Involvement of Impaired Angiogenesis and Myelosuppression in Antiresorptive-agent Related Osteonecrosis of the Jaw Mouse Model.

OBJECTIVE: To explore the involvement of bone marrow cells and angiogenesis in the pathogenesis of antiresorptive agent-related osteonecrosis of the jaw (ARONJ). METHODS: We performed micro-computed tomography (CT) and histological analyses in an ARONJ mouse model generated using bisphosphonate (BP) and cyclophosphamide (CY). RESULTS: Micro-CT analysis showed that BP and CY inhibited osteoneogenesis in the extraction socket. Histological analysis at 3 days after tooth extraction showed inhibition of vascular endothelial cell and mesenchymal stem cell mobilization into the extraction socket. When neovascularization of the extraction fossa was observed from as early as 1 day after extraction, it occurred predominantly in the area adjacent to the extraction fossa and close to the bone marrow cavity. In addition, the extraction fossa communicated with the adjacent bone marrow via the vasculature. Histological evaluation of the alveolar bone marrow around the extraction socket showed a decrease in bone marrow cells in the BP + CY group. CONCLUSION: Both inhibition of angiogenesis and suppression of bone marrow cell mobilization are involved in the pathogenesis of ARONJ.

Histochemical and Morphometrical Analyses of Scarless Wound Healing in Mouse Fetal Model.

OBJECTIVE: Scar formation is an inevitable outcome after craniofacial surgery in the congenital facial anomaly. Scarless healing is the ultimate treatment after the surgery. Therefore, we elucidate the mechanism underlying scarless healing during fetal development. METHODS: A full-thickness back skin excision (1 × 0.5 mm) was made at embryonic day 16.5 (E16.5) and 18.5 (E18.5) in fetal C57BL/6J mice and examined the histochemical and morphometrical findings of wound healing after 48 hours. RESULTS: The wound made at E16.5 showed almost complete re-epithelialization with fine reticular dermal collagen fibers, but not at E18.5. The ratio of CK5 positive area was significantly higher in the wound of E16.5 operation than in the E18.5. The wounds made at E18.5 showed granulation tissue formation which will lead to subsequent scar formation. The collagen fibers tended to be thinner in wound than in normal skin, while the decrease in the number of fibers but the increase in the straightness of fibers were evident in the wound at E18.5. CONCLUSION: Transition point of scarless healing seemed between E16.5 and E18.5 in mice, which may imply that the potential of epithelial regeneration and matrix formation was changed, possibly due to alteration of cell constitution and decrease in stemness, at that time.

Influence of Damage-Associated Molecular Patterns from Chondrocytes in Tissue-Engineered Cartilage.

To obtain stable outcomes in regenerative medicine, the quality of cells for transplantation is of great importance. Cellular stress potentially results in the release of damage-associated molecular patterns (DAMPs) and activates immunological responses, affecting the outcome of transplanted tissue. In this study, we intentionally prepared necrotic chondrocytes that would gradually die and release DAMPs and investigated how the maturation of tissue-engineered cartilage was affected. Necrotic chondrocytes were prepared by a conventional heat-treatment method, by which their viability started to decrease after 24 h. When tissue-engineered cartilage containing necrotic chondrocytes was subcutaneously transplanted into C57BL/6J mice, accumulation of cartilage matrix was decreased compared to the control. Meanwhile, immunohistochemical staining demonstrated that localization of macrophages and neutrophils was more apparent in the constructs of necrotic chondrocytes, suggesting that DAMPs from necrotic chondrocytes could prompt migration of more immune cells. Two-dimensional electrophoresis and mass spectrometry identified prelamin as a significant biomolecule released from necrotic chondrocytes. Also, when prelamin was added to a culture of RAW264, Inos and Il1b were increased in accordance with the content of added prelamin. It was suggested that DAMPs from dying chondrocytes could induce inflammatory properties in surrounding macrophages, impairing the maturation of tissue-engineered cartilage. In conclusion, maturation of tissue-engineered cartilage was hampered when less viable chondrocytes releasing DAMPs were included. Impact statement In regenerative medicine, the quality of cells is of great importance to secure clinical safety. During culture, damage of cells could occur, if not critical enough to cause immediate cell death, but still inducing a less viable status. Damage-associated molecular patterns (DAMPs) are released from necrotic cells, but their influence in regenerative medicine has yet to be clarified. In this study, we elucidated how DAMPs from chondrocytes could affect the maturation of tissue-engineered cartilage. Also, possible DAMPs from necrotic chondrocytes were comprehensively analyzed, and prelamin was identified as a significant molecule, which may serve for detecting the existence of necrotic chondrocytes.

Molecular mechanism and potential target indication of TAK-931, a novel CDC7-selective inhibitor.

Replication stress (RS) is a cancer hallmark; chemotherapeutic drugs targeting RS are widely used as treatments for various cancers. To develop next-generation RS-inducing anticancer drugs, cell division cycle 7 (CDC7) has recently attracted attention as a target. We have developed an oral CDC7-selective inhibitor, TAK-931, as a candidate clinical anticancer drug. TAK-931 induced S phase delay and RS. TAK-931-induced RS caused mitotic aberrations through centrosome dysregulation and chromosome missegregation, resulting in irreversible antiproliferative effects in cancer cells. TAK-931 exhibited significant antiproliferative activity in preclinical animal models. Furthermore, in indication-seeking studies using large-scale cell panel data, TAK-931 exhibited higher antiproliferative activities in RAS-mutant versus RAS-wild-type cells; this finding was confirmed in pancreatic patient-derived xenografts. Comparison analysis of cell panel data also demonstrated a unique efficacy spectrum for TAK-931 compared with currently used chemotherapeutic drugs. Our findings help to elucidate the molecular mechanisms for TAK-931 and identify potential target indications.

Application of induced pluripotent stem cells for cartilage regeneration in CLAWN miniature pig osteochondral replacement model.

INTRODUCTION: Pluripotent stem cells have an advantage that they can proliferate without reduction of the quality, while they have risk of tumorigenesis. It is desirable that pluripotent stem cells can be utilized safely with minimal effort in cartilage regenerative medicine. To accomplish this, we examined the potential usefulness of induced pluripotent stem cells (iPS cells) after minimal treatment via cell isolation and hydrogel embedding for cartilage regeneration using a large animal model. METHODS: Porcine iPS-like cells were established from the CLAWN miniature pig. In vitro differentiation was examined for porcine iPS-like cells with minimal treatment. For the osteochondral replacement model, osteochondral defect was made in the quarters of the anteromedial sides of the proximal tibias in pigs. Porcine iPS-like cells and human iPS cells with minimal treatment were seeded on scaffold made of thermo-compression-bonded beta-TCP and poly-L-lactic acid and transplanted to the defect, and cartilage regeneration and tumorigenesis were evaluated. RESULTS: The in vitro analysis indicated that the minimal treatment was sufficient to weaken the pluripotency of the porcine iPS-like cells, while chondrogenic differentiation did not occur in vitro. When porcine iPS-like cells were transplanted into osteochondral replacement model after minimal treatment in vitro, cartilage regeneration was observed without tumor formation. Additionally, fluorescent in situ hybridization (FISH) indicated that the chondrocytes in the regenerative cartilage originated from transplanted porcine iPS-like cells. Transplantation of human iPS cells also showed the regeneration of cartilage in miniature pigs under immunosuppressive treatment. CONCLUSION: Minimally-treated iPS cells will be a useful cell source for cartilage regenerative medicine.

Low tumor glutathione level as a sensitivity marker for glutamate-cysteine ligase inhibitors.

Previous metabolomic analyses of cancer have revealed elevated glutathione levels in tumors. An inhibitor of cystine uptake was identified to suppress glutathione biosynthesis, leading to ferroptosis, a novel iron-dependent form of cell death that differs from apoptosis and necrosis. Glutamate-cysteine ligase (GCL) is the rate-limiting enzyme in the glutathione biosynthesis pathway. Buthionine sulfoximine (BSO), a GCL inhibitor, has previously demonstrated limited clinical benefits. Therefore, selecting patients who respond well to the inhibitor is a key approach for successful future drug development. Ferroptosis induction by BSO has not been fully examined in prior studies. Therefore, the present study investigated the pharmacological effects of BSO and the association between basal intracellular glutathione levels and sensitivity to BSO in cultured cell lines derived from various types of cancer, including those of the kidney [769P, 786-O, A-498, A704, ACHN, Caki-1, Caki-2, G401, G402, RCC4 VHL(-/-), RCC4 VHL(+/+), SK-NEP-1 and SW156] and ovaries (A2780 and A2780/CDDP). BSO was demonstrated to suppress glutathione levels and induce lipid peroxidation, thereby inhibiting cell viability. The viability-reducing effects of BSO were attenuated by ferroptosis inhibition and enhanced by iron, indicating that BSO induced ferroptosis in cancer cells. The cell lines sensitive to BSO, including G402, tended to exhibit non-significantly lower levels of glutathione compared with the BSO-insensitive cell lines, including Caki-2 (P=0.08). Patient sample data indicated the existence of a population of colorectal tumors with lower glutathione levels compared with those of matched normal tissues that might be suitable for the clinical testing of sensitivity to GCLC inhibitors. Collectively, these data suggest that GCL inhibition leads to ferroptosis in cancer cells, and that low glutathione tumor levels may be a patient selection marker for the use of GCL inhibitors in the treatment of tumors.

In vitro and in vivo antitumor activities of T-3764518, a novel and orally available small molecule stearoyl-CoA desaturase 1 inhibitor.

Most cancer cells are characterized by elevated lipid biosynthesis. The rapid proliferation of cancer cells requires de novo synthesis of fatty acids. Stearoyl-CoA desaturase-1 (SCD1), a key enzyme for lipogenesis, is overexpressed in various types of cancer and plays an important role in cancer cell proliferation. Therefore, it has been studied as a candidate target for cancer therapy. In this study, we demonstrate the pharmacological properties of T-3764518, a novel and orally available small molecule inhibitor of SCD1. T-3764518 inhibited stearoyl-CoA desaturase-catalyzed conversion of stearoyl-CoA to oleoyl-CoA in colorectal cancer HCT-116 cells and their growth. Further, it slowed tumor growth in an HCT-116 and a mesothelioma MSTO-211H mouse xenograft model. Comprehensive lipidomic analyses revealed that T-3764518 increases the membrane ratio of saturated: unsaturated fatty acids in various lipid species such as phosphatidylcholines and diacylglycerols in both cultured cells and HCT-116 xenografts. Treatment-associated lipidomic changes were followed by activated endoplasmic reticulum (ER) stress responses such as increased immunoglobulin heavy chain-binding protein expression in HCT-116 cells. These T-3764518-induced changes led to an increase in cleaved poly (ADP-ribose) polymerase 1 (PARP1), a marker of apoptosis. Additionally, bovine serum albumin conjugated with oleic acid, an SCD1 product, prevented cell growth inhibition and ER stress responses by T-3764518, indicating that these outcomes were not attributable to off-target effects. These results indicate that T-3764518 is a promising new anticancer drug candidate.

Intratumor Heterogeneity in Primary Kidney Cancer Revealed by Metabolic Profiling of Multiple Spatially Separated Samples within Tumors.

Metabolic alteration constitutes a hallmark of cancer. Glycolysis and antioxidant pathways in kidney cancer are elevated, with frequent mutation of the VHL gene. Intratumor genetic heterogeneity has been recently demonstrated in kidney cancer. However, intratumor metabolic heterogeneity has not been investigated. Here, we used global metabolomics analysis and tissue slice tracer studies to demonstrate that different portions of a human primary kidney tumor possess different metabolic characteristics and drug sensitivity. Pyruvate levels were elevated and pyruvate metabolism was altered in some tumor sections. These observations indicated that pyruvate metabolism may constitute a possible vulnerability of kidney cancer; indeed, pyruvate stimulated the growth of primary kidney cancer cells and pharmacological inhibition of pyruvate transporters slowed the growth of patient-derived kidney tumors in mice. These findings deepen our understanding of the intratumor metabolic heterogeneity of kidney cancer and may inform novel therapeutic approaches in human kidney cancer.

Cancer with low cathepsin D levels is susceptible to vacuolar (H+ )-ATPase inhibition.

Vacuolar (H+ )-ATPases (V-ATPases) have important roles in the supply of nutrients to tumors by mediating autophagy and the endocytic uptake of extracellular fluids. Accordingly, V-ATPases are attractive therapeutic targets for cancer. However, the clinical use of V-ATPase inhibitors as anticancer drugs has not been realized, possibly owing to their high toxicity in humans. Inhibition of V-ATPase may be an appropriate strategy in highly susceptible cancers. In this study, we explored markers of V-ATPase inhibitor sensitivity. V-ATPase inhibitors led to pH impairment in acidic intracellular compartments, suppression of macropinocytosis, and decreased intracellular amino acid levels. The sensitivity of cells to V-ATPase inhibitors was correlated with low cathepsin D expression, and cancer cells showed increased sensitivity to V-ATPase inhibitors after pretreatment with a cathepsin D inhibitor and siRNA targeting the cathepsin D gene (CTSD). In addition, V-ATPase inhibitor treatment led to the induction of the amino acid starvation response, upregulation of endoplasmic reticulum stress markers, and suppression of mammalian target of rapamycin (mTOR) signaling in cells expressing low levels of cathepsin D. Some colorectal cancer patients showed the downregulation of cathepsin D in tumor tissues compared with matched normal tissues. These findings indicate that V-ATPase inhibitors are promising therapeutic options for cancers with downregulated cathepsin D.

Impairment of the transition from proliferative stage to prehypertrophic stage in chondrogenic differentiation of human induced pluripotent stem cells harboring the causative mutation of achondroplasia in fibroblast growth factor receptor 3.

INTRODUCTION: Achondroplasia (ACH) is a congenital disease which causes dwarfism and many symptoms resulting from skeletal dysplasia. Because present therapeutic strategies are mainly surgical procedures as symptomatic treatments, development of a radical treatment is desired. Clarification of the ACH pathology is essential for creating a new remedy. However, there are many questions about the disease mechanisms that have not been answered. METHODS: As a single base substitution of the FGFR3 gene had been proved to be the ACH causing genome mutation, our group established disease specific iPS cells by introducing the causative mutation of achondroplasia into human iPS cells by CRISPR/Cas9 based genome editing. These cells were differentiated towards chondrocytes, then the gene and protein expressions were examined by real time RT-PCR and Western blotting, respectively. RESULTS: Based on the western blotting analysis, the FGFR3 protein and phosphorylated ERK were increased in the FGFR3 mutated iPS cells compared to the control cells, while the FGFR3 gene expression was suppressed in the FGFR3 mutated iPS cells. According to chondrogenic differentiation experiments, the IHH expression level was increased in the control cells as the differentiation progressed. On the other hand, up-regulation of the IHH gene expression was suppressed in the FGFR3 mutated iPS cells. CONCLUSIONS: These results suggested that chondrocyte maturation was impaired between the proliferative stage and prehypertrophic stage in the chondrocytes of ACH. The development of chemical compounds which affect the specific maturation stage of chondrocytes is expected to contribute to the ACH treatment, and FGFR3 genome-edited hiPSCs will be a valuable tool in such research studies.

Three-dimensional changes of noses after transplantation of implant-type tissue-engineered cartilage for secondary correction of cleft lip-nose patients.

INTRODUCTION: We have developed an implant-type tissue-engineered cartilage using a poly-l-lactide scaffold. In a clinical study, it was inserted into subcutaneous areas of nasal dorsum in three patients, to correct cleft lip-nose deformity. The aim of this study was to helping evaluation on the efficacy of the regenerative cartilage. METHODS: 3D data of nasal shapes were compared between before and after surgery in computed tomography (CT) images. Morphological and qualitative changes of transplants in the body were also evaluated on MRI, for one year. RESULTS: The 3D data from CT images showed effective augmentation (>2 mm) of nasal dorsum in almost whole length, observed on the medial line of faces. It was maintained by 1 year post-surgery in all patients, while affected curves of nasal dorsum was not detected throughout the observation period. In magnetic resonance imaging (MRI), the images of transplanted cartilage had been observed until 1 year post-surgery. Those images were seemingly not straight when viewed from the longitudinal plain, and may have shown gentle adaptation to the surrounding nasal bones and alar cartilage tissues. CONCLUSION: Those findings suggested the potential efficacy of this cartilage on improvement of cleft lip-nose deformity. A clinical trial is now being performed for industrialization.

The in vivo effect of esculetin ointment and esculetin-mixed Zyderm for Zyderm.

BACKGROUND: Injectable collagen is often used for treatment of wrinkles or scars in cosmetic surgery. However, it is degraded within a short period after subcutaneous injection. The authors aimed to achieve a long-lasting effect of the filler with a new collagenase inhibitor, esculetin (6,7-dihydroxy-2H-chromen-2-one). METHOD: Nude mice were divided into two study groups and a control group (35 mg cattle collagen): (1) those implanted with Zyderm 0.3 g subcutaneously into the dorsal region followed by daily topical application of 5% esculetin ointment (0.5 g/day) to the skin of the implanted area (the 5% esculetin ointment group), and (2) those implanted with a mixture of Zyderm 0.3 g and esculetin (1 to 4 mM) (the esculetin-mixed Zyderm groups). In each group, Zyderm was removed at different time points to measure the wet weight and hydroxyproline level. Furthermore, each removed Zyderm specimen was sectioned for histologic examination with Azan staining and immunostaining. RESULTS: In the esculetin ointment group and the 2 mM esculetin-mixed Zyderm group, the hydroxyproline levels at 30, 60, and 90 days were significantly higher than those in the control group, suggesting that esculetin suppresses the biodegradation of Zyderm. There was no significant difference in hydroxyproline level between the esculetin ointment group and the 2 mM esculetin-mixed Zyderm group; biodegradation occurred to a similar extent with either method of application. CONCLUSIONS: An atelocollagen implant is used as a safe and effective scaffold material for tissue regeneration. Future applications of the present study are expected.

Bone and cartilage repair by transplantation of induced pluripotent stem cells in murine joint defect model.

The establishment of cartilage regenerative medicine has been an important issue in the clinical field, because cartilage has the poor ability of self-repair. Currently, tissue engineering using autologous chondrocytes has risen, but we should investigate more appropriate cell sources that can be obtained without any quantitative limitation. In this study, we focused on induced pluripotent stem (iPS) cells, in which the ethical hurdle does not seem higher than that of embryonic stem cells. Mouse iPS cells were transplanted into the mouse joint defect model of the knee. Strains of the transplants and hosts were arranged to be either closest (homology 75% in genetic background) or identical (100%). For transplantation, we embedded the iPS cells within the collagen hydrogel in order to obtain the effective administration of the cells into defects, which induced the differentiation of the iPS cells. At 8 weeks of transplantation, although the iPS cells with a 75% homology to the host in the genetic background tended to form teratoma, those of 100% showed a joint regeneration. GFP immunohistochemistry proved that the transplanted iPS cells were responsible for the bone and cartilage repair. Taking these results together, the iPS cells are regarded as a promising cell source for the cartilage tissue engineering.

Effects of original crystalloid cardioplegia followed by additional blood cardioplegia: treatments for prolonged cardiac arrest.

PURPOSE: The efficacy of combination treatment of original cold crystalloid cardioplegia (SHA solution; St. Thomas + Histidine + ATP + oxygen) and additional blood cardioplegia was studied in patients who required cardiac arrest time of 120 minutes or longer. METHODS: One hundred and thirty-six patients were included in this study. Patients were divided into two groups according to the cardiac arrest time: S group (cardiac arrest time: 120-149 minutes, n = 81); L group (150-180 minutes, n = 55). Just after cross-clamping of the ascending aorta, 800 ml of SHA solution was infused in an antegrade fashion. Cold-blood cardioplegia was initiated after two hours of cardiac arrest. RESULTS: Six (4%) of the 136 patients died after surgery, 3 in each group. Two critical patients with ischemic cardiomyopathy died of cardiac failure after coronary artery bypass grafting (CABG), and 4 died of noncardiac morbidity. The mean value of postoperative maximum creatine phosphokinase-MB (CPK-MB) in dead patients was 47 IU/L in the S group and 75 IU/L in the L group. The peak CPK-MB values exceeded 100 IU/L in one out of 6 patients who died after surgery. CONCLUSIONS: Combination treatment using original SHA solution and additional blood cardioplegia was effective in patients who required prolonged cardiac arrest.

The optimal conditions of chondrocyte isolation and its seeding in the preparation for cartilage tissue engineering.

To optimize the chondrocyte numbers obtained after collagenase digestion for cartilage tissue engineering, we examined the enzyme concentration and incubation time for collagenase digestion. The appropriate cell density in the chondrocyte primary culture was also verified. The collagenase digestion conditions that maximized the viable cell numbers were 24 h in 0.15% and 0.3% collagenase, 6 h in 0.6%, and 4 h in 1.2%, leading to ∼5 × 105 cells from 0.05 g. When seeded at 10,000 cells/cm², all of these cells became almost confluent within 1 week. Cells digested in 0.3% for 24 h or 0.6% for 6 h and seeded at 3000 cells/cm² may also be acceptable, and similarly reached confluence within 1 week. Results regarding expression of the p53, tumor necrosis factor-α, and interleukin-1ß genes, as well as apoptosis enzyme-linked immunosorbent assay results, show that excessive collagenase exposure may decrease chondrocyte viability or activity. We recommend a 24-h incubation in 0.3% collagenase or 6 h in 0.6% collagenase, and a cell-seeding density of 3000-10,000 cells/cm². These conditions maximize the harvest of isolated chondrocytes from a small amount of biopsied tissue and significantly aid in obtaining a large quantity of cultured cells in a short period.

Selection and effect of ointment bases for preparing collagenase inhibitor ointment using high-performance liquid chromatography and Franz cell apparatus.

After the dorsal subcutaneous administration of injectable collagen implant derived from bovine dermis (Zyderm; INAMED, Santa Barbara, CA) to mice, ointments that contain 3 types of collagenase inhibitors, Esculetin (6,7-dihydroxy-2H-chromen-2-one), ONO-4817 [(2S,4S)-N-hydroxy-5-ethoxymethyloxy-2-methyl-4-(4-phenoxybenzoyl) amino-pentanamide], and MMI270 (CGS27023A) {N-hydroxy-2(R)-[(4-methoxysulfonyl)-(3-picolyl)-amino]-3-methylbutanamide hydrochloride monohydrate} were applied daily on the dorsal region of mice (injection site), and intradermal Zyderm was extirpated after 30, 60, and 90 days to measure the level of hydroxyproline. Furthermore, dermal tissue was examined by Azan staining and immunostaining. A significant difference was observed in the level of hydroxyproline in the Esculetin and the ONO-4817 ointment groups compared with that in the control group after 30 days. A significant difference was also observed in the level of hydroxyproline in the Esculetin ointment group compared with that in the control group after 60 and 90 days. Histologically, 90 days after the application of the ointment, dense localization of type III collagen was observed around the injected Zyderm in the group applied Esculetin ointment compared with the control group. Therefore, it was indicated that Esculetin suppressed the degradation of collagen, and further facilitated the qualitative changes that increased neo-collagen, and that the collagen implant with hypodermic injection remained on behalf of ointments contained within the collagenase inhibitors that were applied on the skin surface.

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.