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Ferroptosis, a novel regulated cell death induced by iron-dependent lipid peroxidation, plays an important role in tumor development and drug resistance. Long noncoding RNAs (lncRNAs) are associated with various types of cancer. However, the precise roles of many lncRNAs in tumorigenesis remain elusive. Here we explored the transcriptomic profiles of lncRNAs in primary CRC tissues and corresponding paired adjacent non-tumor tissues by RNA-seq and found that LINC00239 was significantly overexpressed in colorectal cancer tissues. Abnormally high expression of LINC00239 predicts poorer survival and prognosis in colorectal cancer patients. Concurrently, we elucidated the role of LINC00239 as a tumor-promoting factor in CRC through in vitro functional studies and in vivo tumor xenograft models. Importantly, overexpression of LINC00239 decreased the anti-tumor activity of erastin and RSL3 by inhibiting ferroptosis. Collectively, these data suggest that LINC00239 plays a novel and indispensable role in ferroptosis by nucleotides 1–315 of LINC00239 to interact with the Kelch domain (Nrf2-binding site) of Keap1, inhibiting Nrf2 ubiquitination and increasing Nrf2 protein stability. Considering the recurrence and chemoresistance constitute the leading cause of death in colorectal cancer (CRC), ferroptosis induction may be a promising therapeutic strategy for CRC patients with low LINC00239 expression.

Cells with the desired phenotype and number are critical for regenerative medicine and tissue engineering. Uniparental parthenogenetic embryonic stem cells (pESCs) share fundamental properties with embryonic stem cells. This study aims to determine the viability of pESC-based tissue engineering for bone and cartilage reconstruction. The mouse pESCs were cultured in suspension to form embryoid bodies. An adherent cultivation approach was employed to obtain parthenogenetic embryonic mesenchymal stem cells (pMSCs) from the embryoid bodies. Then, the pMSCs were cultured in conditional media to differentiate into osteogenic and chondrogenic lineages. The pESC-derived osteoblasts and chondroblasts were seeded into coral and sodium alginate scaffolds, respectively. The cell-seeded scaffolds were implanted into dorsal subcutaneous pockets of nude mice to evaluate ectopic reconstruction of bone and cartilage. We demonstrated that pESCs display the capacity to differentiate into all three germ layers. The generated pMSCs were able to differentiate into osteogenic and chondrogenic lineages, which survived well after seeding into coral and alginate acid scaffolds. Six weeks after cell-scaffold implantation, gross inspection and histological examination revealed that ectopic bone and cartilage tissues had successfully regenerated in the specimen. According to the findings of this study, pESC derivatives have a high potential for bone and cartilage regeneration.

Silk fibroin (SF) is considered biocompatible and biodegradable for osteochondral repair. However, it lacks a bioactive domain for cell adhesion, proliferation and differentiation, limiting its therapeutic efficacy. To revamp SF as a biomimicking and bioactive microenvironment to regulate cell behaviours, we engineered an elastin-like polypeptide (ELP, Val-Pro-Gly-Xaa-Gly) to modify SF fibers via simple and green dehydrothermal (DHT) treatment. Our results demonstrated that the ELP successfully bound to SF, and the scaffold was reinforced by the fusion of the silk fiber intersections with ELP (S-ELP-DHT) via the DHT treatment. Both bone mesenchymal stem cells (BMSCs) and chondrocytes exhibited improved spreading and proliferation on the S-ELP-DHT scaffolds. The ex vivo and in vivo experiments further demonstrated enhanced mature bone and cartilage tissue formation using the S-ELP-DHT scaffolds compared to the naked SF scaffolds. These results indicated that a recombinant ELP-modified silk scaffold can mimic three-dimensional (3D) cell microenvironment, and improve bone and cartilage regeneration. We envision that our scaffolds have huge clinical potential for osteochondral repair., Graphical abstract Schematic illustration showing the S-ELP-DHT scaffolds can be fabricated into fibrocartilage or bone mimic by seeding with chondrocytes or BMSCs for osteochondral regeneration.Image 1, Highlights • Elastin-like polypeptide (ELP) modified silk fibroin (SF) scaffold was developed via dehydrothermal treatment (S-ELP-DHT). • The S-ELP-DHT scaffold provided a beneficial cell microenvironment for osteochondral repair. • Greater mature bone and cartilage tissue formation were achieved. • Improved repair efficacy for articular osteochondral defects was confirmed.

We have developed CRISPR-assisted RNA-protein interaction detection method (CARPID), which leverages CRISPR-CasRx-based RNA targeting and proximity labeling to identify binding proteins of specific long non-coding RNAs (lncRNAs) in the native cellular context. We applied CARPID to the nuclear lncRNA XIST, and it captured a list of known interacting proteins and multiple previously uncharacterized binding proteins. We generalized CARPID to explore binders of the lncRNAs DANCR and MALAT1, revealing the method's wide applicability in identifying RNA-binding proteins.

Fibrosis exists in almost all organs/tissues of the human body, plays an important role in the occurrence and development of diseases and is also a hallmark of the aging process. However, there is no effective prevention or therapeutic method for fibrogenesis. As a serine/threonine (Ser/Thr)-protein kinase, glycogen synthase kinase-3β (GSK-3β) is a vital signaling mediator that participates in a variety of biological events and can inhibit extracellular matrix (ECM) accumulation and the epithelial-mesenchymal transition (EMT) process, thereby exerting its protective role against the fibrosis of various organs/tissues, including the heart, lung, liver, and kidney. Moreover, we further present the upstream regulators and downstream effectors of the GSK-3β pathway during fibrosis and comprehensively summarize the roles of GSK-3β in the regulation of fibrosis and provide several potential targets for research. Collectively, the information reviewed here highlights recent advances vital for experimental research and clinical development, illuminating the possibility of GSK-3β as a novel therapeutic target for the management of tissue fibrosis in the future.

Phosphatidylinositol 3-kinase (PI3K) signaling plays a central role in various biological processes, and its abnormality leads to a broad spectrum of human diseases, such as cancer, fibrosis, and immunological disorders. However, the mechanisms by which PI3K signaling regulates the behavior of stem cells during regeneration are poorly understood. Planarian flatworms possess abundant adult stem cells (called neoblasts) allowing them to develop remarkable regenerative capabilities, thus the animals represent an ideal model for studying stem cells and regenerative medicine in vivo. In this study, the spatiotemporal expression pattern of Djpi3k, a PI3K ortholog in the planarian Dugesia japonica, was investigated and suggests its potential role in wound response and tissue regeneration. A loss-of-function study was conducted using small molecules and RNA interference technique, providing evidence that PI3K signaling is required for blastema regrowth and cilia maintenance during planarian regeneration and homeostasis. Interestingly, the mitotic and apoptotic responses to amputation are substantially abated in PI3K inhibitor-treated regenerating animals, while knockdown of Djpi3k alleviates the mitotic response and postpones the peak of apoptotic cell death, which may contribute to the varying degrees of regenerative defects induced by the pharmacological and genetic approaches. These observations reveal novel roles for PI3K signaling in the regulation of the cellular responses to amputation during planarian regeneration and provide insights for investigating the disease-related genes in the regeneration-competent organism in vivo.

Protein glycosylation is an important posttranslational modification that plays a crucial role in cellular function. However, its biological roles in tissue regeneration remain interesting and primarily ambiguous. In this study, we profiled protein glycosylation during head regeneration in planarian Dugesia japonica using a lectin microarray. We found that 6 kinds of lectins showed increased signals and 16 kinds showed decreased signals. Interestingly, we found that protein core fucosylation, manifested by Lens culinaris agglutinin (LCA) staining, was significantly upregulated during planarian head regeneration. Lectin histochemistry indicated that the LCA signal was intensified within the wound and blastemal areas. Furthermore, we found that treatment with a fucosylation inhibitor, 2F-peracetyl-fucose, significantly retarded planarian head regeneration, while supplement with L-fucose could improve DjFut8 expression and stimulate planarian head regeneration. In addition, 53 glycoproteins that bound to LCA were selectively isolated by LCA-magnetic particle conjugates and identified by LC-MS/MS, including the neoblast markers DjpiwiA, DjpiwiB, DjvlgA, and DjvlgB. Overall, our study provides direct evidence for the involvement of protein core fucosylation in planarian regeneration.

The basic fibroblast growth factor (bFGF) plays an important role in the wound repair process. However, lacking of better biomaterials to carry bFGF still is a challenge in skin repair and regeneration. In this study, the human-like collagen (HLC) cross-linked with transglutaminase (TG) to fabricate a HLC/TG hydrogel to load bFGF. The physical properties of hydrogel, such as interior structure, mechanical property, were characterized

Forkhead box O4 (FOXO4), a member of FOXO family, has been highlighted as an essential transcriptional regulator in many diverse carcinomas. Accumulated studies have demonstrated that FOXO4 is downregulated and associated with tumorigenesis, invasiveness, and metastasis of most human cancer. FOXO4 alteration is also closely linked to the prognosis of various types of cancer. The aim of this review is to comprehensively present the clinical and pathological significance of FOXO4 in human cancer. Additionally, the potential clinical applications of future FOXO4 research are discussed. Collectively, the information reviewed here should increase the potential of FOXO4 as a therapeutic target for cancer.

DEAD (Asp-Glu-Ala-Asp) cassette helicase 21 (DDX21) is an ATP-dependent RNA helicase that is overexpressed in various malignancies. There is increasing evidence that DDX21 is involved in carcinogenesis and cancer progression by promoting cell proliferation. However, the functional role of DDX21 in gastric cancer is largely unknown. In this study, we observed that DDX21 was significantly up-regulated in gastric cancer tissues compared to paired adjacent normal tissues. The expression of DDX21 was closely related to the pathological stage of gastric cancer. In vitro and in vivo studies had shown that knockdown of DDX21 inhibited gastric cancer cell proliferation, colony formation, G1/S cell cycle transition and xenograft growth, while ectopic expression of DDX21 promoted these cell functions. Mechanically, DDX21 induced gastric cancer cell growth by up-regulating levels of Cyclin D1 and CDK2. Taken together, these results revealed a novel role for DDX21 in the proliferation of gastric cancer cells via the Cyclin D1 and CDK2 pathways. Therefore, DDX21 can be used as a therapeutic target for gastric cancer.

c-Fos is an immediate-early gene that modulates cellular responses to a wide variety of stimuli and also plays an important role in tissue regeneration. However, the sequence and functions of c-Fos are still poorly understood in newts. This study describes the molecular cloning and characterization of the c-Fos gene (Co-c-Fos) of the Chinese fire-bellied newt, Cynops orientalis. The full-length Co-c-Fos cDNA sequence consists of a 1290 bp coding sequence that encoded 429 amino acids. The alignment and phylogenetic analyses reveal that the amino acid sequence of Co-c-Fos shared a conserved basic leucine zipper domain, including a nuclear localization sequence and a leucine heptad repeat. The Co-c-Fos mRNA is widely expressed in various tissues and is highly and uniformly expressed along the newt limb. After limb amputation, the expression of Co-c-Fos mRNA was immediately upregulated, but rapidly declined. However, the significant upregulation of Co-c-Fos protein expression was sustained for 24 h, overlapping with the wound healing stage of C. orientalis limb regeneration. To investigate if Co-c-Fos participate in newt wound healing, a skin wound healing model is employed. The results show that the treatment of T-5224, a selective c-Fos inhibitor, could largely impair the healing process of newt&rsquo, s skin wound, as well as the injury-induced matrix metalloproteinase-3 upregulation, which is fundamental to wound epithelium formation. These data suggest that Co-c-Fos might participate in wound healing by modulating the expression of its potential target gene matrix metalloproteinase-3. Our study provides important insights into mechanisms that are responsible for the initiation of newt limb regeneration.

Treating serious bone trauma with an osteo-inductive agent such as bone morphogenetic proteins (BMPs) has been considered as an optimized option when delivered via a collagen sponge (CS). Previous works have shown that the BMP concentration and release rate from approved CS carriers is difficult to control with precision. Here we presented the fabrication of a recombinant fusion protein from recombinant human-like collagen (HLC) and human BMP-2 (hBMP2). The fusion protein preserved the characteristic of HLC allowing the recombinant protein to be expressed in Yeast (such as Pichia pastoris GS115) and purified rapidly and easily with mass production after methanol induction. It also kept the stable properties of HLC and hBMP2 in the body fluid environment with good biocompatibility and no cytotoxicity. Moreover, the recombinant fusion protein fabricated a vertical through-hole structure with improved mechanical properties, and thus facilitated migration of bone marrow mesenchymal stem cells (MSCs) into the fusion materials. Furthermore, the fusion protein degraded and released hBMP-2 in vivo allowing osteoinductive activity and the enhancement of utilization rate and the precise control of the hBMP2 release. This fusion protein when applied to cranial defects in rats was osteoinductively active and improved bone repairing enhancing the repairing rate 3.5- fold and 4.2- fold when compared to the HLC alone and the control, respectively. There were no visible inflammatory reactions, infections or extrusions around the implantation sites observed. Our data strongly suggests that this novel recombinant fusion protein could be more beneficial in the treatment of bone defects than the simple superposition of the hBMP2/collagen sponge.

A massively parallel array of monoclonal antibodies enables rapid antibody development and target discovery across species., Antibodies are essential for elucidating gene function. However, affordable technology for proteome-scale antibody generation does not exist. To address this, we developed Proteome Epitope Tag Antibody Library (PETAL) and its array. PETAL consists of 62,208 monoclonal antibodies (mAbs) against 15,199 peptides from diverse proteomes. PETAL harbors binders for a great multitude of proteins in nature due to antibody multispecificity, an intrinsic antibody feature. Distinctive combinations of 10,000 to 20,000 mAbs were found to target specific proteomes by array screening. Phenotype-specific mAb-protein pairs were found for maize and zebrafish samples. Immunofluorescence and flow cytometry mAbs for membrane proteins and chromatin immunoprecipitation–sequencing mAbs for transcription factors were identified from respective proteome-binding PETAL mAbs. Differential screening of cell surface proteomes of tumor and normal tissues identified internalizing tumor antigens for antibody-drug conjugates. By finding high-affinity mAbs at a fraction of current time and cost, PETAL enables proteome-scale antibody generation and target discovery.

Macrophages are a specific mononuclear cell group abundant in almost every organ of higher animals. This group is a pivotal part of the immune system and is involved in immune responses against exogenous antigen invasion. Recently, accumulating evidence has demonstrated that macrophages participate in wound repair and tissue regeneration. In this review, we will first introduce the influences of regeneration after injury in various tissues and organs among macrophage-depleted animal models. Second, the possible relationship between macrophages and reparation capacities will be discussed. Finally, we provide a general idea about the roles of macrophages in the injury- regeneration process and then discuss the current challenges and prospects of their clinical application. The information compiled here may be useful for regenerative research and may promote macrophages as a therapeutic target in regenerative medicine.

Glycans are known to play important roles in molecular recognition, cell-cell adhesion, molecular trafficking, receptor activation, and signal transduction during development and regeneration. Despite numerous investigations of regenerating salamander limbs, global analysis of the precise variation of glycans during the limb regeneration process has received little attention. Here, we have used lectin microarrays and lectin histochemistry to analyze the alterations and distribution of glycans during the early stages leading to blastema formation during Cynops orientalis limb regeneration in response to limb amputation. Compared with the control group, analysis at several time points (3, 7, and 14 days postamputation) using microarrays containing 37 lectins showed that limb tissues expressed significantly different complements of glycans recognized by 9 different lectins. Postamputation limb tissues showed higher expression of two glycan structures recognized by the lectins STL and LTL and lower expression of seven glycan structures recognized by PHA-E, MAL-I, SNA, UEA-I, PHA-E + L, VVA, and GNA. We also observed significant changes in glycans specifically at 7 days postamputation, including higher binding capacity by WFA, GSL-I, and NPA and lower binding capacity by PNA, HHL, ConA, LCA, GSL-II, and PWM. Next, we validated our lectin microarray data using lectin histochemistry in limb tissues. Glycans recognized by STL and GNA showed similar changes in signal intensity to those found in the lectin microarrays, with STL staining in the cytoplasm and GNA in the cytoplasm and nucleus. Our findings are the first report of significant postamputation changes in glycans in limb tissues and suggest that those glycans perform potentially important functions during the early stages of C. orientalis limb regeneration.

Background Salamanders regenerate their limbs after amputation. However, the molecular mechanism of this unique regeneration remains unclear. In this study, isobaric tags for relative and absolute quantification (iTRAQ) coupled with liquid chromatography tandem mass spectrometry (LC-MS/MS) was employed to quantitatively identify differentially expressed proteins in regenerating limbs 3, 7, 14, 30 and 42 days post amputation (dpa). Results Of 2636 proteins detected in total, 253 proteins were differentially expressed during different regeneration stages. Among these proteins, Asporin, Cadherin-13, Keratin, Collagen alpha-1(XI) and Titin were down-regulated. CAPG, Coronin-1A, AnnexinA1, Cathepsin B were up-regulated compared with the control. The identified proteins were further analyzed to obtain information about their expression patterns and functions in limb regeneration. Functional analysis indicated that the differentially expressed proteins were associated with wound healing, immune response, cellular process, metabolism and binding. Conclusions This work indicated that significant proteome alternations occurred during salamander limb regeneration. The results may provide fundamental knowledge to understand the mechanism of limb regeneration. Electronic supplementary material The online version of this article (10.1186/s12864-017-4125-4) contains supplementary material, which is available to authorized users.

Background Embryonic stem cell (ESC) derivatives hold great promise for the construction of tissue-engineered skin equivalents (TESE). However, harvesting of ESCs destroys viable embryos and may lead to political and ethical concerns over their application. In the current study, we directed mouse parthenogenetic embryonic stem cells (pESCs) to differentiate into fibroblasts, constructed TESE, and evaluated its function in vivo. Methods The stemness marker expression and the pluripotent differentiation ability of pESCs were tested. After embryoid body (EB) formation and adherence culture, mesenchymal stem cells (MSCs) were enriched and directed to differentiate into fibroblastic lineage. Characteristics of derived fibroblasts were assessed by quantitative real-time PCR and ELISA. Functional ability of the constructed TESE was tested by a mouse skin defects repair model. Results Mouse pESCs expressed stemness marker and could form teratoma containing three germ layers. MSCs could be enriched from outgrowths of EBs and directed to differentiate into fibroblastic lineage. These cells express a high level of growth factors including FGF, EGF, VEGF, TGF, PDGF, and IGF1, similar to those of ESC-derived fibroblasts and mouse fibroblasts. Seeded into collagen gels, the fibroblasts derived from pESCs could form TESE. Mouse skin defects could be successfully repaired 15 days after transplantation of TESE constructed by fibroblasts derived from pESCs. Conclusions pESCs could be induced to differentiate into fibroblastic lineage, which could be applied to the construction of TESE and skin defect repair. Particularly, pESC derivatives avoid the limitations of political and ethical concerns, and provide a promising source for regenerative medicine.

Uniparental parthenogenesis yields pluripotent stem cells without the political and ethical concerns surrounding the use of embryonic stem cells (ESCs) for biomedical applications. In the current study, we hypothesized that parthenogenetic stem cells (pSCs) could be directed to differentiate into tenocytes and applied for tissue-engineered tendon. We showed that pSCs displayed fundamental properties similar to those of ESCs, including pluripotency, clonogenicity, and self-renewal capacity. pSCs spontaneously differentiated into parthenogenetic mesenchymal stem cells (pMSCs), which were positive for mesenchymal stem cell surface markers and possessed osteogenic, chondrogenic, and adipogenic potential. Then, mechanical stretch was applied to improve the tenogenic differentiation of pMSCs, as indicated by the expression of tenogenic-specific markers and an increasing COL1A1:3A1 ratio. The pSC-derived tenocytes could proliferate and secrete extracellular matrix on the surface of poly(lactic-co-glycolic) acid scaffolds. Finally, engineered tendon-like tissue was successfully generated after in vivo heterotopic implantation of a tenocyte-scaffold composite. In conclusion, our experiment introduced an effective and practical strategy for applying pSCs for tendon regeneration.

The understanding of adipose tissue development is crucial for the treatment of obesity-related diseases. Adipogenesis has been extensively investigated at the gene and protein levels in recent years. However, the alterations in protein glycosylation during this process remains unknown, particularly that of parthenogenetic embryonic stem cells (pESCs), a type of ESCs with low immunogenicity and no ethical concerns regarding their use. Protein glycosylation markedly affects cell growth and development, cell-to-cell communication, tumour growth and metastasis. In the present study, the adipogenic potentials of J1 ESCs and pESCs were first compared and the results demonstrated that pESCs had lower adipogenic potential compared with J1 ESCs. Lectin microarray was then used to screen the alteration of protein glycosylation during adipogenesis. The results revealed that protein modification of GlcNAc and α-1-2-fucosylation increased, whereas α-1-6-fucosylation, α-2-6-sialylation and α-1-6-mannosylation decreased in J1 ESCs and pESCs during this process. In addition, α-1-3-mannosylation decreased only in pESCs. Lectin histochemistry and quantitative polymerase chain reaction of glycosyltransferase confirmed the results obtained by lectin microarray. Therefore, protein glycosylation of ESCs was significantly altered during adipogenesis, indicating that protein glycosylation analysis is not only helpful for studying the mechanism of adipogenesis, but may also be used as a marker to monitor adipogenic development.

The selection of appropriate seed cells is crucial for adipose tissue engineering. Here, we reported the stepwise induction of parthenogenetic embryonic stem cells (pESCs) to differentiate into adipogenic cells and its application in engineering injectable adipose tissue with Pluronic F-127. pESCs had pluripotent differentiation capacity and could form teratomas that include the three primary germ layers. Cells that migrated from the embryoid bodies (EBs) were selectively separated and expanded to obtain embryonic mesenchymal stem cells (eMSCs). The eMSCs exhibited similar cell surface marker expression profiles with bone morrow mesenchymal stem cells (BMSCs) and had multipotent differentiation capacity. Under the induction of dexamethasone, indomethacin, and insulin, eMSCs could differentiate into adipogenic cells with increased expression of adipose-specific genes and oil droplet depositions within the cytoplasm. To evaluate their suitability as seed cells for adipose tissue engineering, the CM-Dil labelled adipogenic cells derived from eMSCs were seeded into Pluronic F-127 hydrogel and injected subcutaneously into nude mice. Four weeks after injection, glistering and semitransparent constructs formed in the subcutaneous site. Histological observations demonstrated that new adipose tissue was successfully fabricated in the specimen by the labelled cells. The results of the current study indicated that pESCs have great potential in the fabrication of injectable adipose tissue.

Colon cancer is a type of malignant tumor that causes considerable mortality worldwide. Epithelial cellular adhesion molecule (EpCAM), a tumor-associated antigen of colon tumors, is a target for colon cancer therapy. EpCAM-specific monoclonal antibodies (mAbs) have been applied in human colon cancer since the 1990s; however, the therapeutic effects are limited. EpCAM activates nuclear signaling pathways by releasing its intracellular domain (EpICD). The released EpICD stimulates the Wnt/β-catenin signaling pathway, which is also strongly associated with tumorigenesis. EpCAM is also a target gene of the Wnt/β-catenin signaling pathway. EpCAM and the Wnt/β-catenin signaling pathway form a functional interaction cycle in colon cancer. Thus, we propose a new therapeutic drug for colon cancer: an EpCAM single-chain fragment variable antibody (scFv)-truncated protamine-siRNA. EpCAM scFv can recognize and bind colon cancer cells through its EpCAM antigen activity. Furthermore, the specific siRNA transferred into colon cancer cells specifically inhibits Wnt/β-catenin signal transmission. Therefore, this new drug may efficiently interrupt the functional cycle between EpCAM and Wnt/β-catenin signaling and be an effective therapeutic strategy for colon cancer.

Background Marrow mesenchymal stem cells (MSCs) can differentiate into specific phenotypes, including chondrocytes, and have been widely used for cartilage tissue engineering. However, cartilage grafts from MSCs exhibit phenotypic alternations after implantation, including matrix calcification and vascular ingrowth. Methods We compared chondromodulin-1 (Chm-1) expression between chondrocytes and MSCs. We found that chondrocytes expressed a high level of Chm-1. We then adenovirally transduced MSCs with Chm-1 and applied modified cells to engineer cartilage in vivo. Results A gross inspection and histological observation indicated that the chondrogenic phenotype of the tissue-engineered cartilage graft was well maintained, and the stable expression of Chm-1 was detected by immunohistological staining in the cartilage graft derived from the Chm-1 gene-modified MSCs. Conclusions Our findings defined an essential role for Chm-1 in maintaining chondrogenic phenotype and demonstrated that Chm-1 gene-modified MSCs may be used in cartilage tissue engineering.

With the increasing popularity of minimally invasive surgery, to develop an injectable bone would be highly preferable for the repair of bone nonunions and defects. However, the use of dissociated cells and exogenous carriers to construct injectable bone faces several drawbacks. To circumvent these limitations, we first harvested a cell sheet from rabbit bone marrow stromal cells using a continuous culture method and a scraping technique. The obtained sheet was then cut into fragments of multicellular aggregates, each of which was composed of a certain number of cells, extracellular matrix, and intercellular connections. The aggregates showed apparent mineralization properties, high alkaline phosphatase activity, increased osteocalcin content, and upregulated bone markers, implying their in vitro osteogenic potential. Then, serum-free medium (the control group), dissociated cell suspension (the cell group), and suspension of multicellular aggregates (the aggregate group) were injected subcutaneously on the back of the nude mice to evaluate ectopic bone formation. The results revealed that the aggregate group showed significantly larger and denser bone at the injection sites than the cell group, whereas bone formation did not occur in the control group. Additionally, when injecting them locally into the mandibular fracture gap of delayed healing in a rabbit model, we observed the most improved bone healing in the aggregate group. More cells survive and retain at the injection sites in the aggregate group than that in the cell group postoperatively. Our study indicates that the multicellular aggregates might be considered a promising strategy to generate injectable bone tissue and improve the efficacy of cell therapy.

Objectives: The periosteum plays an important role in bone regeneration. However, the harvesting of autogenous periosteum is associated with disadvantages such as donor site morbidity and limited donor sources. This study uses an osteogenic predifferentiated cell sheet to fabricate a scaffold-free tissue-engineered periosteum (TEP). Material and methods: We generated an osteogenic predifferentiated cell sheet from rabbit bone marrow stromal cells (BMSCs) using a continuous culture system and harvested it using a scraping technique. Then, the in vitro characterization of the sheet was investigated using microscopy investigation, quantitative analysis of alkaline phosphatase (ALP) activity, and RT-PCR. Next, we demonstrated the in vivo osteogenic potential of the engineered sheet in ectopic sites together with a porous β-tricalcium phosphate ceramic. Finally, we evaluated its efficiency in treating delayed fracture healing after wrapping the cell sheet around the mandible in a rabbit model. Results: The engineered periosteum showed sporadic mineralized nodules, elevated ALP activity, and up-regulated gene expression of osteogenic markers. After implantation in the subcutaneous pockets of the donor rabbits, the in vivo bone-forming capability of the engineered periosteum was confirmed by histological examinations. Additionally, when wrapping the engineered periosteum around a mandibular fracture gap, we observed improved bone healing and reduced amounts of fibrous tissue at the fracture site. Conclusion: The osteogenic predifferentiated BMSC sheet can act as a scaffold-free TEP to facilitate bone regeneration. Hence, our study provides a promising strategy for enhancing bone regeneration in clinical settings. To cite this article: Ma D, Yao H, Tian W, Chen F, Liu Y, Mao T, Ren L. Enhancing bone formation by transplantation of a scaffold-free tissue-engineered periosteum in a rabbit model. Clin. Oral Impl. Res. 22, 2011; 1193–1199. doi: 10.1111/j.1600-0501.2010.02091.x

Hypoxia is a common phenomenon in human solid tumors and has been considered as an important, independent negative prognostic factor for response to treatment and survival of tumor patients. Hypoxia-inducible factor-1 (HIF-1) is the central transcription factor which is activated by hypoxia and modulates the expression of many genes involved in cell metabolism, proliferation, apoptosis, angiogenesis. Recently, it has been reported that HIF-1 contributes to tumor radioresistance by upregulating survivin expression under hypoxic conditions. Moreover, in hypoxic tumor cells, HIF-1 dependent signal transduction pathway is activated and could be further enhanced by radiation, thereby providing survival signals to adjacent vascular endothelial cells by upregulation of VEGF and bFGF and resulting in tumor radioresistance through vascular radioprotection. Recent research revealed that the stability of HIF-1alpha, one of the two subunits of HIF-1, determines the whole HIF-1 activity and the C-terminal transactivation domain of HIF-1alpha could reduce HIF-1 activity when overexpressed in tumor cells by disruption of the assembly of HIF-1 transcription complex. Therefore, we postulate that fusion with protein transduction domains would overcome the inability of C-terminal transactivation domain of HIF-1alpha to cross cellular membrane. Thus the recombinant fusion proteins could serve as cell-permeable HIF-1 antagonists, function as both inhibitors of tumor angiogenesis and tumor radiosensitizers, and would be widely used in clinical settings to improve tumor response to radiotherapy.

The development of bone tissue engineering has provided new solutions for bone defects. However, the cell-scaffold-based approaches currently in use have several limitations, including low cell seeding rates and poor bone formation capacity. In the present study, we developed a novel strategy to engineer bone grafts using mesenchymal stem cell sheets and coral particles. Rabbit bone marrow mesenchymal stem cells were continuously cultured to form a cell sheet with osteogenic potential and coral particles were integrated into the sheet. The composite sheet was then wrapped around a cylindrical mandrel to fabricate a tubular construct. The resultant tubular construct was cultured in a spinner-flask bioreactor and subsequently implanted into a subcutaneous pocket in a nude mouse for assessment of its histological characteristics, radiological density and mechanical property. A similar construct assembled from a cell sheet alone acted as a control. In vitro observations demonstrated that the composite construct maintained its tubular shape, and exhibited higher radiological density, compressive strength and greater extracellular matrix deposition than did the control construct. In vivo experiments further revealed that new bone formed ectopically on the composite constructs, so that the 8-week explants of the composite sheets displayed radiological density similar to that of native bone. These results indicate that the strategy of using a combination of a cell sheet and coral particles has great potential for bone tissue engineering and repairing bone defects.

Osteoporosis, a metabolic bone disease, threatens postmenopausal women globally. Hormone replacement therapy (HTR), especially estrogen replacement therapy (ERT), is used widely in the clinic because it has been generally accepted that postmenopausal osteoporosis is caused by estrogen deficiency. However, hypogonadal α and β estrogen receptor null mice were only mildly osteopenic, and mice with either receptor deleted had normal bone mass, indicating that estrogen may not be the only mediator that induces osteoporosis. Recently, follicle-stimulating hormone (FSH), the serum concentration of which increases from the very beginning of menopause, has been found to play a key role in postmenopausal osteoporosis by promoting osteoclastogenesis. In this article, we confirmed that exogenous FSH can enhance osteoclast differentiation in vitro and that this effect can be neutralized by either an anti-FSH monoclonal antibody or anti-FSH polyclonal sera raised by immunizing animals with a recombinant GST-FSHβ fusion protein antigen. Moreover, immunizing ovariectomized rats with the GST-FSHβ antigen does significantly prevent trabecular bone loss and thereby enhance the bone strength, indicating that a FSH-based vaccine may be a promising therapeutic strategy to slow down bone loss in postmenopausal women.

Clinical application of platelet-rich plasma (PRP)-based injectable tissue engineering is limited by weak mechanical properties and a rapid fibrinolytic rate. We proposed a new strategy, a cell bricks-stabilized PRP injectable system, to engineer and regenerate cartilage with stable morphology and structure in vivo. Chondrocytes from the auricular cartilage of rabbits were isolated and cultured to form cell bricks (fragmented cell sheet) or cell expansions. Fifteen nude mice were divided evenly (n = 5) into cells-PRP (C-P), cell bricks-PRP (CB-P) and cell bricks-cells-PRP (CB-C-P) groups. Cells, cell bricks or a cell bricks/cells mixture were suspended in PRP and were injected subcutaneously in animals. After 8 weeks, all the constructs were replaced by white resilient tissue; however, specimens from the CB-P and CB-C-P groups were well maintained in shape, while the C-P group appeared distorted, with a compressed outline. Histologically, all groups presented lacuna-like structures, glycosaminoglycan-enriched matrices and positive immunostaining of collagen type II. Different from the uniform structure presented in CB-C-P samples, CB-P presented interrupted, island-like chondrogenesis and contracted structure; fibrous interruption was shown in the C-P group. The highest percentage of matrix was presented in CB-C-P samples. Collagen and sGAG quantification confirmed that the CB-C-P constructs had statistically higher amounts than the C-P and CB-P groups; statistical differences were also found among the groups in terms of biomechanical properties and gene expression. We concluded that cell bricks-enriched PRP gel sufficiently enhanced the morphological stability of the constructs, maintained chondrocyte phenotypes and favoured chondrogenesis in vivo, which suggests that such an injectable, completely biological system is a suitable cell carrier for cell-based cartilage repair.

The transcription factors (Oct4, Sox2, c-Myc, and Klf4) play an important role in the generation of induced pluripotent stem cells. These factors are expressed in metaphase II oocytes and embryonic stem cells (ESCs). The mechanisms responsible for the reprogramming of ooplasm during nuclear transfer are expected to be associated with the four factors. Here, we show that different paternal genetic backgrounds are able to influence the in vitro development of parthenogenetic and cloned embryos. Using real- time polymerase chain reaction (PCR) we found that the expression level of Oct4 in oocytes was less than that of ESCs, whereas oocytes from KM x C3H females showed the highest expression level of Sox2 than the other strains tested or in G1 ESCs. c-Myc mRNA levels in oocytes from KM mice were greater than those found in ESCs or oocytes of KM x C3H mice. These data demonstrate that the expression of the four transcription factors was different among the oocytes, which may be a contributing factor for the different efficiencies of parthenogenesis and the development of cloned embryos in vitro.

Our strategy entails investigating the influence of varied concentrations (0, 10, 100 and 1000 ng/ml) of human recombinant bone morphogenetic protein-2 (rhBMP-2) on the osteogenic expression of canine osteoblasts, seeded onto poly-caprolactone 20% tricalcium phosphate (PCL-TCP) scaffolds in vitro. Biochemical assay revealed that groups with rhBMP-2 displayed an initial burst in cell growth that was not dose-dependent. However, after 13 days, cell growth declined to a value similar to control. Significantly less cell growth was observed for construct with 1000 ng/ml of rhBMP-2 from 20 days onwards. Confocal microscopy confirmed viability of osteoblasts and at day 20, groups seeded with rhBMP-2 displayed heightened cell death as compared to control. Phase contrast and scanning electron microscopy revealed that osteoblasts heavily colonized surfaces, rods and pores of the PCL-TCP scaffolds. This was consistent for all groups. Finally, Von Kossa and osteocalcin assays demonstrated that cells from all groups maintained their osteogenic phenotype throughout the experiment. Calcification was observed as early as four days after stimulation for groups seeded with rhBMP-2. In conclusion, rhBMP-2 seems to enhance the differentiated function of canine osteoblasts in a non-dose dependent manner. This resulted in accelerated mineralization, followed by death of osteoblasts as they underwent terminal differentiation. Notably, PCL-TCP scaffolds seeded only with canine osteoblasts could sustain excellent osteogenic expression in vitro. Hence, the synergy of PCL with bioactive TCP and rhBMP-2 in a novel composite scaffold, could offer an exciting approach for bone regeneration.