Biomaterials for craniofacial bone engineering.

Conditions such as congenital anomalies, cancers, and trauma can all result in devastating deficits of bone in the craniofacial skeleton. This can lead to significant alteration in function and appearance that may have significant implications for patients. In addition, large bone defects in this area can pose serious clinical dilemmas, which prove difficult to remedy, even with current gold standard surgical treatments. The craniofacial skeleton is complex and serves important functional demands. The necessity to develop new approaches for craniofacial reconstruction arises from the fact that traditional therapeutic modalities, such as autologous bone grafting, present myriad limitations and carry with them the potential for significant complications. While the optimal bone construct for tissue regeneration remains to be elucidated, much progress has been made in the past decade. Advances in tissue engineering have led to innovative scaffold design, complemented by progress in the understanding of stem cell-based therapy and growth factor enhancement of the healing cascade. This review focuses on the role of biomaterials for craniofacial bone engineering, highlighting key advances in scaffold design and development.

Novel bile acid derivatives induce apoptosis via a p53-independent pathway in human breast carcinoma cells.

We have compared the anti-proliferative effects of ursodeoxycholic acid (UDCA), chenodeoxycholic acid (CDCA) and their derivatives, HS-1183, HS-1199 and HS-1200, on MCF-7 (wild-type p53) and MDA-MB-231 (mutant p53) cells. While UDCA and CDCA exhibited no significant effect, their novel derivatives inhibited the proliferation of both cell lines in a concentration-dependent manner, concomitant with apoptotic nuclear changes and the increase of a sub-G1 population and DNA fragmentation. Furthermore, we also observed an increase in the ratio of pro-apoptotic protein Bax to anti-apoptotic protein Bcl-2 and cleavages of lamin B and poly(ADP-ribose) polymerase (PARP) in MCF-7 and MDA-MB-231 cells. Cell cycle related proteins, cyclin D1 and D3, as well as retinoblastoma protein (pRb) were down-regulated, while the level of cyclin-dependent kinase inhibitor p21(WAF1/CIP1) was increased in both cancer cells after treatment with novel bile acids. These findings suggest that these cytotoxic effects of novel bile acid derivatives on human breast carcinoma cells were mediated via apoptosis through a p53-independent pathway.

Inhibition of NFkappaB by methyl chlorogenate from Eriobotrya japonica.

Methylchlorogenic acid (MC) is one of the main components in the leaves of Eriobotrya japonica. We previously reported that MC is the most potent antioxidant among several components of Eriobotrya japonica, and its antioxidant activity is stronger than that of chlorogenic acid. Antioxidants are expected to inhibit redox-sensitive NFkappaB activation since NFkappaB is readily influenced by cellular oxidative state. Based on these findings, in vivo experiments with MC were conducted to determine its ability to downregulate the NFkappaB activation in mouse liver. Results clearly showed that MC is a potent suppressor of BHP-induced NFkappaB activation. We observed a significant reduction by MC on BHP-induced translocation of p65 subunit of NFkappaB. This may be due to formation of p50/p65 heterodimer, which is mainly inducible NFkappaB. MC slightly blocked the BHP-induced IkappaB alpha degradation. There is a possibility of IkappaB alpha resynthesis via activated NFkappaB during a 5 h waiting period following BHP injection. The present results suggest that MC may inhibit NFkappaB activation, exhibiting its ability to downregulate the NFkappaB-dependent gene expression. Thus, it can be expected that MC may have potential for therapeutic intervention on various NFkappaB-dependent pathological conditions such as inflammatory or possibly mutagenic processes.

All-trans retinoic acid induced differentiation of rat mammary epithelial cells cultured in serum-free medium.

Retinoids are applied to not only cancer prevention but also cancer chemotherapy by stimulating differentiation of cells. We studied differentiation inducing effect of all-trans retinoic acid (ATRA) by studying proportion of high dense fractions of stem-like cells and the size of S phase fraction in cell cycle. From mammary organoids obtained from 7- to 8-week old F344 female rat mammary gland, we cultured rat mammary epithelial cells (RMEC) and treated physiological doses of 10(-6), 10(-7), and 10(-8) M ATRA from the first day and then cultured for 4, 7, and 14 days. After that, immunostaining was performed using peanut agglutinin (PNA) and anti-Thy-1.1 monoclonal antibody (Thy-1.1) that can be used as markers of differentiation. We separated four different cell subpopulations by flow cytometry: cells negative to both reagents (B-), PNA-positive cells (PNA+), Thy-1.1-positive cells (Thy-1.1+), and cells positive to both reagents (B+). We observed continuous decreases of high dense fractions of stem-like cells (PNA+ subpopulations) for 14 days and as much decreases as high doses of ATRA, which were thought to be proportional to doses of ATRA. We labeled RMEC with bromodeoxyuridine and investigated cell cycle fractions that went through S phase. We observed a tendency of decrease of S phase fraction with time in culture, which is thought to be related to continuous decreases of PNA+ subpopulations and inhibitory role of ATRA on cell cycle. These results suggest that physiological doses of ATRA could stimulate differentiation of RMEC and convert stem-like RMEC to differentiated cells in SFM for a relatively long period of 14 days.

Induction of differentiation of the cultured rat mammary epithelial cells by triterpene acids.

We investigated the effects of triterpene acids (TAs), ursolic acid (UA) and oleanolic acid (OA), on the induction of proliferation and differentiation of normal rat mammary epithelial cells (RMEC) or organoids cultured in Matrigel or primary culture system. To elucidate the effects, we tested their differentiation inducing activities with intercellular communication ability, cell cycle patterns, induction of apoptosis, and morphological differentiation in the three dimensional extracellular culture system. To study the changes of RMEC subpopulation in culture, the cultured cells were isolated, immunostained with peanut lectin (PNA) and anti-Thy-1.1 antibody and then analyzed with flow cytometry. Four different subpopulations, such as PNA and Thy-1.1 negative cells (B-), PNA positive cells (PNA+), Thy-1.1 positive cells (Thy-1.1+), PNA and Thy-1.1 positive cells (B+), were obtained and the size of each subpopulation was changed in culture with time in the presence of TAs. Intercellular communication was observed in culture for 7 days in TAs-treated cells, but not in culture for 4 days with scrape-loading dye transfer technique. G2/M phase cells and the number of apoptotic population were increased in TAs-treated groups in cell cycle analyses. S phase fractions were reduced and the change of G1 phase cells was not observed. The colonies with distinct multicellular structures, such as stellate, ductal, webbed, squamous, lobulo-ductal colonies, were observed in Matrigel culture and the frequencies of each colony were changed in the presence of TAs. These results suggest that UA and OA have differentiation inducing effects on rat mammary epithelial cells in primary or in Matrigel culture.

Inhibitory effects of retinoids on development of squamous metaplasia in rat mammary epithelial organoids cultured in Matrigel.

Squamous metaplasia (SQM) developed in cultures of rat mammary organoids in reconstituted basement membrane, Matrigel, under either a complete hormone medium (CHM) or a serum-free mammary epithelium growth medium (MEGM). Organoids cultured in CHM gave rise to fewer such SQM (approximately 5%) than those in MEGM (approximately 16%). Formation of SQM was completely suppressed when retinoids were added to CHM. However, a few SQM were still observed in cultures in MEGM with added retinoids. Addition of 5% fetal bovine serum suppressed development of SQM cultured in MEGM. Delayed addition of retinoids also inhibited further development of SQM. Development of SQM from mammary epithelial cells is not common, and regulatory molecules other than retinoids apparently are involved in their formation and prevention.