Three-dimensional bioprinting of osteochondral composite tissue with innovative bio-ink and PCL to repair articular cartilage defects / 中华骨科杂志

Objective:A new type of bio-ink and polycaprolactone (PCL) were used to construct an integrated osteochon-dral composite tissue block by multi-nozzle 3D bioprinter. And the repair results to osteochondral defects were evaluated.Methods:In freeze-drying group: Freeze-dried composite scaffold made by silk fibroin (SF) and β-tricalcium phosphate was used to repair osteochondral defects, as control. In the 3D printing group: PCL was used to printed a hollow multi-layer cylinder frame by 3D biological printer. Extracellular matrix, SF and bone marrow mesenchymal stem cells were used as chondral bio-ink. Then, chon-dral bio-ink was used to print tissue-engineered cartilage on top of PCL frame. Before implantation of cartilage defect, autogenous cancellous bone was filled in PCL frame, then the tissue-engineered osteochondral composite was used to repair osteochondral defects. In mosaic group: Autologous osteochondral transplantation was performed. The repair results of the above three groups were compared by histological score, biochemical analysis and biomechanical test to evaluate the effect of repairing rabbit cartilage defects.Results:The compression modulus of neo-cartilage in the 3D print group 2.56±0.30 MPa was close to that of the mosaic group 2.51±0.13 MPa ( P>0.05), and significantly higher than that of freeze-dried group 1.37±0.14 MPa ( F=11.058, P<0.05). The sGAG contents in the 3D print group 14.49±0.7 μg/mg was close to that of the mosaic group 14.98±0.81 μg/mg ( P>0.05), and significantly higher than that of freeze-dried group 8.72±0.73 μg/mg ( F=20.973, P<0.05). However, there was no significant difference in collagen content between the three groups ( P>0.05). The results of ICRS cartilage repair histology score showed that the scores of the 3D print group were close to those of the mosaic group in the matrix, cell distribution, cell viability and subchondral bone ( P>0.05), and were significantly higher than those of freeze-dried group in the surface and cartilage mineralization scores ( F=19.544, P<0.05). Conclusion:Using the new bio-ink to make bone cartilage composite scaffold by 3D bio printing can simplify the construction of tissue-engineered bone cartilage composite tissue in vitro, and can repair cartilage defects in vivo.

Wound healing effects of silk fibroin-bone morphogenetic protein-2 scaffolds on inflammatory pulp in rats / 北京大学学报(医学版)

Objective:To identify the healing effect of electrospun silk fibroin-BMP-2 as a biologic pulp capping agent to inflammatory pulp in rat caused by lipopolysaccharide ( LPS) .Methods:A total of 30 healthy adult male Wistar rats were randomly divided into five groups:(1) normal control group without operation;(2) blank control group without capping agents;(3) calcium hydroxide capping group;(4) electrospun silk fibroin capping group;(5) electrospun silk fibroin-BMP-2 capping group .Bilateral up-per first molars of each rat in group 2-5 were drilled to expose the pulp to LPS which was used to estab-lish a model of inflammatory pulp .The exposed pulp was capped with different capping agents or without capping agents.Then the hole was sealed.The animals were sacrificed on days 3, 7, and 14 post-opera-tion and histological analysis was carried out , including HE stain and CD 14 immunohistochemical stain . Results:On day 7 and 14 , the lowest inflammatory reaction score in HE stain among pulp capping groups was that of silk fibroin-BMP-2 group .The next were calcium hydroxide group and silk fibroin group .That of blank control group was the highest .The ranking of reparative dentine scores of those groups was just reversed.The D values of immunohistochemical stain of CD 14 were not significantly different in groups applied pulp capping agents but significantly lower than blank control group on days 3 and 7.However, the D value of silk fibroin-BMP-2 group ( 0 .145 ±0 .011 ) was significantly lower than blank control group (0.287 ±0.019), calcium hydroxide group (0.170 ±0.017) and silk fibroin group (0.175 ± 0 .018 ) on day 14 .Conclusion:Electrospun silk fibroin compounded with BMP-2 promoted wound hea-ling of exposed pulp and had better potential to stimulate formation of reparative dentine to establish a suitable environment for pulp recovery .

Clinical Outcomes of Silk Patch in Acute Tympanic Membrane Perforation

OBJECTIVES: The silk patch is a thin transparent patch that is produced from silk fibroin. In this study, we investigated the treatment effects of the silk patch in patients with traumatic tympanic membrane perforation (TTMP). METHODS: The closure rate, otorrhea rate, and closure time in all patients and the closure time in successful patients were compared between the paper patch and silk patch groups. RESULTS: Demographic data (gender, site, age, traumatic duration, preoperative air-bone gap, and perforation size and location) were not significantly different between the two groups. The closure rate and otorrhea rate were not significantly different between the two groups. However, the closure time was different between the two groups (closure time of all patients, P=0.031; closure time of successful patients, P=0.037). CONCLUSION: The silk patch which has transparent, elastic, adhesive, and hyper-keratinizing properties results in a more efficient closure time than the paper patch in the treatment of TTMP patients. We therefore believe that the silk patch should be recommended for the treatment of acute tympanic membrane perforation.

Caracterização e avaliação da citocompatibilidade de nanocompósitos baseados em celulose bacteriana e fibroína para aplicação em regeneração tecidual / Characterization and cytocompatibility evaluation of cellulose and fibroin based nanocomposites for tissue regeneration applications

A engenharia de tecidos tem como perspectiva desenvolver opções terapêuticas para quadros clínicos com prognóstico limitado, objetivando substituir ou regenerar os tecidos lesados por meio do uso de biomateriais. A metodologia baseia-se no cultivo de células sobre uma matriz ou scaffold que deve ser otimizado para minimizar o risco de infecções e permitir a liberação das moléculas bioativas no local pretendido. As propriedades dos diversos biomateriais são essenciais para o sucesso da metodologia e estes podem ser manipulados de forma a mimetizar a arquitetura da matriz nativa. Assim, nos propomos a desenvolver um novo biocompósito baseado em celulose bacteriana (CB)/ fibroína (FB) visando aplicações em regeneração tecidual na odontologia, em 3 concentrações diferentes (CB/FB25%, CB/FB50% e CB/FB75%). Estes foram caracterizados por análise termogravimétrica, Espectroscopia Vibracional na Região do Infravermelho (FT-IR), Difração de Raios-X, e Microscopia Eletrônica de Varredura (MEV), demonstrando boa estabilidade físico-química, inclusive resitência à degradação térmica em altas temperaturas. A investigação da citocompatibilidade foi conduzida por meio de testes in vitro apenas com as amostras de CB/FB50%. Os testes aplicados foram MTT, azul de tripano, adesão e proliferação celular. As análises estatísticas foram realizadas por meio do software Graph Prism 5.0. Os resultados demonstraram que o material desenvolvido é biocompatível e não citotóxico. As imagens de MEV revelaram um número muito maior de células aderidas à superfície dos scaffolds de CB/FB quando comparado ao de CB pura. Com base nos dados obtidos é possível sugerir que o nanocompósito baseados em CB/FB50% configura uma excelente alternativa como scaffold para a reparação de tecidos.

Tissue engineering has the purpose of developping therapeutic options especially designed to be used on limited clinical conditions, aiming to replace or regenerate damaged tissues applying biomaterials for that. The method is based on the cultivation of cells on a matrix or scaffold that should be optimized to offer minimum risk of infections and allow the release of bioactive molecules at the desired location. The success of the methodology depends on Biomaterials' properties that can be manipulated to mimic the three-dimensional architecture of native tissues extracellular matrix. Thus, we purpose to prepare a new nanocomposite based on bacterial cellulose (BC) / fibroin (SF) aiming at applications in the process of tissue regeneration in dentistry. Three different material's compositions were prepared (BC/SF 25 % BC/SF 50 % and BC/SF 75 %) and all of them were characterized by thermogravimetric analysis, Vibrational Spectroscopy in the Infrared Region (FT-IR), X-ray diffraction, and Scanning Electron Microscopy (SEM). The material showed good physical and chemical stability including resistance to thermal degradation at high temperatures. The investigation of the cytocompatibility was realized just using the samples BC/SF 50 % and the in vitro tests conducted were MTT, Trypan Blue, cell adhesion and proliferation assays. The statistical analysis was done applying the software Graph Pad Prism 5.0. The results showed that the developed nanocomposite is biocompatible and non-cytotoxic. SEM images revealed a greater number of cells attached to the scaffold CB / FB surface when compared to pure CB scaffolds. Based on the data obtained, it is possible to suggest that the nanocomposite based on CB/FB50% configures an excellent alternative as a scaffold for tissue repair

Curative effect of porous silk fibroin scaffolds combined with chondroitinase ABC in treatment of spinal cord injury / 中华创伤杂志

Objective To investigate the effect of porous silk fibroin scaffolds (PSFSs) combined with chondroitinase ABC (ChABC)for treatment of rats with spinal cord injury (SCI).Methods After exposed to T9 spinal cord transection injury,96 SD rats were divided into control group,PSFSs group,ChABC group,and PSFSs plus ChABC group according to random number table.BBB scoring system was used to evaluate hindlimb motor function in rats.Immunohistochemistry and Western blot analysis were performed to detect expression levels of neurofilament-200 (NF-200),glial fibrillary acidic protein (GFAP),and growth associated protein-43 (GAP-43) of the injured spinal cord.Immuno-fluorescence staining was carried out to evaluate regeneration of nerve fiber.Results BBB score improved in PSFSs group (8.1 ± 0.8),ChABC group (9.0 ± 1.1),and PSFSs plus ChABC group (13.7 ± 1.3) compared with control group 4 weeks after injury (5.3 ±0.7,P ＜0.05).Immunohistochemistry showed higher integral absorbance (IA) values of NF-200 and GAP-43 in those treatment groups,but smaller GFAP-positive area was observed compared with control group (P ＜ 0.05).Immuno-fluorescence staining indicated more GAP-43 growth at injury sites in PSFSs plus ChABC group in contrast with other 3 groups.Western blotting showed levels of NF-200,GFAP,and GAP-43 differed among groups (P ＜ 0.05).Conclusion PSFSs combined with chondroitinase ABC transplantation can enhance axonal regeneration,inhibit glial scar proliferation and hence promote motor function recovery.

Preliminary Construction of Tissue Engineering Nucleus Pulposus Combining Silk Fibroin Porous Scaffold with Rabbit Nucleus Pulposus Cells / 天津医药

Objective To investigate the feasibility of construction of tissue engineering nucleus pulposus by com?bining the novel silk fibroin porous scaffold with PKH26 labeled rabbit nucleus pulposus cells. Methods Rabbit nucleus pulposus cells were isolated and cultured, then the passage 1 nucleus pulposus cells were stained with safranin O and typeⅡcollagen immunohistochemical staining. The isolated rabbit nucleus pulposus cells were labeled with PKH26. MTT assay was used for examining the proliferation of the nucleus pulposus cells before and after labeling. Labeled cells were inoculat?ed in the scaffold, cultured for 4 days and then the cell-scaffold complexes were implanted subcutaneously into nude mice. After 12 weeks of in vivo culture, the cell-scaffold complexes were detected by in vivo imaging technology, H&E staining, toluidine blue staining, safranin O staining and collagen typeⅡimmunohistochemical staining. Results Safranin O stain?ing and typeⅡcollagen immunohistochemical staining of the passage 1 nucleus pulposus cells were positive. The fluores?cence intensity of labeled cell was distributed, and the difference of OD value of nucleus pulposus cells was not statistically significant before and after labeling (P>0.05). The in vivo imaging technique showed a strong fluorescencea in porous scaf?fold. H&E staining of cell-scaffold complexes showed that the scaffolds were filled with a large number of nucleus pulposus cells and large amount of extracellular matrix. Toluidine blue staining, safranin O staining and typeⅡcollagen immunohisto?chemical staining were positive, and large amount of extracellular matrix was secreted around the cells. Conclusion The new silk fibroin porous scaffold with rabbit nucleus pulposus cells in vivo culture formed nucleus pulposus like tissue, which can be used for construction of tissue engineering nucleus.

The effect of silk fibroin and rhBMP-2 on bone regeneration in rat calvarial defect model

INTRODUCTION: This study evaluated the capability of silk fibroin (SF) and recombinant human bone morphogenetic protein-2 loaded SF (SF-BMP) as a bone defect replacement matrix when grafted in a calvarial bone defect of rats in vivo. MATERIALS AND METHODS: A total 70 calvarial critical size defects (5.0 mm in diameter) made on 35 adult female Sprague-Dawley rats were used in this study. The defects were transplanted with (1) rhBMP-2 loaded silk fibroin graft (SF-BMP: 0.8+10 microg), (2) Silk fibroin (SF: 10 microg), and (3) no graft material (Raw). The samples were evaluated with soft x-rays, alkaline phosphatase activity, calcium/phosphate quantification, histological and histomorphometric analysis at postoperative 4 and 8 weeks. RESULTS: The SF-BMP group (48.86+/-14.92%) had a significantly higher mean percentage bone area than the SF group (24.96+/-11.01%) at postoperative 4 weeks.(P<0.05) In addition, the SF-BMP group (40.01+/-12.43%) had a higher % bone area at postoperative 8 weeks than the SF group (33.26+/-5.15%). The mean ratio of gray scale levels to the host bone showed that the SF-BMP group (0.67+/-0.08) had a higher mean ratio level than the SF group (0.61+/-0.09) at postoperative 8 weeks. These differences were not statistically significant.(P=0.168 and P=0.243, respectively) CONCLUSION: The rhBMP-2 loaded silk fibroin graft revealed fewer immunoreactions and inflammation as well as more new bone formation than the pure silk fibroin graft. Therefore, silk fibroin may be a candidate scaffold for tissue engineered bone regeneration.