflammatory processes. The present study investigated the effects of SP on γ‐irradiated bone marrow stem cells (BMSCs). In mouse bone marrow extracts, SP prolonged activation of Erk1/2 and enhanced Bcl‐2 expression, but attenuated the activation of apoptotic molecules (e.g., p38 and cleaved caspase‐3) and down‐regulated Bax. We also observed that SP‐decreased apoptotic cell death and stimulated cell proliferation in γ‐irradiated mouse bone marrow tissues through TUNEL assay and PCNA analysis. To determine how SP affects bone marrow stem cell populations, mouse bone marrow cells were isolated and colony‐forming unit (CFU) of mesenchymal stem cells (MSCs) and hematopoietic stem cells (HSCs) was estimated. SP‐pretreated ones showed higher CFUs of MSC and HSC than untreated ones. Furthermore, when SP was pretreated in cultured human MSC, it significantly decreased apoptotic cells at 48 and 72 h after γ‐irradiation. Compared with untreated cells, SP‐treated human MSCs showed reduced cleavage of apoptotic molecules such as caspase‐8, ‐9, ‐3, and poly ADP‐ribose polymerase (PARP). Thus, our results suggest that SP alleviates γ‐radiation‐induced damage to mouse BMSCs and human MSCs via regulation of the apoptotic pathway. J. Cell. Physiol. 226: 1204–1213, 2011.

Transforming growth factorβ1 transactivates EGFR via an H2O2-dependent mechanism in squamous carcinoma cell line

TGFbeta is known to transactivate EGFR. However, the signaling component involved in this crosstalk has yet to be revealed. Here, we found that TGFbeta(1) phosphorylated EGFR in a dose-dependent manner in SCC13 and A431 cells, and it was not blocked by EGF-neutralizing antibody. H(2)O(2) was increased by TGFbeta(1) treatment in the same time-kinetics as EGFR activation. Pretreatment of N-acetyl cysteine abolished TGFbeta(1)-induced H(2)O(2) induction and EGFR activation. Direct treatment of H(2)O(2) phosphorylated EGFR and catalase inhibitor prolonged TGFbeta(1)-induced EGFR activation. These results show that TGFbeta(1) activates EGFR via an H(2)O(2)-dependent mechanism, which subsequently leads to the activation of Erk(1/2).

Xiphoid Process-Derived Chondrocytes: A Novel Cell Source for Elastic Cartilage Regeneration

Reconstruction of elastic cartilage requires a source of chondrocytes that display a reliable differentiation tendency. Predetermined tissue progenitor cells are ideal candidates for meeting this need; however, it is difficult to obtain donor elastic cartilage tissue because most elastic cartilage serves important functions or forms external structures, making these tissues indispensable. We found vestigial cartilage tissue in xiphoid processes and characterized it as hyaline cartilage in the proximal region and elastic cartilage in the distal region. Xiphoid process‐derived chondrocytes (XCs) showed superb in vitro expansion ability based on colony‐forming unit fibroblast assays, cell yield, and cumulative cell growth. On induction of differentiation into mesenchymal lineages, XCs showed a strong tendency toward chondrogenic differentiation. An examination of the tissue‐specific regeneration capacity of XCs in a subcutaneous‐transplantation model and autologous chondrocyte implantation model confirmed reliable regeneration of elastic cartilage regardless of the implantation environment. On the basis of these observations, we conclude that xiphoid process cartilage, the only elastic cartilage tissue source that can be obtained without destroying external shape or function, is a source of elastic chondrocytes that show superb in vitro expansion and reliable differentiation capacity. These findings indicate that XCs could be a valuable cell source for reconstruction of elastic cartilage.

Fully Dedifferentiated Chondrocytes Expanded in Specific Mesenchymal Stem Cell Growth Medium with FGF2 Obtains Mesenchymal Stem Cell Phenotype In Vitro but Retains Chondrocyte Phenotype In Vivo

Given recent progress in regenerative medicine, we need a means to expand chondrocytes in quantity without losing their regenerative capability. Although many reports have shown that growth factor supplementation can have beneficial effects, the use of growth factor–supplemented basal media has widespread effect on the characteristics of chondrocytes. Chondrocytes were in vitro cultured in the 2 most widely used chondrocyte growth media, conventional chondrocyte culture medium and mesenchymal stem cell (MSC) culture medium, both with and without fibroblast growth factor-2 (FGF2) supplementation. Their expansion rates, expressions of extracellular matrix–related factors, senescence, and differentiation potentials were examined in vitro and in vivo. Our results revealed that chondrocytes quickly dedifferentiated during expansion in all tested media, as assessed by the loss of type II collagen expression. The 2 basal media (chondrocyte culture medium vs. MSC culture medium) were associated with distinct differences in cell senescence. Consistent with the literature, FGF2 was associated with accelerated dedifferentiation during expansion culture and superior redifferentiation upon induction. However, chondrocytes expanded in FGF2-containing conventional chondrocyte culture medium showed MSC-like features, as indicated by their ability to direct ectopic bone formation and cartilage formation. In contrast, chondrocytes cultured in FGF2-supplemented MSC culture medium showed potent chondrogenesis and almost no bone formation. The present findings show that the chosen basal medium can exert profound effects on the characteristics and activity of in vitro–expanded chondrocytes and indicate that right growth factor/medium combination can help chondrocytes retain a high-level chondrogenic potential without undergoing hypertrophic transition.

Disc-type hyaline cartilage reconstruction using 3D-cell sheet culture of human bone marrow stromal cells and human costal chondrocytes and maintenance of its shape and phenotype after transplantation

In this study, we developed the disc-type bio-cartilage reconstruction strategies for transplantable hyaline cartilage for reconstructive surgery using 3D-cell sheet culture of human bone marrow stromal cells and human costal chondrocytes. We compared chondrogenesis efficiency between different chondrogenic-induction methods such as micromass culture, pellet culture, and 3D-cell sheet culture. Among them, the 3D-cell sheet culture resulted in the best chondrogenesis with the disc-type bio-cartilage (>12 mm diameter in size) in vitro, but sometimes spontaneous curling and contraction of 3D-cell sheet culture resulted in the formation of bead-type cartilage, which was prevented by type I collagen coating or by culturing on amniotic membrane. Previously, it was reported that tissue-engineered cartilage reconstructed in vitro does not maintain its cartilage phenotype after transplantation but tends to transform to other tissue type such as bone or connective tissue. However, the disc-type bio-cartilage of 3D-cell sheet culture maintained its hyaline cartilage phenotype even after exposure to the osteogenic-induction condition in vitro for 3 weeks or after the transplantation for 4 weeks in mouse subcutaneous. Collectively, the disc-type bio-cartilage with 12 mm diameter can be reproducibly reconstructed by the 3D-cell sheet culture, whose hyaline cartilage phenotype and shape can be maintained under the osteogenic-induction condition as well as after the transplantation. This disc-type bio-cartilage can be proposed for the application to reconstructive surgery and repair of disc-type cartilage such as mandibular cartilage and digits.

A Pathophysiological Validation of Collagenase II-Induced Biochemical Osteoarthritis Animal Model in Rabbit

BACKGROUND:Current dilemma working with surgically-induced OA (osteoarthritis) model include inconsistent pathological state due to various influence from surrounding tissues. On the contrary, biochemical induction of OA using collagenase II has several advantageous points in a sense that it does not involve surgery to induce model and the extent of induced cartilage degeneration is almost uniform. However, concerns still exists because biochemical OA model induce abrupt destruction of cartilage tissues through enzymatic digestion in a short period of time, and this might accompany systemic inflammatory response, which is rather a trait of RA (rheumatoid arthritis) than being a trait of OA.METHODS:To clear the concern about the systemic inflammatory response that might be caused by abrupt destruction of cartilage tissue, OA was induced to only one leg of an animal and the other leg was examined to confirm the presence of systemic degenerative effect.RESULTS:Although the cartilage tissues were rapidly degenerated during short period of time upon biochemical induction of OA, they did not accompanied with RA-like process based on the histology data showing degeneration of articular cartilage occurred only in the collagenase-injected knee joint. Scoring evaluation data indicated that the cartilage tissues in non-induced joint remained intact. Neutrophil count transiently increase between day 8 and day 16, and there were no significant change in other complete blood count profile showing a characteristics of OA disease.CONCLUSION:These study shows that biochemically induced cartilage degeneration truly represented uniform and reliable OA state.

Non‐destructive label‐free continuous monitoring of in vitro chondrogenesis via electrical conductivity and its anisotropy

Non-destructive label-free continuous monitoring of in vitro tissue culture is an unmet demand in tissue engineering. Noting that different compositions of cartilage lead to different electrical tissue properties, we propose a new method to measure the electrical conductivity and its anisotropy during in vitro chondrogenesis. We used a conductivity tensor probe with 17 electrodes and a bio-impedance spectroscopy (BIS) device to measure the conductivity values and the anisotropy ratios at the bottom and top surfaces of the tissue samples during the culture period of 6 weeks. Clearly distinguishing glycosaminoglycans (GAGs), collagen, and also various mixtures of them, the measured conductivity value and the estimated tissue anisotropy provide diagnostic information of the depth-dependent tissue structure and compositions. Continuously monitoring the individual tissue during the entire chondrogenesis process without any adverse effect, the proposed method may significantly increase the productivity of cartilage tissue engineering.