Silk Sponge Research Articles

Silk Sponges Ornamented with a Placenta-Derived Extracellular Matrix Augment Full-Thickness Cutaneous Wound Healing by Stimulating Neovascularization and Cellular Migration.

Regeneration of full-thickness wounds without scar formation is a multifaceted process, which depends on in situ dynamic interactions between the tissue-engineered skin substitutes and a newly formed reparative tissue. However, the majority of the tissue-engineered skin substitutes used so far in full-thickness wound healing cannot mimic the natural extracellular matrix (ECM) complexity and thus are incapable of providing a suitable niche for endogenous tissue repair. Herein, we demonstrated a simple approach to fabricate porous hybrid ECM sponges (HEMS) using a placental ECM and silk fibroin for full-thickness wound healing. HEMS with retained cytokines/growth factors provided a noncytotoxic environment in vitro for human foreskin fibroblasts (HFFs), human epidermal keratinocytes (HEKs), and human amniotic membrane-derived stem cells to adhere, infiltrate, and proliferate. Interestingly, HEMS-conditioned media accelerated the migration of HFFs and HEKs owing to the presence of cytokines/growth factors. Also, the ex vivo chick chorioallantoic membrane assay of HEMS demonstrated its excellent vascularization potential by inducing and supporting blood vessels. Additionally, HEMS when subcutaneously implanted demonstrated no severe immune response to the host. Furthermore, HEMS implanted in full-thickness wounds in a rat model showed augmented healing progression with well-organized epidermal-dermal junctions via pronounced angiogenesis, accelerated migration of HFFs/HEKs, enhanced granulation tissue formation, and early re-epithelialization. Taken together, these findings show that porous HEMS ornamented with cytokines/growth factors having superior physicomechanical properties may be an appropriate skin substitute for full-thickness cutaneous wounds.

An Insight on Silk Protein Sericin: From Processing to Biomedical Application.

Silks are naturally occurring polymers that have been used clinically as sutures for hundreds of years. It's so for obtained from insects or worms, silk consists of a filament core protein, termed fibroin, and a glue-like coating made up of sericin proteins. An important component of silk has an extended history of being discarded as a waste in the course of silk processing. The cost of sericin for tissue engineering is underestimated and its capability in using as regenerative remedy has simply began to be explored. Its variable amino acid composition and various functional groups confer upon it attractive bioactive proteins, which are particularly interesting for biomedical programs. Because of its antioxidant properties, moisturizing ability, and mitogenic effect on mammalian cells, sericin is beneficial in cell regeneration and tissue engineering. Research shows that keratinocytes and fibroblasts have brought about the improvement of sericin-primarily based biomaterials for skin tissue repair, in particular as wound dressings. Moreover, sericin may be used for bone tissue engineering due to its ability to set off nucleation of bone-like hydroxyapatite. Stable silk sericin biomaterials, as films, sponges, and hydrogels, are obtained by means of cross-linking, ethanol precipitation, or mixing with different polymers. Now a day, sericin may also be used for delivery of drugs due to its chemical reactivity and pH-responsiveness which facilitate the fabrication of nano and microparticles, hydrogels, and conjugated molecules, enhancing the bioactivity of drugs. In this review, we outlined the current headways from extraction of sericin till its physical properties and biomedical applications.

Modular flow chamber for engineering bone marrow architecture and function.

The bone marrow is a soft, spongy, gelatinous tissue found in the hollow cavities of flat and long bones that support hematopoiesis in order to maintain the physiologic turnover of all blood cells. Silk fibroin, derived from Bombyx mori silkworm cocoons, is a promising biomaterial for bone marrow engineering, because of its tunable architecture and mechanical properties, the capacity of incorporating labile compounds without loss of bioactivity and demonstrated ability to support blood cell formation. In this study, we developed a bone marrow scaffold consisting of a modular flow chamber made of polydimethylsiloxane, holding a silk sponge, prepared with salt leaching methods and functionalized with extracellular matrix components. The silk sponge was able to support efficient platelet formation when megakaryocytes were seeded in the system. Perfusion of the chamber allowed the recovery of functional platelets based on multiple activation tests. Further, inhibition of AKT signaling molecule, which has been shown to be crucial in regulating physiologic platelet formation, significantly reduced the number of collected platelets, suggesting the applicability of this tissue model for evaluation of the effects of bone marrow exposure to compounds that may affect platelet formation. In conclusion, we have bioengineered a novel modular system that, along with multi-porous silk sponges, can provide a useful technology for reproducing a simplified bone marrow scaffold for blood cell production exvivo.

In vivo evaluation of microporous silk sponges with modified pulp for bone tissue engineering

In vivo evaluation of microporous silk sponges with modified pulp for bone tissue engineering

Shape Memory Silk Protein Sponges for Minimally Invasive Tissue Regeneration.

Porous silk protein scaffolds are designed to display shape memory characteristics and volumetric recovery following compression. Two strategies are utilized to realize shape recovery: addition of hygroscopic plasticizers like glycerol, and tyrosine modifications with hydrophilic sulfonic acid chemistries. Silk sponges are evaluated for recovery following 80% compressive strain, total porosity, pore size distribution, secondary structure development, in vivo volume retention, cell infiltration, and inflammatory responses. Glycerol-modified sponges recover up to 98.3% of their original dimensions following compression, while sulfonic acid/glycerol modified sponges swell in water up to 71 times their compressed volume, well in excess of their original size. Longer silk extraction times (lower silk molecular weights) and higher glycerol concentrations yielded greater flexibility and shape fidelity, with no loss in modulus following compression. Sponges are over 95% porous, with secondary structure analysis indicating glycerol-induced β-sheet physical crosslinking. Tyrosine modifications with sulfonic acid interfere with β-sheet formation. Glycerol-modified sponges exhibit improved rates of cellular infiltration at subcutaneous implant sites with minimal immune response in mice. They also degrade more rapidly than unmodified sponges, a result posited to be cell-mediated. Overall, this work suggests that silk sponges may be useful for minimally invasive deployment in soft tissue augmentation procedures.

Silk fibroin based carrier system for delivery of fibrinogen and thrombin as coagulant supplements.

The control of bleeding is one of the most important interventions after a traumatic injury. Hemostatic devices delivering blood clotting accelerating agents such as fibrinogen are increasingly used due to their efficacy and their ease of application. In the present study, we describe a method to incorporate the coagulant supplements fibrinogen and thrombin in silk protein sponges by mixing the coagulants with an aqueous silk solution, followed by molding, freeze-drying, and water annealing. In this combination system, we demonstrate the delivery of fibrinogen while maintaining its hemostatic potential. Concentration ratios of silk to fibrinogen of 1.0%/2.8%, 2.3%/1.5%, and 3.0%/0.8% were used. The thrombin-induced fibrin polymeric network filled the space in and next to the silk spongy structure but also remained interconnected to the silk, providing an intact network. The mechanical characterization of the fibrinogen-releasing silk sponges before and after the induction of the fibrinogen polymerization demonstrated that the fibrin network resulted in reduced permanent deformation from 21.1% to 6.5%, 19.6% to 5.7%, and 12.7% to 9.4% for the 2.8%, 1.5%, and 0.8% fibrinogen-containing silk sponges, respectively. Moreover, the fibrin formation lead to a more linear elastic behavior over longer strain ranges. In combination, the Calcein-AM/PI staining and MTT assay results indicate uniform cell adhesion on the surface and cytocompatibility of the silk/fibrin sponges, respectively. Moreover, the co-delivery of thrombin with fibrinogen via silk as carrier material is described, offering a more mechanically robust and durable system while preserving hemostatic features of the coagulant substances for the generation of hemostatic devices. © 2016 Wiley Periodicals, Inc. J Biomed Mater Res Part A: 105A: 687-696, 2017.

Spider Silk-CBD-Cellulose Nanocrystal Composites: Mechanism of Assembly.

The fabrication of cellulose-spider silk bio-nanocomposites comprised of cellulose nanocrystals (CNCs) and recombinant spider silk protein fused to a cellulose binding domain (CBD) is described. Silk-CBD successfully binds cellulose, and unlike recombinant silk alone, silk-CBD self-assembles into microfibrils even in the absence of CNCs. Silk-CBD-CNC composite sponges and films show changes in internal structure and CNC alignment related to the addition of silk-CBD. The silk-CBD sponges exhibit improved thermal and structural characteristics in comparison to control recombinant spider silk sponges. The glass transition temperature (Tg) of the silk-CBD sponge was higher than the control silk sponge and similar to native dragline spider silk fibers. Gel filtration analysis, dynamic light scattering (DLS), small angle X-ray scattering (SAXS) and cryo-transmission electron microscopy (TEM) indicated that silk-CBD, but not the recombinant silk control, formed a nematic liquid crystalline phase similar to that observed in native spider silk during the silk spinning process. Silk-CBD microfibrils spontaneously formed in solution upon ultrasonication. We suggest a model for silk-CBD assembly that implicates CBD in the central role of driving the dimerization of spider silk monomers, a process essential to the molecular assembly of spider-silk nanofibers and silk-CNC composites.

Fabrication and characterization of Eri silk fibers-based sponges for biomedical application

Cocoon-derived semi-domesticated Eri silk fibers still lack exploitation for tissue engineering applications due to their poor solubility using conventional methods. The present work explores the ability to process cocoon fibers of non-mulberry Eri silk (Samia/Philosamia ricini) into sponges through a green approach using ionic liquid (IL) – 1-buthyl-imidazolium acetate as a solvent. The formation of β-sheet structures during Eri silk/IL gelation was acquired by exposing the Eri silk/IL gels to a saturated atmosphere composed of two different solvents: (i) isopropanol/ethanol (physical stabilization) and (ii) genipin, a natural crosslinker, dissolved in ethanol (chemical crosslinking). The sponges were then obtained by freeze-drying. This approach promotes the formation of both stable and ordered non-crosslinked Eri silk fibroin matrices. Moreover, genipin-crosslinked silk fibroin sponges presenting high height recovery capacity after compression, high swelling degree and suitable mechanical properties for tissue engineering applications were produced. The incorporation of a model drug – ibuprofen – and the corresponding release study from the loaded sponges demonstrated the potential of using these matrices as effective drug delivery systems. The assessment of the biological performance of ATDC5 chondrocyte-like cells in contact with the developed sponges showed the promotion of cell adhesion and proliferation, as well as extracellular matrix production within 2weeks of culture. Sponges’ intrinsic properties and biological findings open up their potential use for biomedical applications. Statement of SignificanceThis work addresses the preparation and characterization of non-mulberry cocoon-derived Eri silk sponges. The insolubility of cocoons-derived non-mulberry silkworms impairs their processability and applications in the healthcare field. We used a green approach with ionic liquids to overcome the lack solubility of such silk fibers. The formation of beta-sheet structures into Eri-based sponges was physically and chemically induced. The sponges were obtained by freeze-drying. The developed structures exhibited flexibility to adapt and recover their shapes upon application and subsequent removal of load, high swelling degree, ability to load an anti-inflammatory drug and to promote its sustained release. They promoted in vitro cellular adhesion, proliferation and extracellular matrix production of a chondrocyte-like cell line, opening up their potential application for biomedical applications.

Freezing Temperature Effects on Silk Sponge Formation

Freezing Temperature Effects on Silk Sponge Formation

Silk-extracellular matrix sponges for striated muscle tissue engineering: Regeneration after injury and disease

Event Abstract Back to Event Silk-extracellular matrix sponges for striated muscle tissue engineering: Regeneration after injury and disease Whitney Stoppel1, Kelly E. Sullivan1, David L. Kaplan1 and Lauren D. Black1, 2 1 Tufts University, Biomedical Engineering, United States 2 Sackler School of Graduate Biomedical Sciences at Tufts University, Cell, Molecular, and Developmental Biology, United States Introduction: Repair and regeneration of critically sized defects or injuries to striated muscle tissue, such as cardiac and skeletal muscles, necessitates the use of a complex biomaterial capable of providing structural support that mimics native tissue mechanics and architecture, while promoting host cell integration, vascularization, and remodeling. Silk fibroin protein sponges containing decellularized extracellular matrix (ECM) make an excellent candidate material for the support and repair of injured tissue due to the tunability of the silk scaffold properties, the ease of incorporation of ECM components[1], and demonstrated efficacy in vitro[3],[4]. Materials and Methods: Silk fibroin protein was extracted from B. mori cocoons[2]. ECM was isolated from either adult or fetal porcine cardiac muscle. Anisotropic sponges were formed via directional freezing, followed by lyophilization and water annealing (Fig. 1). Primary and stem cell-derived cardiac progenitors were used to evaluate scaffold formulations in vitro via Western blot analysis and histology. Optimized scaffolds were implanted subcutaneously in Sprague Dawley rats to evaluate cell infiltration and vascularization[1]. In addition, scaffolds were implanted at the site of injury (left ventricle following permanent ligation of the left anterior descending coronary artery to induce myocardial infarction (MI)). Ejection fraction and left ventricle diameters were used to monitor animal health and assess functional outcomes over 8 weeks (echocardiography). Pressure-volume loop analyses, echocardiography, histology, and gene expression of cells in the infarct region and boarder zones were used to determine the degree of repair, cell infiltration, vascularization, and immune response at the termination of the study. All silk-ECM sponges were compared to a silk only control as well as a sham (no repair) condition. Results and Discussion: Addition of ECM components improved cell infiltration in silk sponges implanted subcutaneously[1]. Silk-ECM composite patches also showed improved function of cardiac progenitors in vitro. In a pilot study, trends in ejection fraction and fractional area shortening calculated from echocardiography data suggested that silk-ECM patches were able to restore function similar to pre-injury values after 6 and 8 weeks (p < 0.15) (Fig. 2). Ongoing experiments are evaluating silk-ECM sponges loaded with soluble ECM for their ability to improve cardiac function following myocardial infarction (1 week post-MI implant). Conclusion: Silk fibroin-ECM sponges improve host cell infiltration and remodeling, promote angiogenesis, and improve functional measures in rodent models of cardiac injury. Future work aims to develop materials for the eventual use in the repair of congenital heart defects in pediatric patients. This work was funded in part by the National Institutes of Health P41 program (EB002520).; WLS would like to acknowledge funding from the National Institutes of Health Institutional Research and Academic Career Development Awards program at Tufts University (K12GM074869, Training in Education and Critical Research Skills (TEACRS)).

Intratumoral Implantation of Vincristine-Loaded Sustained-Release Silk Sponge Is Effective in Tumor Control in an Orthotopic Neuroblastoma Murine Model

Harris, Jamie MD; Coburn, Jeannine M. PhD; Poirier, Jennifer PhD; Kaplan, David L. PhD; Chiu, Bill MD, FACS Author Information

In vitro bioengineered model of cortical brain tissue.

A bioengineered model of 3D brain-like tissue was developed using silk-collagen protein scaffolds seeded with primary cortical neurons. The scaffold design provides compartmentalized control for spatial separation of neuronal cell bodies and neural projections, which resembles the layered structure of the brain (cerebral cortex). Neurons seeded in a donut-shaped porous silk sponge grow robust neuronal projections within a collagen-filled central region, generating 3D neural networks with structural and functional connectivity. The silk scaffold preserves the mechanical stability of the engineered tissues, allowing for ease of handling, long-term culture in vitro and anchoring of the central collagen gel to avoid shrinkage, and enabling neural network maturation. This protocol describes the preparation and manipulation of silk-collagen constructs, including the isolation and seeding of primary rat cortical neurons. This 3D technique is useful for mechanical injury studies and as a drug screening tool, and it could serve as a foundation for brain-related disease models. The protocol of construct assembly takes 2 d, and the resulting tissues can be maintained in culture for several weeks.

Lyophilized Silk Sponges: A Versatile Biomaterial Platform for Soft Tissue Engineering.

We present a silk biomaterial platform with highly tunable mechanical and degradation properties for engineering and regeneration of soft tissues such as, skin, adipose, and neural tissue, with elasticity properties in the kilopascal range. Lyophilized silk sponges were prepared under different process conditions and the effect of silk molecular weight, concentration and crystallinity on 3D scaffold formation, structural integrity, morphology, mechanical and degradation properties, and cell interactions in vitro and in vivo were studied. Tuning the molecular weight distribution (via degumming time) of silk allowed the formation of stable, highly porous, 3D scaffolds that held form with silk concentrations as low as 0.5% wt/v. Mechanical properties were a function of silk concentration and scaffold degradation was driven by beta-sheet content. Lyophilized silk sponges supported the adhesion of mesenchymal stem cells throughout 3D scaffolds, cell proliferation in vitro, and cell infiltration and scaffold remodeling when implanted subcutaneously in vivo.

Primary fabrication of a tissue-engineered mesh for pelvic floor reconstruction and research in vivo

To explore the fabrication of a tissue-engineered mesh fabricated with autologous adipose-derived mesenchymal stem cells (ADSC) and silk fibroin scaffold for female pelvic reconstruction. Sprague-Dawley rat adipose tissue was obtained from inguinal region. Mesenchymal stem cells were isolated and proliferated in vitro. The sericin of silk was removed after knitting in to web. Then a compound scaffold freeze-dried of silk fibroin web and silk fibroin solution was prepared for ADSC seeding. after 1-week culturing, the cellular morphology and extracellular matrix secretion on scaffold were observed by scanning electronic microscopy. The tissue-engineered meshes were implanted subcutaneously into the homologous rats while silk fibroin scaffolds served as control. Then the biomechanics, collagen matrix and local tissue response to tissue-engineered meshes in vivo with that to silk fibroin scaffolds were estimated. ADSC were fibroblast-like and proliferated well in vitro. SEM showed that the scaffold had an interwoven structure with a smooth surface and the silk sponges in scaffold had a uniform and porous structure. ADSC adhered firmly to the scaffold, secreted extracellular matrix and formed cell sheets on scaffold. In vivo studies showed that the tissue matrix of tissue-engineered meshes was better organized than silk fibroin scaffolds. They also had higher failure force (3.081 ± 0.121 vs 2.167 ± 0.148 N) and Young's modulus (3.191 ± 0.146 vs 2.263 ± 0.213 MPa) and more collagen content (18.648 ± 0.867 vs 14.123 ± 0.989 µg/mg) than silk fibroin scaffolds (P < 0.05). As a suitable cell type for tissue engineering, ADSC may be successfully isolated and stably proliferated in vitro. The tissue-engineered meshes have an excellent biocompatibility and appropriate properties for pelvic floor reconstruction.

작잠 실크 피브로인에 의한 in vitro 상처 회복 효과 및 에탄올 처리에 따른 작잠 실크 피브로인 스폰지의 구조 전이

작잠 누에고치를 정련한 후 질산칼슘4수화물의 용융액을 사용하여 재생 작잠 실크피브로인 스펀지를 제조하였다. 작잠 실크피브로인은 280 nm에서 tyrosine 잔기 등에 기인한 흡광대를 나타내었다. 작잠 실크피브로인 스펀지를 에탄올 농도별로 처리한 후 구조 전이를 관찰한 결과 80% 에탄올 처리시에는 <TEX>${\beta}$</TEX>-sheet 구조(<TEX>$700cm^{-1}$</TEX>), <TEX>${\alpha}$</TEX>-helix 구조(<TEX>$625cm^{-1}$</TEX>), 그리고 random coil (<TEX>$660cm^{-1}$</TEX>) 구조가 공존하는 것으로 나타났다. 또한 작잠 실크피브로인을 이용한 in vitro 상처회복실험 결과 실크피브로인의 첨가에 의하여 상처회복 효과가 인정되었다. Regenerated Antheraea pernyi silk sponge was prepared using calcium nitrate 4 hydrate melt and examined the conformational changes treated with aqueous ethanol solution. The conformation of silk sponges was changed from random coil structure to <TEX>${\beta}$</TEX>-sheet and <TEX>${\alpha}$</TEX>-helix conformation with low ethanol concentration (50 ~ 70%). On the other hand, that of silk sponges with 80% ethanol treatment showed <TEX>${\beta}$</TEX>-sheet (<TEX>$700cm^{-1}$</TEX>), <TEX>${\alpha}$</TEX>-helix (<TEX>$625cm^{-1}$</TEX>), and random coil (<TEX>$660cm^{-1}$</TEX>) specific peaks. Wound healing effect in vitro was observed by cytoslective wound healing kit. Therefore, regenerated Antheraea pernyi silk sponges might be used as promising wound dressing materials.

Characterization of silk sponge in the wet state using13C solid state NMR for development of a porous silk vascular graft with small diameter

The 13C solid state NMR spectra of the sponge prepared from Bombyx mori silk fibroin with different 13C labeling sites, [3-13C]Ser, [3-13C]Tyr and [3-13C]Ala were observed in dry and wet states. Here the silk fibroin sponge was prepared using poly(ethylene) glycol diglycidyl ether (PGDE) as porogen. Two broad peaks assigned to β-sheet and random coil were observed for each 13C-labeled site in the dry state. Moreover, in the wet state, one additional sharp peak assigned to random coil with high mobility due to the presence of water was observed. When the silk fibroin aqueous solution and PGDE was mixed in the sponge preparation process, the slow structural change from random coil to β-sheet structure occurs together with decrease in the motion of the silk fibroin backbone chain. The silk fibroin graft with small diameter was prepared with a knitted silk fiber tube coated with this silk fibroin sponge. The high porosity and suture retention strength which are comparable to an ePTFE vascular graft were attained. The excellent results, high patency and high tissue ingrowth, were obtained with the porous silk fibroin graft with small diameter by a rat abdominal aorta implantation experiment.

Development Of 3D Models For Studying Megakaryopoiesis In Vitro

IntroductionSilk fibroin, derived from Bombyx mori silkworm cocoons, is used extensively in biomaterials and regenerative medicine. The useful characteristics of this protein include self-assembly, robust mechanical properties, biocompatibility and biodegradability. Moreover, silk can be enhanced through a variety of chemical modifications that affect cell attachment, growth and differentiation. Thrombocytopenia occurs when a patient suffers from an abnormally low platelet count in the peripheral blood; usually a result of disease, trauma, or cancer treatment. To treat these patients, it is estimated that two million platelet transfusions are performed in the U.S. each year. This high demand for platelets has created a clinical demand for studying the causes of thrombocytopenia and alternative routes for treatment. Platelets are anuclear cells that are released into the bloodstream in the bone marrow by megakaryocytes via the extension of long filaments called proplatelets. It is hypothesized that platelet production from megakaryocytes is regulated by environmental factors at the site of bone marrow vascular niche. Studies of megakaryopoiesis are typically performed on extracellular matrix protein-coated culture plates and transwell membranes. While these initial studies have provided invaluable insight into the process of megakaryopoiesis, the goal of the present project was to create a bone marrow model that mimics the vascular niche for functional in vitro platelet production. We hypothesized that a silk-based in vitro tissue model would allow the effects of substrate surface properties and endothelial co-culture on megakaryopoiesis to be studied in a holistic manner, thereby enabling further elucidation of the mechanisms involved in the process of platelet production. ResultsIn order to more closely mimic the bone marrow vascular niche structure, a porous silk sponge was assembled around the silk vessel-like tubes. Megakarycytes seeded in the porous silk sponge migrated toward the silk tube and released platelets into the tube lumen. The perfusion bioreactor moved the platelets into the platelet collecting bags. After perfusion the platelets were collected and analyzed by flow cytometry. The bioreactor platelets exhibited similar morphology, CD41 positive staining, and activation compared to peripheral blood platelet controls. Megakaryocyte attachment and proplatelet formation through the silk vascular wall were improved by altering the silk properties. Silk functionalized by entrapping extracellular matrix proteins within the tube membrane resulted in increased megakaryocyte attachment and proplatelet compared to unfunctionalized silk tube controls. Silk surface roughness improved megakaryocyte attachment compared to the control but did not affect proplatelets. Decreasing the silk stiffness improved proplatelets, but did not significantly affect megakaryocyte attachment. Co-culture with endothelial cells improved megakaryocyte attachment while maintaining a high level of proplatelet formation. Additionally, megakaryocyte and endothelial cell co-culture on the silk vessel model resulted in an icreased platelet production compared to megakaryocytes cultured alone. ConclusionsThe goal of this project was to develop an in vitro model of megakaryopoiesis using a tissue engineering approach. Using human megakaryocytes and endothelial cells, we demonstrate the following advanced features of the silk-based model: (1) immobilization of extracellular matrix components within the membrane, (2) tunable surface topography, (3) tunable mechanical properties, (4) physiologically relevant thickness for appropriate proplatelet extension, and (5) controlled localization of a vascular endothelium. Thus, by functionalizing silk, we can control megakaryocyte function on silk. The broader impact of this work offers a versatile new tool for studying megakaryocyte development and platelet production in vitro. Disclosures:No relevant conflicts of interest to declare.

Three-dimensional spatial configuration of tumour cells confers resistance to chemotherapy independent of drug delivery

Anticancer drug discovery has been hampered by the lack of reliable preclinical models, which routinely use cells grown in two-dimensional (2D) culture systems. However, many of the characteristics of cells in 2D culture do not translate into the findings in animal xenografts. Three-dimensional (3D) growth may be responsible for some of these changes, and models using cells grown in 3D may form a more representative step in tumouricidal validation prior to animal implantation and human testing. For the 3D model, we cultured 143.98.2, SaOS2 or U2OS osteosarcoma cells seeded in porous Bombyx mori silk sponges. We conducted real-time PCR on cells grown in 2D culture and 3D scaffolds for the proliferation markers cyclin B1 and E2F1 and the actin regulator RhoA, and found a significant decrease in expression levels for the 3D tumour models (p = 0.02, < 0.001 and 0.008 for cyclin B1, E2F1 and RhoA for 143.98.2; p = 0.02, 0.002 and 0.02 for cyclin B1, E2F1 and RhoA for U2OS, respectively). In contrast, p21 was upregulated when SaOS2 and U2OS were cultured in the 3D scaffolds (p < 0.001) and there was no increase in DNA quantity during the culture period. We correspondingly observed G1 arrest when cell cycle analysis was conducted. Cytotoxicity results for cells treated with serial dilutions of doxorubicin and cisplatin showed that cells in 3D scaffolds were less sensitive to drug treatment than in 2D culture, and the difference was more pronounced for cell cycle specific agents. Copyright © 2013 John Wiley & Sons, Ltd.

인체적용을 위한 실크 스펀지의 제조 및 특성

인체적용을 위한 실크 스펀지의 제조 및 특성

A novel in vivo platform for studying alveolar bone regeneration in rat

Alveolar bone regeneration is a significant challenge in dental implantation. Novel biomaterials and tissue-engineered constructs are under extensive development and awaiting in vivo animal tests to find clinical endpoint. Here, we establish a novel in vivo model, modifying gingivoperiosteoplasty in rat for the alveolar bone regeneration. Rat premaxillary bone defects were filled with silk scaffold or remained empty during the implantation period (up to 6 weeks), and harvested samples were analyzed by micro-computed tomography and histopathology. Empty defects showed increased but limited new bone formation with increasing implantation period. In defects implanted with silk sponge, the bone formation was significantly greater than that of empty defect, indicating an effective role of silk scaffold in the defect model. The modified premaxillary defect model in rat is simple to perform, while mimicking the clinical conditions, finding usefulness for the development of biomaterials and tissue-engineered constructs targeting alveolar bone regeneration in dental implantation.