Monosialotetrahexosylganglioside in the treatment of chronic oxaliplatin-induced peripheral neurotoxicity: TJMUCH-GI-001, a randomised controlled trial.

BACKGROUND: Chronic oxaliplatin-induced peripheral neurotoxicity (OIPN) is the most troublesome and dose-limiting side effect of oxaliplatin. There is no effective treatment for chronic OIPN. We conducted a randomised controlled trial to investigate the efficacy of monosialotetrahexosylganglioside (GM1) in treating chronic OIPN. METHODS: In this single-centre, double-blind, phase Ⅲ trial, gastrointestinal cancer patients with persistent chronic OIPN were randomised in 1:1 ratio to receive either GM1 or placebo at Tianjin Medical University Cancer Institute and Hospital, China. GM1 was dosed at 60 mg daily for every 3 weeks or 40 mg daily for every 2 weeks. Seven- and fourteen- day infusions were administered to concurrent oxaliplatin users and oxaliplatin discontinuation patients, respectively. The primary endpoint was the relief of neurotoxicity (≥30% improvement), measured by a newly developed patient reported outcome measure (MCIPN) based on prior questionnaires including the European Organization for Research and Treatment of Cancer Quality of Life Chemotherapy Induced Peripheral Neuropathy Questionnaire twenty-item scale. Visual analogue score (VAS) was used as another instrument for patients to evaluate the total Chronic OIPN treatment effect. VAS responders (≥30% improvement), double responders (≥30% improvement in both MCIPN and VAS), and high responders (≥50% improvement in the MCIPN total score) were also calculated. The secondary endpoints were safety and quality of life. The additional endpoints are progression-free survival (PFS), disease-free survival (DFS), overall survival (OS), and tumour response. (Trial registration number: NCT02486198 at ClinicalTrials.gov). FINDINGS: Between May 2015 to December 2017, 145 patients were randomly assigned to receive either GM1 (n=73) and placebo (n=72). Majority of the patients in both arms (90% in GM1 and 83% in placebo) continued receiving oxaliplatin on the trial. More patients responded in the GM1 group than in the placebo group (MCIPN responders: 53% vs 14%, VAS responders: 49% vs 22%, double responders: 41% vs 7%, and high responders: 32% vs 13%, all P < ·01). Analyses were also performed in concurrent oxaliplatin users. The results were consistent with those of the whole group. No deleterious effects of GM1 on survival or tumour response were found. There were no ≥G3 GM1-related adverse events. INTERPRETATION: In patients with chronic OIPN, the use of GM1 reduces the severity of chronic OIPN compared with placebo. FUNDING: This work was supported by clinical trial development fund of Tianjin Medical University Cancer Institute and Hospital (No.C1706).

Combined exome sequencing and deep phenotyping in highly selected fetuses with skeletal dysplasia during the first and second trimesters improves diagnostic yield.

OBJECTIVE: To investigate the genetic etiology of skeletal dysplasia in highly selected fetuses during the first and second trimesters using deep phenotyping and exome sequencing (ES). METHOD: Fetuses with short femurs were identified using the established prenatal diagnostic approach. A multidisciplinary team reviewed fetal phenotypic information (prenatal ultrasound findings, fetal postmortem, and radiographs) in a cohort of highly selected fetuses with skeletal dysplasia during the first and second trimesters. The affected families underwent multiplatform genetic tests. RESULTS: Of the 27 affected fetuses, 21 (77.8%) had pathogenic or potential pathogenic variations in the following genes: COL1A1, FGFR3, COL2A1, COL1A2, FLNB, DYNC2LI1, and TRIP11. Two fetuses had compound heterozygous mutations in DYNC2LI1 and TRIP11, respectively, and the other 19 carried de novo autosomal dominant variants. Novel variants were identified in COL1A1, COL2A1, COL1A2, DYNC2LI1, and TRIP11 in 11 fetuses. We also included the first description of the phenotype of odontochondrodysplasia in a prenatal setting. CONCLUSIONS: ES or panel sequencing offers a high diagnostic yield for fetal skeletal dysplasia during the first and second trimesters. Comprehensive and complete phenotypic information is indispensable for genetic analysis and the expansion of genotype-phenotype correlations in fetal skeletal abnormalities.

A nomogram for prediction of stage III/IV gastric cancer outcome after surgery: A multicenter population-based study.

Most patients with gastric cancer (GC) are first diagnosed at stage III-IV and surgery resection remains the primary therapeutic modality for these patients. However, clinical staging used for prediction of those patients provides limited information. We collected clinicopathological data and disease-progression information from 508 patients with stage III-IV GC at three Chinese hospitals and 1298 patients from the Surveillance, Epidemiology, and End Results database. Based on the stepwise multivariate regression model, we constructed a novel nomogram to predict overall survival (OS). The performance of discrimination for this model was measured using Harrell's concordance index (C-index) and receiver-operating characteristic curve (ROC), and was validated using calibration plots. Multivariate Cox regression analyses showed that tumor size, age at diagnosis, N stage, tumor grade, and distant metastases were outstanding independent prognostic factors of stage III-IV GC. We developed a nomogram based on these five prognostic predictors. In the training set, the C-index of the nomogram was 0.645 (95% CI: 0.611-0.679), which was higher than that of the American Joint Committee on Cancer TNM system alone (sixth TNM: 0.544; seventh TNM: 0.575; eighth TNM: 0.568). Similar results were observed in validation cohort. Moreover, calibration blots demonstrated good consistency between the actual and predicted OS probabilities. According to the nomogram, GC individuals could be classified into three groups (low-, middle-, and high-risk) (P < .001). Our nomogram complements the current staging system for prediction of individual prognosis with stage III-IV GC, and may be helpful for making individualized treatment decisions.

The c-Myc/miR-27b-3p/ATG10 regulatory axis regulates chemoresistance in colorectal cancer.

Oxaliplatin (OXA) resistance is the major obstacle to the anticancer effects of chemotherapy in colorectal cancer (CRC) patients. MicroRNAs (miRNAs) play an important role in the chemoresistance of various tumors. Our objective is to clarify the underlying mechanism of miRNAs in chemoresistance and provide a potential strategy to improve the response of CRC patients to chemotherapeutics. Methods: MiRNA microarray and Real-time PCR were performed to compare changes in miRNA expression between oxaliplatin-resistant and the parental cells. CCK8, apoptosis assay, immunofluorescence and xenograft studies were used to elucidate the impact of miR-27b-3p on regulating chemoresistance. Luciferase reporter assay and western blot were carried to assess the regulatory role of miR-27b-3p in ATG10 expression. The effects of miR-27b-3p and ATG10 on autophagy were investigated by GFP-LC3 fluorescence microscopy, transmission electron microscopy, and western blot. ChIP assay and luciferase assay were performed to test the c-Myc's occupancy on the miR-27B promoter. Results: We observed that miR-27b-3p expression was significantly downregulated in oxaliplatin-resistant cell lines (SW480-OxR and HCT116-OxR) compared to the corresponding parental cell lines and that miR-27b-3p expression was positively correlated with disease-free survival (DFS) time in colorectal cancer patients. MiR-27b-3p could sensitize colorectal cancer cells to oxaliplatin in vitro and in vivo. Under oxaliplatin treatment, chemoresistant cells showed a higher autophagy level than parental cells. Moreover, we also identified that miR-27b-3p inhibited the expression of ATG10 at the posttranscriptional level, thus inhibiting autophagy. Further study demonstrated that c-Myc can inhibit the expression of miR-27b-3p via binding to the promoter region of miR-27B gene. Conclusions: Our study identifies a novel c-Myc/miR-27b-3p/ATG10 signaling pathway that regulates colorectal cancer chemoresistance. These results suggest that miR-27b-3p is not only a potential indicator for evaluating efficiency of chemotherapy, but also a valuable therapeutic target for CRC, especially for patients with chemoresistance.

Circular RNA circ_0008450 upregulates CXCL9 expression by targeting miR-577 to regulate cell proliferation and invasion in nasopharyngeal carcinoma.

As a kind of malignant tumor, nasopharyngeal carcinoma (NPC) has attracted increasing attention from researchers. As a member of the circular RNA (circRNA) family, circ_0008450 has been investigated in hepatocellular carcinoma but not in NPC. This study aims to reveal the special biologic role and mechanism of circ_0008450 in NPC. qRT-PCR analysis was conducted to test the level of circ_0008450 in different tissues and cells. Loss/Gain of function assay was utilized to detect the influence of silenced/overexpressed circ_0008450 on the proliferation, apoptosis, migration, and invasion of NPC cells. The mechanism of circ_0008450 was assessed by performing qRT-PCR and luciferase reporter experiments. The results showed that circ_0008450 was elevated in NPC tissues and cells. Silenced circ_0008450 could inhibit cell proliferation, and metastatic properties and increased the number of apoptotic cells. Ectopically expressed circ_0008450 strengthened the abovementioned malignant biological behaviors. Mechanistically, circ_0008450 reduced miR-577-mediated repression of CXCL9, resulting in facilitating the oncogenic functions of NPC. In conclusion, circ_0008450 acts as an oncogene in NPC cells through regulating miR-577/CXCL9 signaling. Our findings might provide a new therapeutic target for treating NPC.

Fucosyltransferase 8 deficiency suppresses breast cancer cell migration by interference of the FAK/integrin pathway.

BACKGROUND AND OBJECTIVE: Fucosyltranferase 8 (FUT8), which catalyzes core fucosylation of glycopeptides, plays important roles in cancer development. In this study, we aimed to explore the influence of FUT8 expression on migration ability of human breast cancer cells and its potential mechanisms. METHODS: The core fucosylation levels in normal and FUT8 deficient MCF-7 cells were analyzed by lectin LCA blots. Then, the cell adhesion assay, transwell and wound healing experiments were conducted. The phosphorylation of FAK and core fucosylation of E-cadherin and its downstream integrins in the FAK/integrin pathway were measured. Moreover, the expression levels of nuclear ß-catenin, MMP-2, and MMP-9 were also measured. RESULTS: The core fucosylation levels were significantly reduced by inhibited FUT8. FUT8 deficiency suppressed the adhesion, migration and invasion of MCF-7 cells; the potential mechanisms might involve three aspects. FUT8 deficiency inhibited FAK/integrin pathway by suppressing core fucosylation of E-cadherin. In addition, FUT8 deficiency reduced nuclear ß-catenin accumulation. The suppression of MMP-2 and MMP-9 expression also accounted for FUT8 deficiency inhibiting breast cancer cells migration. CONCLUSIONS: FUT8 deficiency suppressed migration of MCF-7 cells by impacting core fucosylation of E-cadherin and the downstream FAK/integrin pathway. Therefore, FUT8 is a potential biomarker for breast cancer detection and treatment.

Preferential selection and transfer of euploid noncarrier embryos in preimplantation genetic diagnosis cycles for reciprocal translocations.

OBJECTIVE: To develop and validate a new strategy to distinguish between balanced/euploid carrier and noncarrier embryos in preimplantation genetic diagnosis (PGD) cycles for reciprocal translocations and to successfully achieve a live birth after selective transfer of a noncarrier embryo. DESIGN: Retrospective and prospective study. SETTING: In vitro fertilization (IVF) units. PATIENT(S): Eleven patients undergoing mate pair sequencing for identification of translocation breakpoints, followed by clinical PGD cycles. INTERVENTION(S): Embryo biopsy with 24-chromosome testing to determine carrier status of balanced/euploid embryos. MAIN OUTCOME MEASURE(S): Definition of translocation breakpoints and polymerase chain reaction (PCR) diagnostic primers, correct diagnosis of euploid embryos for carrier status, and a live birth with a normal karyotype after transfer of a noncarrier embryo. RESULT(S): In 9 of 11 patients (82%), translocation breakpoints were successfully identified. In four patients with a term PGD pregnancy established with a balanced/euploid embryo of unknown carrier status, the correct carrier status was retrospectively determined, matching with the cytogenetic karyotype of the resulting newborns. In a prospective PGD cycle undertaken by a patient with a 46,XY,t(7;14)(q22;q24.3) translocation, the four balanced/euploid embryos identified comprised three carriers and one noncarrier. Transfer of the noncarrier embryo resulted in birth of a healthy girl who was subsequently confirmed with a normal 46,XX karyotype. CONCLUSION(S): The combination of mate pair sequencing and PCR breakpoint analysis of balanced reciprocal translocation derivatives is a novel, reliable, and accurate strategy for distinguishing between carrier and noncarrier balanced/euploid embryos. The method has potential application in clinical PGD cycles for patients with reciprocal translocations or other structural rearrangements.

Successful surgery in lesional epilepsy secondary to posterior quandrant ulegyria coexisting with benign childhood focal epilepsy: A case report.

The present study reports, for the first time, a rare case of benign childhood focal epilepsy(BCFE) coexisting with lesional epilepsy secondary to parietooccipital ulegyria. The patient underwent right parietooccipital lobe disconnection plus tailored resection of temporooccipitoparietal junction cortex under electrocorticography (ECoG) monitoring. Post-operatively, there was no impairment of neurological function and the patient only experiences a few breakthrough benign partial seizures during sleep.

Upregulation of nuclear factor-κB activity mediates CYP24 expression and reactive oxygen species production in indoxyl sulfate-induced chronic kidney disease.

AIM: Chronic kidney disease (CKD) is associated with an inflammation-mediated process, and the vitamin D (3) catabolizing enzyme, CYP24, is frequently overexpressed in CKD, where it may play a crucial role in kidney disease. METHODS: Herein, in this study, we investigated CYP24, reactive oxygen species (ROS), and inflammatory responses in an indoxyl sulfate (IS)-induced CKD model to elucidate the role of CYP24 in CKD. RESULTS: Our results showed that IS upregulates proinflammatory cytokine, CYP24 and nuclear factor-κB (NF-κB) expression in human renal proximal tubule epithelial cells. In addition, IS treatment increased ROS production and simultaneously upregulated CYP24 expression and NF-κB translocation. Moreover, the IS-induced upregulation of CYP24 expression was alleviated by an inhibitor of NF-κB, as well as a siRNA specific to NF-κB p65. Furthermore, the renal cortex of DN (Dahl salt-resistant normotensive) + IS, DH (Dahl salt-sensitive hypertensive), and DH + IS rats showed increased expression of NF-κB p65, CYP24, 8-hydroxydeoxyguanosine (8-OHdG), a marker of ROS and macrophage infiltration compared with DN rats. CONCLUSIONS: These results provide evidence that administration of IS in human renal tubular epithelial cells upregulates NF-κB, which leads to increase CYP24 expression and ROS production. They also suggest that suppressing NF-κB signalling is promising for the development into a strategy for CKD treatment.

Analyzing 395,793 samples shows significant association between rs999737 polymorphism and breast cancer.

Large-scale genome-wide association studies (GWAS) have been conducted and reported the association between rs999737 polymorphism at 14q24.1 (RAD51L1) and breast cancer risk. Following studies investigated rs999737 polymorphism in European and Asian populations. However, some of these studies reported weak and no significant association. Here, we reevaluated this association using large-scale samples from previous 11 studies (n=395,793; 162,261 cases and 233,532 controls) from the PubMed database. We evaluated the genetic heterogeneity among the selected studies. The pooled odds ratio (OR) is calculated by the fixed effect model. All statistical tests for heterogeneity and meta-analysis were computed using R package. We did not identify significant heterogeneity among the included studies using the allele model (P=0.1314 and I (2)=33.4 %). We observed significant association between rs999737 and breast cancer using the allele model (P=2.47E - 35, OR=0.92, 95 % confidence interval (CI) 0.91-0.93). Our analysis further supports previous findings that the rs999737 polymorphism contributes to breast cancer susceptibility. We believe that our finding will be very useful for future genetic studies in breast cancer.

The human leukocyte antigen G promotes trophoblast fusion and ß-hCG production through the Erk1/2 pathway in human choriocarcinoma cell lines.

The human leukocyte antigen G (HLA-G) is expressed on the fetal-maternal interface and plays a role in protecting fetal-derived trophoblasts from the maternal immune response, allowing trophoblasts to invade the uterus. However, HLA-G also possesses immune suppressing-independent functions. We found that HLA-G expressing BeWo choriocarcinoma cells increased cell-cell fusion compared to control BeWo cells under forskolin treatment. Regardless of forskolin treatment, the expression of fusogenic gene mRNAs, including syncytin-1, the transcription factor glial cell missing 1 (Gcm1), and beta human chorionic gonadotropin (ß-hCG) were elevated. HLA-G up-regulates ß-hCG production in human choriocarcinoma cells because HLA-G knockdown in JEG-3 cells induces a dramatic decrease in ß-hCG compared with control cells. The defect in ß-hCG production in HLA-G knocked-down cells could not be completely overcome by stimulating hCG production through increasing intracellular cAMP levels. HLA-G expressing cells have increased phosphorylation levels for extracellular signal-regulated kinase1/2 (Erk1/2) in BeWo cells. The Erk1/2 pathway is inactivated after the inhibition of HLA-G expression in JEG-3 cells. Finally, Erk1/2 inhibition was able to suppress the increased hCG production induced by HLA-G expression. Together, these data suggest novel roles for HLA-G in regulating ß-hCG production via the modulation of the Erk1/2 pathway and by inducing trophoblast cell fusion.

Proprotein convertase furin regulates apoptosis and proliferation of granulosa cells in the rat ovary.

Folliculogenesis is tightly controlled by a series of hormones, growth factors and cytokines, many of which are secreted as proproteins and require processing by proteases before becoming functional. Furin is a member of the subtilisin-like proteases that activate large numbers of proprotein substrates and is ubiquitously expressed and implicated in many physiological and pathological processes. However, the precise role of furin during folliculogenesis has not been thoroughly investigated. The goal of the present work is to identify the role of furin in the development of granulosa cells during folliculogenesis, using immunohistochemistry, RT-PCR, Western blot and functional studies in primary cultured rat granulosa cells. Our results demonstrate that furin is highly expressed in granulosa cells and oocytes of the ovary with very limited expression in other ovarian cells such as the epithelial, stromal or theca cells. Furin siRNA significantly increases apoptosis of the granulosa cells from large antral/preovulatory follicles, in part via downregulation of the anti-apoptotic proteins, XIAP and p-AKT. On the contrary, furin siRNA markedly decreases proliferation of granulosa cells based on the downregulation of proliferation cell nuclear antigen (PCNA). Taken together, these data suggest that furin may play an important role in regulating apoptosis and proliferation of granulosa cells.

Characteristics of gas-liquid pulsed discharge plasma reactor and dye decoloration efficiency.

The pulsed high-voltage discharge is a new advanced oxidation technology for water treatment. Methyl Orange (MO) dye wastewater was chosen as the target object. Some investigations were conducted on MO decoloration including the discharge characteristics of the multi-needle reactor, parameter optimization, and the degradation mechanism. The following results were obtained. The color group of the azo dye MO was effectively decomposed by water surface plasma. The decoloration rate was promoted with the increase of treatment time, peak voltage, and pulse frequency. When the initial conductivity was 1700 microS/cm, the decoloration rate was the highest. The optimum distance between the needle electrodes and the water surface was 1 mm, the distance between the grounding electrode and the water surface was 28 mm, and the number of needle electrodes and spacing between needles were 24 and 7.5 mm, respectively. The decoloration rate of MO was affected by the gas in the reactor and varied in the order oxygen > air> argon > nitrogen, and the energy yield obtained in this investigation was 0.45 g/kWh.

[Value of detection of cell-free fetal DNA in maternal plasma in the prenatal diagnosis of chromosomal abnormalities].

OBJECTIVE: To investigate the value of detection of fetal cell-free fetal DNA (cff-DNA) in maternal plasma in the prenatal diagnosis of chromosomal abnormalities. METHODS: The plasma from 3200 gravidas (singleton with 20.3 ± 3.8 gestational weeks) was collected from April 1(st) 2011 to May 30(th) 2012. They were divided into 3 groups: (1) To tally 1720 cases were included in the high-risk serological screening group, in which women were younger than 35 years and got high-risk results in serological screening; (2) To tally 1310 cases were included in the advanced age group, in which women's age was more than 35 years; (3) To tally 170 cases were included in the supplementary group, in which women were younger than 35 years and got low-risk results in serological screening, or women who didn't take serological screening tests. All the 3030 gravidas in group 1 and 2 didn't take invasive prenatal diagnosis because of fear of abortion or short of prenatal diagnosis. Cff-DNA were detected by next generation sequencing in Shenzhen BGI Genomics Center for clinical laboratory. Amniocentesis and karyotype analysis were provided to the positive cases and women with negative results were followed-up by telephone. RESULTS: (1) The 3200 cases took cff-DNA detection, and 31 cases got positive results, including 27 cases of trisomy 21 and 4 cases of trisomy 18. Sixteen cases of trisomy 21 and 1 case of trisomy 18 were in the high-risk serological screening group. 7 cases of trisomy 21 and 2 cases of trisomy 18 were in the advanced age group. Four cases of trisomy 21 and 1 case of trisomy 18 were in the supplementary group. (2) And the 84% (26/31) cff-DNA detecting positive cases received amniocentesis. In the 27 trisomy 21 positive cases, 23 received amniocentesis and got karyotype of 47XN, +21, with the diagnostic accordance rate of 100%. In the 4 cases who didn't take karyotype analysis, fetal anomaly (ventricular septal defect, dextrocardia and choroid plexus cyst) was found in 1 case before 20 gestational weeks; intrauterine fetal demise happened in 1 case before getting the result; 2 other cases who already had healthy children took abortion in the local hospital without taking amniocentesis. In the 4 trisomy 18 positive cases, 3 took amniocentesis, 2 of which were trisomy 18 and took abortion, the other was chimera (46, XN/47, XN, +18) with only 2% cells of trisomy 18, with no malformation found after delivery. Hypoevolutism (3 weeks less than gestational week), general hydropsy and intrauterine fetal demise happened before the other case took amniocentesis. (3) Follow up of cff-DNA negative cases:until May 30(th) 2012, no Down's baby was found in the 1230 cases with cff-DNA test negative results. CONCLUSIONS: (1) The non-invasive fetal trisomy test (NIFTY) by next generation sequencing is a safe, accurate and high throughput method for the prenatal diagnosis of trisomy-21. (2) Use NIFTY as a further screening for pregnant women with high-risk serological screening results could lower invasive prenatal diagnosis rate. (3) Cases with positive NIFTY test results should receive amniocentesis and karyotype analysis to confirm the diagnosis before abortion.

Circulating microRNAs are elevated in plasma from severe preeclamptic pregnancies.

Until recently, the molecular pathogenesis of preeclampsia (PE) remained largely unknown. Reports have shown that circulating microRNAs (miRNAs) are promising novel biomarkers for cancer, pregnancy, tissue injury, and other conditions. The objective of this study was to identify differentially expressed miRNAs in plasma from severe preeclamptic pregnancies compared with plasma from normal pregnancies. By mature miRNA microarray analysis, 15 miRNAs, including 13 up- and two downregulated miRNAs, were screened to be differentially expressed in plasma from women with severe PE (sPE). Seven miRNAs, namely miR-24, miR-26a, miR-103, miR-130b, miR-181a, miR-342-3p, and miR-574-5p, were validated to be elevated in plasma from severe preeclamptic pregnancies by real-time quantitative stem-loop RT-PCR analysis. Gene ontology and pathway enrichment analyses revealed that these miRNAs were involved in specific biological process categories (including regulation of metabolic processes, regulation of transcription, and cell cycle) and signaling pathways (including the MAP kinase signaling pathway, the transforming growth factor-ß signaling pathway, and pathways in cancer metastasis). This study presents, for the first time, the differential expression profile of circulating miRNAs in sPE patients. The seven elevated circulating miRNAs may play critical roles in the pathogenesis of sPE, and one or more of them may become potential markers for diagnosing sPE.

Effects of cidofovir on human papillomavirus-positive cervical cancer cells xenografts in nude mice.

Cidofovir (CDV) is an acyclic nucleoside phosphonate analog that shows broad spectrum anti-DNA virus activity. In this study, we have investigated the influence of cidofovir on the tumor xenografts derived from HeLa and SiHa cells on nude mice. The HeLa/SiHa xenografts in nude mice were established by inoculating cells subcutaneously. Administration of cidofovir by intratumoral injection led to significant tumor reduction. Enhanced protein levels of p53 and p-pRb within the tumor samples were observed. Immunohistology analysis of the tumor sections indicated decreased PCNA index and increased apoptosis index. Our study gives more evidence and explanation on in vivo inhibition effect of cidofovir on HPV genome-positive cervical carcinoma cell line xenografts.

Direct chemical cross-linking of platelet-derived growth factor-BB to the demineralized bone matrix improves cellularization and vascularization.

In previous studies, we have described the use of demineralized bone matrix (DBM) as a carrier for the localized delivery system of growth factors in vitro and in vivo. The aim of the present work was to develop a direct chemical approach to immobilize the platelet-derived growth factor-BB (PDGF-BB) on DBM with cross-linking reagents. The amount of PDGF-BB covalently immobilized on DBM was significantly increased. The increased proliferation of fibroblasts demonstrated that the biological activity of PDGF-BB was not significantly reduced by cross-linking. Compared with control groups, there was a statistically significant increase in blood vessel density in the PDGF-C-DBM group after having been subcutaneously implanted into the dorsal side of the rats. The surface bioactivity of scaffolds on stimulation cell and new blood vessel invasion was improved. Therefore, the direct chemical cross-linking approach could be used to retain growth factors on collagen scaffolds effectively to develop functional biomaterials.