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3D printing of silk fibroin-based hybrid scaffold treated with platelet rich plasma for bone tissue engineering.

ABSTRACT: 3D printing/bioprinting are promising techniques to fabricate scaffolds with well controlled and patient-specific structures and architectures for bone tissue engineering. In this study, we developed a composite bioink consisting of silk fibroin (SF), gelatin (GEL), hyaluronic acid (HA), and tricalcium phosphate (TCP) and 3D bioprinted the silk fibroin-based hybrid scaffolds. The 3D bioprinted scaffolds with dual crosslinking were further treated with human platelet-rich plasma (PRP) to generate PRP coated scaffolds. Live/Dead and MTT assays demonstrated that PRP treatment could obviously promote the cell growth and proliferation of human adipose derived mesenchymal stem cells (HADMSC). In addition, the treatment of PRP did not significantly affect alkaline phosphatase (ALP) activity and expression, but significantly upregulated the gene expression levels of late osteogenic markers. This study demonstrated that the 3D printing of silk fibroin-based hybrid scaffolds, in combination with PRP post-treatment, might be a more efficient strategy to promote osteogenic differentiation of adult stem cells and has significant potential to be used for bone tissue engineering.

SUBMITTER: Wei L

PROVIDER: S-EPMC6812411 | biostudies-literature | 2019 Dec

REPOSITORIES: biostudies-literature

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3D printing of silk fibroin-based hybrid scaffold treated with platelet rich plasma for bone tissue engineering.

Wei Liang L Wu Shaohua S Kuss Mitchell M Jiang Xiping X Sun Runjun R Reid Patrick P Qin Xiaohong X Duan Bin B

Bioactive materials 20190925

3D printing/bioprinting are promising techniques to fabricate scaffolds with well controlled and patient-specific structures and architectures for bone tissue engineering. In this study, we developed a composite bioink consisting of silk fibroin (SF), gelatin (GEL), hyaluronic acid (HA), and tricalcium phosphate (TCP) and 3D bioprinted the silk fibroin-based hybrid scaffolds. The 3D bioprinted scaffolds with dual crosslinking were further treated with human platelet-rich plasma (PRP) to generate ...[more]

PMID: 31667442

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Project description:BACKGROUND:Cartilage tissue engineering is a promising technique for repairing cartilage defect. Due to the limitation of cell number and proliferation, mesenchymal stem cells (MSCs) have been developed as a substitute to chondrocytes as a cartilage cell-source. This study aimed to develop cartilage tissue from human adipose-derived stem cells (ADSCs) cultured on a Bombyx mori silk fibroin scaffold and supplemented with 10% platelet-rich plasma (PRP). METHODS:Human ADSCs and PRP were characterized. A silk fibroin scaffold with 500 μm pore size was fabricated through salt leaching. ADSCs were then cultured on the scaffold (ADSC-SS) and supplemented with 10% PRP for 21 days to examine cell proliferation, chondrogenesis, osteogenesis, and surface marker expression. The messenger ribonucleic acid (mRNA) expression of type 2 collagen, aggrecan, and type 1 collagen was analysed. The presence of type 2 collagen confirming chondrogenesis was validated using immunocytochemistry. The negative and positive controls were ADSC-SS supplemented with 10% foetal bovine serum (FBS) and ADSC-SS supplemented with commercial chondrogenesis medium, respectively. RESULTS:Cells isolated from adipose tissue were characterized as ADSCs. Proliferation of the ADSC-SS PRP was significantly increased (p < 0.05) compared to that of controls. Chondrogenesis was observed in ADSC-SS PRP and was confirmed through the increase in glycosaminoglycans (GAG) and transforming growth factor-β1 (TGF-β1) secretion, the absence of mineral deposition, and increased surface marker proteins on chondrogenic progenitors. The mRNA expression of type 2 collagen in ADSC-SS PRP was significantly increased (p < 0.05) compared to that in the negative control on days 7 and 21; however, aggrecan was significantly increased on day 14 compared to the controls. ADSC-SS PRP showed stable mRNA expression of type 1 collagen up to 14 days and it was significantly decreased on day 21. Confocal analysis showed the presence of type 2 collagen in the ADSC-SS PRP and positive control groups, with high distribution outside the cells forming the extracellular matrix (ECM) on day 21. CONCLUSION:Our study showed that ADSC-SS with supplemented 10% PRP medium can effectively support chondrogenesis of ADSCs in vitro and promising for further development as an alternative for cartilage tissue engineering in vivo.

Project description:The repair of bone defects remains a major challenge in the clinic, and treatment requires bone grafts or bone replacement materials. Existing biomaterials have many limitations and cannot meet the various needs of clinical applications. To treat bone defects, we constructed a nanohydroxyapatite (nHA)/methylacrylylated silk fibroin (MASF) composite biological scaffold using photocurable 3D printing technology. In this study, scanning electron microscopy (SEM) was used to detect the changes in the morphological structure of the composite scaffold with different contents of nanohydroxyapatite, and FTIR was used to detect the functional groups and chemical bonds in the composite scaffold to determine the specific components of the scaffold. In in vitro experiments, bone marrow mesenchymal stem cells from SD rats were cocultured with scaffolds soaking solution, and the cytotoxicity, cell proliferation, Western blot analysis, Quantitative real-time PCR analysis, bone alkaline phosphatase activity and alizarin red staining of scaffolds were detected to determine the biocompatibility of scaffolds and the effect of promoting proliferation and osteogenesis of bone marrow mesenchymal stem cells in vitro. In the in vivo experiment, the skull defect was constructed by adult SD rats, and the scaffold was implanted into the skull defect site. After 4 weeks and 8 weeks of culture, the specific osteogenic effect of the scaffold in the skull defect site was detected by animal micro-CT, hematoxylin and eosin (HE) staining and Masson's staining. Through the analysis of the morphological structure of the scaffold, we found that the frame supported good retention of the lamellar structure of silk fibroin, when mixed with nHA, the surface of the stent was rougher, the cell contact area increased, and cell adhesion and lamellar microstructure for cell migration and proliferation of the microenvironment provided a better space. FTIR results showed that the scaffold completely retained the β -folded structure of silk fibroin, and the scaffold composite was present without obvious impurities. The staining results of live/dead cells showed that the constructed scaffolds had no significant cytotoxicity, and thw CCK-8 assay also showed that the constructed scaffolds had good biocompatibility. The results of osteogenic induction showed that the scaffold had good osteogenic induction ability. Moreover, the results also showed that the scaffold with a MASF: nHA ratio of 1: 0.5 (SFH) showed better osteogenic ability. The micro-CT and bone histometric results were consistent with the in vitro results after stent implantation, and there was more bone formation at the bone defect site in the SFH group.This research used photocurable 3D printing technology to successfully build an osteogenesis bracket. The results show that the constructed nHA/MASF biological composite material, has good biocompatibility and good osteogenesis function. At the same time, in the microenvironment, the material can also promote bone defect repair and can potentially be used as a bone defect filling material for bone regeneration applications.

Project description:Articular cartilage is an avascular tissue with limited regenerative property. Therefore, a defect or trauma in articular cartilage due to disease or accident can lead to progressive tissue deterioration. Cartilage tissue engineering, by replacing defective cartilage tissue, is a method for repairing such a problem. In this research, three main aspects-cell, biomaterial scaffold, and bioactive factors-that support tissue engineering study were optimized. Adipose-derived mesenchymal stem cells (ADSC) that become cartilage were grown in an optimized growth medium supplemented with either platelet rich plasma (PRP) or L-ascorbic acid (LAA). As the characterization result, the ADSC used in this experiment could be classified as Mesenchymal Stem Cell (MSC) based on multipotency analysis and cell surface marker analysis. The biomaterial scaffold was fabricated from the Bombyx morii cocoon using silk fibroin by salt leaching method and was engineered to form different sizes of pores to provide optimized support for cell adhesion and growth. Biocompatibility and cytotoxicity evaluation was done using MTT assay to optimize silk fibroin concentration and pore size. Characterized ADSC were grown on the optimized scaffold. LAA and PRP were chosen as bioactive factors to induce ADSC differentiation to become chondrocytes. The concentration optimization of LAA and PRP was analyzed by cell proliferation using MTT assay and chondrogenic differentiation by measuring glycosaminoglycan (GAG) using Alcian Blue at 605 nm wavelength. The optimum silk fibroin concentration, pore size, LAA concentration, and PRP concentration were used to grow and differentiate characterized ADSC for 7, 14, and 21 days. The cell morphology on the scaffold was analyzed using a scanning electron microscope (SEM). The result showed that the ADSC could adhere on plastic, express specific cell surface markers (CD73, CD90, and CD105), and could be differentiated into three types of mature cells. The silk fibroin scaffold made from 12% w/v concentration formed a 500 µm pore diameter (SEM analysis), and was shown by MTT assay to be biocompatible and to facilitate cell growth. The optimum concentrations of the bioactive factors LAA and PRP were 50 µg/mL and 10%, respectively. GAG analysis with Alcian Blue staining suggested that PRP induction medium and LAA induction medium on 12% w/v scaffold could effectively promote not only cell adhesion and cell proliferation but also chondrogenic differentiation of ADSC within 21 days of culture. Therefore, this study provides a new approach to articular tissue engineering with a combination of ADSC as cell source, LAA and PRP as bioactive factors, and silk fibroin as a biocompatible and biodegradable scaffold.

Project description:In this research, hWJ-MSCs were grown on silk scaffolds and induced towards chondrogenesis by supplementation with L-ascorbic acid (LAA) or platelet rich plasma (PRP). Silk scaffolds were fabricated with salt leaching method by mixing silk fibroin (SF) with silk spidroin (SS). The silk fibroin was obtained from Bombyx mori cocoon that had been degummed, and the silk spidroin was obtained from wild-type spider Argiope appensa. The effect of scaffold composition and inducer on cell proliferation was observed through MTT assay. The most optimal treatment then continued to be used to induce hWJ-MSC towards chondrogenic differentiation for 7 and 21 days. Scaffolds characterization showed that the scaffolds produced had 3D structure with interconnected pores, and all were biocompatible with hWJ-MSCs. Scaffold with the addition of 10% SS + 90% SF showed higher compressive strength and better pore interconnectivity in comparison to 100% silk fibroin scaffold. After 48 h, cells seeded on scaffold with spidroin and fibroin mix had flattened morphology in comparison to silk fibroin scaffold which appeared to be more rounded on the scaffold surface. Scaffold with 10% (w/w) of silk spidroin (SS) + 90% (w/w) of silk fibroin (SF) was the most optimal composition for cell proliferation. Immunocytochemistry of integrin β1 and RGD sequence, showed that scaffold with SS 10% provide better cell attachment with the presence of RGD sequence from the spidroin silk which could explain the higher cell proliferation than SF100% scaffold. Based on Alcian Blue staining and Collagen Type II immunocytochemistry (ICC), cells grown on 10% SS + 90% SF scaffold with 10% PRP supplementation were the most optimal to support chondrogenesis of hWJ-MSCs. These results showed that the addition of spidroin silk from A. appensa. had impact on scaffold compressive strength and chondrogenic differentiation of hWJ-MSC and had the potential for further development of bio-based material scaffold in cartilage tissue engineering.

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3D printing of silk fibroin-based hybrid scaffold treated with platelet rich plasma for bone tissue engineering.

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3D printing of silk fibroin-based hybrid scaffold treated with platelet rich plasma for bone tissue engineering.

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