Low-Temperature Photothermal Therapy Platform Based on Pd Nanozyme-Modified Hydrogenated TiO2.

In photothermal treatments (PTTs), normal tissues around cancerous tumors get injured by excessive heat, whereas damaged cancer cells are easily restored by stress-induced heat shock proteins (HSPs) at low temperatures. Therefore, to achieve a unique tumor microenvironment (TME), it is imperative to increase PTT efficiency and reduce normal tissue injury by adopting appropriate reactive oxygen species (ROS) and lipid peroxides (LPO) cross-linked with HSPs. In the present research, a potential strategy for mild photothermal treatments (mPTTs) was proposed by initiating localized catalytic chemical reactions in TME based on Pd nanozyme-modified hydrogenated TiO2 (H-TiO2@Pd). In vitro and in vivo evaluations demonstrated that H-TiO2@Pd had good peroxidase-like activities (POD), glutathione oxidase-like activities (GSHOx), and photodynamic properties and also satisfactory biocompatibility for 4T1 cells. Localized catalytic chemical reactions in H-TiO2@Pd significantly depleted GSH to downregulate the protein expression of GPX4 and promoted the accumulation of LPO and ROS, which consumed HSP70 or inhibited its function in 4T1 cells. Hence, the as-constructed low-temperature photothermal therapeutic platform based on Pd nanozyme-modified H-TiO2 can be a promising candidate to develop a safe and effective mPTT for cancer treatments.

A novel dose fall-off index and preliminary application in brain and lung stereotactic radiotherapy.

BACKGROUND: Stereotactic radiotherapy (SRT) has been widely used for the treatment of brain metastases and early stage non-small-cell lung cancer (NSCLC). Excellent SRT plans are characterized by steep dose fall-off, making it critical to accurately and comprehensively predict and evaluate dose fall-off. PURPOSE: A novel dose fall-off index was proposed to ensure high-quality SRT planning. METHODS: The novel gradient index (NGI) had two different modes: NGIx V for three-dimensions and NGIx r for one-dimension. NGIx V and NGIx r were defined as the ratios of the decreased percentage dose (x%) to the corresponding isodose volume and equivalent sphere radii, respectively. A total of 243 SRT plans at our institution between April 2020 and March 2022 were enrolled, including 126 brain and 117 lung SRT plans. Measurement-based verifications were performed using SRS MapCHECK. Ten plan complexity indexes were calculated. Dosimetric parameters related to radiation injuries were also extracted, including the normal brain volume exposed to 12 Gy (V12 ) and 18 Gy (V18 ) during single-fraction SRT (SF-SRT) and multi-fraction SRT (MF-SRT), respectively, and the normal lung volume exposed to 12 Gy (V12 ). The performance of NGI and other common dose fall-off indexes, gradient index (GI), R50% and D2cm were evaluated using Spearman correlation analysis to explore their correlations with the PTV size, gamma passing rate (GPR), plan complexity indexes, and dosimetric parameters. RESULTS: There were statistically significant correlations between NGI and PTV size (r = -0.98, P < 0.01 for NGI50 V and r = -0.93, P < 0.01 for NGI50 r), which were the strongest correlations compared with GI (r = 0.11, P = 0.13), R50% (r = -0.08, P = 0.19) and D2cm (r = 0.84, P < 0.01). The fitted formulas of NGI50 V = 23.86V-1.00 and NGI50 r = 113.5r-1.05 were established. The GPRs of enrolled SRT plans were 98.6 ± 1.7%, 94.2 ± 4.7% and 97.1 ± 3.1% using the criteria of 3%/2 mm, 3%/1 mm, and 2%/2 mm, respectively. NGI50 V achieved the strongest correlations with various plan complexity indexes (|r| ranged from 0.67 to 0.91, P < 0.01). NGI50 V also showed the highest r values with V12 (r = -0.93, P < 0.01) and V18 (r = -0.96, P < 0.01) of the normal brain during SF-SRT and MF-SRT, respectively, and V12 (r = -0.86, P < 0.01) of the normal lung during lung SRT. CONCLUSIONS: Compared with GI, R50% and D2cm , the proposed dose fall-off index, NGI, had the strongest correlations with the PTV size, plan complexity and V12 /V18 of the normal tissues. These correlations established on NGI are more helpful and reliable for SRT planning, quality control, and reducing the risk of radiation injuries.

Co-assembled Nanocarriers of De Novo Thiol-Activated Hydrogen Sulfide Donors with an RGDFF Pentapeptide for Targeted Therapy of Non-Small-Cell Lung Cancer.

Hydrogen sulfide releasing agents (or H2S donors) have been recognized gasotransmitters with potent cytoprotective and anticancer properties. However, the clinical application of H2S donors has been hampered by their fast H2S-release, instability, and lack of tumor targeting, despite the unclear molecular mechanism of H2S action. Here we rationally designed an amphiphilic pentapeptide (RGDFF) to coassemble with the de novo designed thiol-activated H2S donors (CL2/3) into nanocarriers for targeted therapy of non-small-cell lung cancer, which has been proved as a one-stone-three-birds strategy. The coassembly approach simply solved the solubility issue of CL2/3 by the introduction of electron-donating groups (phenyl rings) to slow down the H2S release while dramatically improving their biocompatible interface, circulation time, slow release of H2S, and tumor targeting. Experimental results confirmed that as-prepared coassembled nanocarriers can significantly induce the intrinsic apoptotic, effectively arrest cell cycle at the G2/M phase, inhibit H2S-producing enzymes, and lead to mitochondrial dysfunction by increasing intracellular ROS production in H1299 cells. The mouse tumorigenesis experiments further confirmed the in vivo anticancer effects of the coassembled nanocarriers, and such treatment made tumors more sensitive to radiotherapy then improved the prognosis of tumor-bearing mice, which holds great promise for developing a new combined approach for NSCLC.

Correlation between the γ passing rates of IMRT plans and the volumes of air cavities and bony structures in head and neck cancer.

BACKGROUND: Both patient-specific dose recalculation and γ passing rate analysis are important for the quality assurance (QA) of intensity modulated radiotherapy (IMRT) plans. The aim of this study was to analyse the correlation between the γ passing rates and the volumes of air cavities (Vair) and bony structures (Vbone) in target volume of head and neck cancer. METHODS: Twenty nasopharyngeal carcinoma and twenty nasal natural killer T-cell lymphoma patients were enrolled in this study. Nine-field sliding window IMRT plans were produced and the dose distributions were calculated by anisotropic analytical algorithm (AAA), Acuros XB algorithm (AXB) and SciMoCa based on the Monte Carlo (MC) technique. The dose distributions and γ passing rates of the targets, organs at risk, air cavities and bony structures were compared among the different algorithms. RESULTS: The γ values obtained with AAA and AXB were 95.6 ± 1.9% and 96.2 ± 1.7%, respectively, with 3%/2 mm criteria (p > 0.05). There were significant differences (p < 0.05) in the γ values between AAA and AXB in the air cavities (86.6 ± 9.4% vs. 98.0 ± 1.7%) and bony structures (82.7 ± 13.5% vs. 99.0 ± 1.7%). Using AAA, the γ values were proportional to the natural logarithm of Vair (R2 = 0.674) and inversely proportional to the natural logarithm of Vbone (R2 = 0.816). When the Vair in the targets was smaller than approximately 80 cc or the Vbone in the targets was larger than approximately 6 cc, the γ values of AAA were below 95%. Using AXB, no significant relationship was found between the γ values and Vair or Vbone. CONCLUSION: In clinical head and neck IMRT QA, greater attention should be paid to the effect of Vair and Vbone in the targets on the γ passing rates when using different dose calculation algorithms.

Activity and Safety of Tegafur, Gimeracil, and Oteracil Potassium for Nasopharyngeal Carcinoma: A Systematic Review and Meta-Analysis.

In clinical practice, tegafur, gimeracil, and oteracil potassium (S-1) therapy is commonly administered to treat nasopharyngeal carcinoma (NPC). However, its efficacy and safety remain controversial in both randomized controlled trials (RCTs) and non-RCTs. We aimed to evaluate the efficacy and safety of S-1 treatment for NPC. We searched PubMed, Ovid, EMBASE, the Cochrane Library, China National Knowledge Infrastructure, Wanfang Database, and VIP databases for RCTs of chemotherapy with or without S-1 for NPC, from 2001 to 2020. A meta-analysis was performed using RevMan5.3 and Stata15. Randomized controlled trials published in journals were included irrespective of blinding and language used. Patients were diagnosed with NPC through a clinicopathological examination; patients of all cancer stages and ages were included. Overall, 25 trials and 1858 patients were included. There were significant differences in the complete remission (OR = 2.42, 95% CI (1.88-3.10), P < 0.05) and overall response rate (OR = 2.68, 95% CI (2.08-3.45), P < 0.05) between the S-1 and non-S-1 groups. However, there was no significant difference in partial remission (OR = 1.10, 95% CI (0.87-1.39), P=0.42) and seven adverse reactions (leukopenia, thrombocytopenia, nausea and vomiting, diarrhea, dermatitis, oral mucositis, and anemia) between the S-1 and non-S-1 groups. Additionally, statistical analyses with six subgroups were performed. S-1 was found to be a satisfactory chemotherapeutic agent combined with radiotherapy, intravenous chemotherapy, or chemoradiotherapy for NPC. As an oral medicine, the adverse reactions of S-1, especially gastrointestinal reactions, can be tolerated by patients, thereby optimizing their quality of life. S-1 may be a better choice for the treatment of NPC. This trial is registered with CRD42019122041.

Fabrication of Biomolecule-Loaded Composite Scaffolds Carried by Extracellular Matrix Hydrogel.

Fabrication of multifunctional scaffolds with biomimicking physical and biological signals play an important role in enhancing tissue regeneration. Multifunctional features come from the composite scaffold with various bioactive molecules. However, simple, biocompatible, and controllable hybridization strategy is still lacking. In this study, we leverage naturally derived extracellular matrix (ECM) as chemically controllable hydrogel carrier to effectively load functional biomolecules. The use of ECM hydrogel takes advantage of both native functionality of ECM components and tunability of hydrogel in controlling release of loaded molecules. As a proof of concept, porous acellular bone scaffold was selected as the solid pristine scaffold to be composited with BMP-2 and VEGF, which are loaded by spinal cord-derived ECM (SC-ECM) hydrogel. Crosslinking degree of SC-ECM hydrogel is tuned by changing genipin concentration, which renders the control over release kinetics of BMP-2 and VEGF. The mechanical strength of scaffold maintained after hybridization and is not significantly decreased in wet condition. In vitro evaluations of scaffolds cocultured with osteoblasts and mesenchymal stem cells (MSCs) demonstrate the biocompatible and bioactive features resulting from the composite scaffolds. Evidenced by alkaline phosphatase test, immunofluorescence, and real-time polymerase chain reaction, differentiation of MSCs towards osteoblast lineage is significantly enhanced by composite scaffolds. Therefore, our strategy in fabricating composite scaffold enabled by biomolecule-loaded ECM hydrogel holds great promise in regenerating diverse tissue types by appropriate combinations of solid pristine scaffolds, ECM, and bioactive molecules. Impact statement We developed a bioactive molecule (e.g., growth factor, protein) loading method using extracellular matrix hydrogel as a carrier. It brings a new strategy to fabricate composite scaffolds with unique biofunctions.

Degradation of petroleum hydrocarbons by embedding immobilized crude oil degrading bacteria.

In this study, the removal effect of free and immobilized bacteria on crude oil was determined. Sodium alginate and polyvinyl alcohol were used as embedding agent, and ramie was modified as an adsorbent to immobilize free bacteria. The conditions for preparing immobilized pellets were optimized using the response surface method, and the best combination was simulated and obtained by Design-Expert 8.0. The best degradation rate of immobilized bacteria was 75.52%. The degradation by free bacteria and immobilized bacteria showed that the selected microorganisms had a good degradation effect on petroleum hydrocarbons.

Carbon Quantum Dots: In vitro and in vivo Studies on Biocompatibility and Biointeractions for Optical Imaging.

BACKGROUND: Understanding the biocompatibility and biointeractions of nano-carbon quantum dots (nano-CQDs) in vitro and in vivo is important for assessing their potential risk to human health. In the previous research, the physical properties of CQDs synthesized by the laser ablation in liquid (LAL) method were analyzed in detail; however, possible bioapplications were not considered. MATERIALS AND METHODS: CQDs were prepared by LAL and characterized by atomic force microscopy, fluorescence lifetime, absorption spectrum, Fourier-transform infrared spectroscopy, and dynamic light scattering. Their biocompatibility was evaluated in vitro using assays for cytotoxicity, apoptosis, and biodistribution and in vivo using immunotoxicity and the relative expression of genes. Cells were measured in vitro using fluorescence-lifetime imaging microscopy to analyze the biointeractions between CQDs and intracellular proteins. RESULTS: There were no significant differences in biocompatibility between the CQDs and the negative control. The intracellular interactions had no impact on the optical imaging of CQDs upon intake by cells. Optical imaging of zebrafish showed the green fluorescence was well dispersed. CONCLUSION: We have demonstrated that the CQDs have an excellent biocompatibility and can be used as efficient optical nanoprobes for cell tracking and biomedical labeling except for L929 and PC-3M cells.

The prognosis analysis of RFWD2 inhibiting the expression of ETV1 in colorectal cancer.

BACKGROUND: The poor prognosis is partly due to the lack of efficient methods for early diagnosis on colorectal cancer (CRC). METHODS: Bioinformatic analysis and Immunohistochemical analysis were used to evaluate E3 ubiquitin ligase Ring finger and WD domain 2 (RFWD2) and ETS variant 1 (ETV1) mRNA and protein expression levels. RESULTS: The abundance of RFWD2 and ETV1 proteins from 76 CRC patients were examined. The relationship between their expression levels and clinic pathological parameters including prognostic significances were also detected. The expression of RFWD2 and ETV1 and the relative genes functions in CRC through bioinformatics methods were further analyzed. CONCLUSIONS: In conclusion, RFWD2 functioning as a tumor suppressor by negative regulating ETV1, which might play an important role in the development and progression of CRC. RFWD2 and ETV1 have the potential to serve as a pair of molecular biomarkers for the early diagnosis of CRC.

Erratum to the prognosis analysis of RFWD2 inhibiting the expression of ETV1 in colorectal cancer.

[This corrects the article DOI: 10.21037/tcr.2019.11.35.].

Synthesis and biological evaluation of arylpiperazine derivatives as potential anti-prostate cancer agents.

A novel scaffold of arylpiperazine derivatives was discovered as potent androgen receptor (AR) antagonist through rational drug designation based on our pre-work, leading to the discovery of a series of new antiproliferative compounds. Compounds 10, 16, 27, 29 and 31 exhibited relatively strong antagonistic potency against AR and exhibited potent AR binding affinities, while compounds 5, 6, 10, 14, 16, 19, 21, 27 and 31 exhibited strong cytotoxic activities against LNCaP cells (AR-rich) as well as also displayed the higher activities than finasteride toward PC-3 (AR-deficient) and DU145 (AR-deficient). Docking study suggested that the most potent antagonist 16 mainly bind to AR ligand binding pocket (LBP) site through hydrogen bonding interactions. The structure-activity relationship (SAR) of these designed arylpiperazine derivatives was rationally explored and discussed. These results indicated that the novel scaffold compounds demonstrated a step towards the development of novel and improved AR antagonists, and promising candidates for future development were identified.

Cancer risk assessment in modern radiotherapy workflow with medical big data.

Modern radiotherapy (RT) is being enriched by big digital data and intensive technology. Multimodality image registration, intelligence-guided planning, real-time tracking, image-guided RT (IGRT), and automatic follow-up surveys are the products of the digital era. Enormous digital data are created in the process of treatment, including benefits and risks. Generally, decision making in RT tries to balance these two aspects, which is based on the archival and retrieving of data from various platforms. However, modern risk-based analysis shows that many errors that occur in radiation oncology are due to failures in workflow. These errors can lead to imbalance between benefits and risks. In addition, the exact mechanism and dose-response relationship for radiation-induced malignancy are not well understood. The cancer risk in modern RT workflow continues to be a problem. Therefore, in this review, we develop risk assessments based on our current knowledge of IGRT and provide strategies for cancer risk reduction. Artificial intelligence (AI) such as machine learning is also discussed because big data are transforming RT via AI.

Synthesis, biological evaluation and SAR of naftopidil-based arylpiperazine derivatives.

For the development of potential anti-prostate cancer agents, 24 kinds of novel naftopidil-based arylpiperazine derivatives have been synthesized and characterized by spectroscopic methods. Their antitumor activities were evaluated against several classical prostate cancer cell lines including PC-3, LNCaP, and DU145. Among all the compounds, 9, 13, 17, 21 and 27 showed strong cytotoxic activities against DU145 cells (IC50 < 1 µM). Further testing confirmed that compound 17 inhibited the growth of DU145 cells by inducing cell cycle arrest at G0/G1 phase. Besides, antagonistic activities of compounds (9, 13, 17, 21 and 27) towards a1-ARs (α1A, α1B, and α1D) were further evaluated using dual-luciferase reporter assays, and the compounds 13 and 17 exhibited better a1-ARs subtype selectivity. The structure-activity relationship (SAR) of these developed arylpiperazine derivatives was rationally discussed. Taken together, these results suggested that further development of such compounds may be of great interest.

Isolation, screening, and crude oil degradation characteristics of hydrocarbons-degrading bacteria for treatment of oily wastewater.

The aim of this study was to isolate hydrocarbons-degrading bacteria for treatment of oily wastewater from long-standing petroleum-polluted sediments in Bohai Bay, China. Six hydrocarbons-degrading bacteria were screened and identified as Pseudomonas sp. and Bacillus sp. A new approach using a combination of various bacterial species in petroleum biodegradation was proposed and evaluated for its degradation characteristics. Gas chromatography-flame ionization detection (GC-FID) analysis showed that mixed bacterial agents (YJ01) degraded 80.64% of crude oil and 76.30% of crude oil alkanes, exhibiting good biodegradation effect. Besides, after 14 days of culture, the biodegradation assessment markers, pristane and phytane, showed significant degradation rates of 46.75% and 78.23%, respectively. Kinetic analysis indicated that the degradation trends followed a single first-order kinetics model and the degradation half-life (t1/2) of 15 g/L crude oil was significantly shorter (5.48 days). These results indicated that YJ01 could degrade a wider range of hydrocarbons as well as some recalcitrant hydrocarbon components, and can be applied for bioremediation and treatment of oil-contaminated environment.

Gd2O3-doped silica @ Au nanoparticles for in vitro imaging cancer biomarkers using surface-enhanced Raman scattering.

There has been an interest in developing multimodal approaches to combine the advantages of individual imaging modalities, as well as to compensate for respective weaknesses. We previously reported a composite nano-system composed of gadolinium-doped mesoporous silica nanoparticle and gold nanoparticle (Gd-Au NPs) as an efficient MRI contrast agent for in vivo cancer imaging. However, MRI lacks sensitivity and is unsuitable for in vitro cancer detection. Thus, here we performed a study to use the Gd-Au NPs for detection and imaging of a widely recognized human cancer biomarker, epidermal growth factor receptor (EGFR), in individual human cancer cells with surface-enhanced Raman scattering (SERS). The Gd-Au NPs were sequentially conjugated with a monoclonal antibody recognizing EGFR and a Raman reporter molecule, 4-meraptobenzoic acid (MBA), to generate a characteristic SERS signal at 1075cm-1. By spatially mapping the SERS intensity at 1075cm-1, cellular distribution of EGFR and its relocalization on the plasma membrane were measured in situ. In addition, the EGFR expression levels in three human cancer cell lines (S18, A431 and A549) were measured using this SERS probe, which were consistent with the comparable measurements using immunoblotting and immunofluorescence. Our SERS results show that functionalized Gd-Au NPs successfully targeted EGFR molecules in three human cancer cell lines and monitored changes in single cell EGFR distribution in situ, demonstrating its potential to study cell activity under physiological conditions. This SERS study, combined with our previous MRI study, suggests the Gd-Au nanocomposite is a promising candidate contrast agent for multimodal cancer imaging.

[Influence of projection data correction on digital breast tomosynthesis imaging].

OBJECTIVE: To investigate the effect of detector performance during digital breast tomography (DBT) projection data acquisition on reconstructed image quality. METHODS: With reference to the traditional detector data correction method and the specific data acquisition pattern in DBT imaging, we utilized dark field correction, light field and its gain correction for processing the projection data collected by the detector. The reconstructed images were evaluated using iterative reconstruction method based on total generalized variation (TGV). RESULTS: In physical breast phantom experiment, the proposed method resulted in a reduced Heel effect caused by nonuniform photon number. The reconstructed DBT images after correction showed obviously improved image quality especially in the details with a low contrast. CONCLUSION: The dark field correction, light field and its gain correction process for DBT image reconstruction can improve the image quality.

Magnetic and fluorescent Gd2O3:Yb3+/Ln3+ nanoparticles for simultaneous upconversion luminescence/MR dual modal imaging and NIR-induced photodynamic therapy.

The development of upconversion nanoparticles (UCNs) for theranostics application is a new strategy toward the accurate diagnosis and efficient treatment of cancer. Here, magnetic and fluorescent lanthanide-doped gadolinium oxide (Gd2O3) UCNs with bright upconversion luminescence (UCL) and high longitudinal relaxivity (r1) are used for simultaneous magnetic resonance imaging (MRI)/UCL dual-modal imaging and photodynamic therapy (PDT). In vitro and in vivo MRI studies show that these products can serve as good MRI contrast agents. The bright upconversion luminescence of the products allows their use as fluorescence nanoprobes for live cells imaging. We also utilized the luminescence-emission capability of the UCNs for the activation of a photosensitizer to achieve significant PDT results. To the best of our knowledge, this study is the first use of lanthanide-doped Gd2O3 UCNs in a theranostics application. This investigation provides a useful platform for the development of Gd2O3-based UCNs for clinical diagnosis, treatment, and imaging-guided therapy of cancer.

High sensitivity of gold nanoparticles co-doped with Gd2O3 mesoporous silica nanocomposite to nasopharyngeal carcinoma cells.

Nanoprobes for combined optical and magnetic resonance imaging have tremendous potential in early cancer diagnosis. Gold nanoparticles (AuNPs) co-doped with Gd2O3 mesoporous silica nanocomposite (Au/Gd@MCM-41) can produce pronounced contrast enhancement for T1 weighted image in magnetic resonance imaging (MRI). Here, we show the remarkably high sensitivity of Au/Gd@MCM-41 to the human poorly differentiated nasopharyngeal carcinoma (NPC) cell line (CNE-2) using fluorescence lifetime imaging (FLIM). The upconversion luminescences from CNE-2 and the normal nasopharyngeal (NP) cells (NP69) after uptake of Au/Gd@MCM-41 show the characteristic of two-photon-induced-radiative recombination of the AuNPs. The presence of the Gd3+ ion induces a much shorter luminescence lifetime in CNE-2 cells. The interaction between AuNPs and Gd3+ ion clearly enhances the optical sensitivity of Au/Gd@MCM-41 to CNE-2. Furthermore, the difference in the autofluorescence between CNE-2 and NP69 cells can be efficiently demonstrated by the emission lifetimes of Au/Gd@MCM-41 through the Forster energy transfers from the endogenous fluorophores to AuNPs. The results suggest that Au/Gd@MCM-41 may impart high optical resolution for the FLIM imaging that differentiates normal and high-grade precancers.

The MRI marker gene MagA attenuates the oxidative damage induced by iron overload in transgenic mice.

We aimed to create transgenic (Tg) mice engineered for magnetic resonance imaging (MRI). To ascertain if MagA expression contributes to oxidative stress and iron metabolism, we report the generation of Tg mice in which ubiquitous expression of MagA can be detected by MRI in vivo. Expression of MagA in diverse tissues of Tg mice was assessed, and iron accumulation and deposition of nanoparticles in tissues were analyzed. Levels of antioxidant enzymes, lipid peroxidation and cytokine production were determined, and iron metabolism-related proteins were also detected. MagA Tg showed no apparent pathologic symptoms and no histologic changes compared with wild-type (WT) mice. Overexpression of MagA resulted in specific alterations of the transverse relaxation rate (R2) of water. Transgene-dependent changes in R2 were detectable by MRI in iron-overloaded mice. We also evaluated antioxidant abilities between WT and Tg groups or two iron-overloaded groups. Together with the data of cytokines and iron metabolism-related proteins, we inferred that MagA could regulate nanoparticle production and thus attenuate the oxidative damage induced by iron overload. The novel MagA Tg mouse, which expresses an MRI reporter in many tissues, would be a valuable model of MagA molecular imaging in which to study diseases related to iron metabolism.

A tubular gelatin scaffold capable of the time-dependent controlled release of epidermal growth factor and mitomycin C.

A tubular gelatin scaffold for the time-dependent controlled release of epidermal growth factor (EGF) and mitomycin C (MMC) was fabricated. EGF was incorporated using silk fibroin carriers, and MMC was planted using polylactide (PLA) microspheres. The relationship between scaffold properties and crosslinking degrees was evaluated. As the crosslinking degree was increased from 23.7% to 65.3%, the mechanical properties of the scaffold obviously improved, and the compressive modulus increased to approximately 65kPa. The mass degradation of the scaffold was also controlled from 9 days to approximately 1 month. In vitro release tests indicated that the scaffold mainly released EGF in the early period and MMC in the later period. Urethral epithelial cells (UECs) and urethral scar derived fibroblast cells (UFCs) were coseeded in the scaffold at a ratio of 1:1. After 9 days of coculture, immunostaining results displayed that the proportion of UECs continuously increased to approximately 71%. These changes in cell proportion were confirmed by the results of Western blot analysis. Therefore, the scaffold promoted the growth but inhibited the regeneration of UFCs. This scaffold for time-dependent controlled release of multiple biofactors may be potentially useful in urethral reconstruction and other tissue engineering studies.