Jeong Eun Song

Fabrication of duck’s feet collagen–silk hybrid biomaterial for tissue engineering

Collagen constituting the extracellular matrix has been widely used as biocompatible material for human use. In this study, we have selected ducks feet for extracting collagen. A simple method not utilizing harsh chemical had been employed to extract collagen from ducks feet. We fabricated ducks feet collagen/silk hybrid scaffold for the purpose of modifying the degradation rate of ducks feet collagen. This study suggests that extracted collagen from ducks feet is biocompatible and resembles collagen extracted from porcine which is commercially used. Ducks feet collagen is also economically feasible and it could therefore be a good candidate as a tissue engineering material. Further, addition of silk to fabricate a ducks feet collagen/silk hybrid scaffold could enhance the biostability of ducks feet collagen scaffold. Ducks feet collagen/silk scaffold increased the cell viability compared to silk alone. Animal studies also showed that ducks feet collagen/silk scaffold was more biocompatible than silk alone and more biostable than ducks feet or porcine collagen alone. Additionally, the results revealed that ducks feet collagen/silk hybrid scaffold had high porosity, cell infiltration and proliferation. We suggest that ducks feet collagen/silk hybrid scaffold could be used as a dermal substitution for full thickness skin defects.

Reduction of inflammatory reaction in the use of purified alginate microcapsules

Alginate, a polysaccharide extracted from brown seaweed, remains the most widely used biomaterial for immobilizing cells to be transplanted, because of the good viability of the encapsulated cells and the relatively ease of processing for cell encapsulation. However, the main drawback is the immune reaction in vivo. To overcome this problem, we have demonstrated a modified Korbutt method for alginate purification. After alginate microcapsules were manufactured, NIH/3T3 fibroblast cells were seeded in purified and non-purified alginate microcapsules, and the cell proliferation was analyzed by 3-(4,5-dimethylthiazol-2-yl)-2,5 diphenyltetrazolium bromide assay. Reverse transcriptase-polymerase chain reaction was performed to assess the mRNA expression of RAW 264.7 macrophage cells for inflammation cytokines such as TNF-α. Purified and non-purified alginate microcapsules were implanted into Wister rats, and subsequently extracted after 1–2 weeks. Tissues surrounding the implants were harvested and underwent histological evaluation through H&E staining and immunohistochemical evaluation through ED-1 staining. In this result, contaminated materials in the purified alginate were eliminated by purification process. Thereby, density of inflammatory cell decreased about 30% more than non-purified alginate and thickness of fibrotic wall decreased about three times. In concluding, the purified alginate is anticipated to be highly potent for numerous biomaterial applications.

Effect of pore sizes of PLGA scaffolds on mechanical properties and cell behaviour for nucleus pulposus regeneration in vivo

This study investigated the influence of pore sizes of poly(lactic‐co‐glycolic acid) (PLGA) scaffolds on the compressive strength of tissue‐engineered biodiscs and selection of the best suitable pore size for cells to grow in vivo. PLGA scaffolds were fabricated by solvent casting/salt‐leaching with pore sizes of 90–180, 180–250, 250–355 and 355–425 µm. Nucleus pulposus (NP) cells were seeded on PLGA scaffolds with various pore sizes. Each sample was harvested at each time point, after retrieval of PLGA scaffolds seeded with NP cells, which were implanted into subcutaneous spaces in nude mice at 4 and 6 weeks. MTT assay, glycosaminoglycan (GAG) assay, haematoxylin and eosin (H&E) staining, safranin O staining and immunohistochemistry (for collagen type II) were performed at each time point. As the pores became smaller, the value of the compressive strength of the scaffold was increased. The group of scaffolds with pore sizes of 90–250 µm showed better cell proliferation and ECM production. These results demonstrated that the compressive strength of the scaffold was improved while the scaffold had pore sizes in the range 90–250 µm and good cell interconnectivity. Suitable space in the scaffold for cell viability is a key factor for cell metabolism. Copyright

Effect of pore sizes of silk scaffolds for cartilage tissue engineering

The aim of this study was to investigate the effects of silk fibroin scaffold, a natural biodegradable polymer scaffold, on the adhesive and proliferative behaviors of chondrocytes. Various silk fibroin scaffolds were produced using the salt extraction method, and scaffolds with different pore sizes (90-180, 180-250, 250-355, and 355-425 μm) were constructed based on the size of the salt particles. Chondrocytes were seeded on the scaffolds and incubated. The produced scaffolds were analyzed with Fourier transform-infrared spectroscopy and exhibited characteristics similar to those of natural silk in terms of chemical composition and structure. Moreover, we found that the mechanical strength decreased as the pore size increased. Scanning electron microscopy images confirmed the existence of pores in the silk fibroin scaffold. Additionally, scaffolds with smaller pore sizes facilitated improved cell adhesion. Using MTT analysis, we found that scaffold with pore sizes of 90-180 and 180-250 μm provided the best environment for cell proliferation. The amount levels of sulfated glycosaminoglycan (sGAG) and collagen were highest for scaffolds with a pore size of 90-180 μm. In gene expression analysis, scaffolds with pore sizes of 90-180 and 180-250 μm showed the highest expression of the chondrocytes marker aggrecan and type II collagen. Collectively, these data suggest that silk fibroin scaffolds with smaller pore sizes (90-250 μm) provide the best environment for adhesion and proliferation of chondrocytes.

Enhanced osteogenesis of β-tricalcium phosphate reinforced silk fibroin scaffold for bone tissue biofabrication.

Scaffolds, used for tissue regeneration are important to preserve their function and morphology during tissue healing. Especially, scaffolds for bone tissue engineering should have high mechanical properties to endure load of bone. Silk fibroin (SF) from Bombyx mori silk cocoon has potency as a type of biomaterials in the tissue engineering. β-tricalcium phosphate (β-TCP) as a type of bioceramics is also critical as biomaterials for bone regeneration because of its biocompatibility, osteoconductivity, and mechanical strength. The aim of this study was to fabricate three-dimensional SF/β-TCP scaffolds and access its availability for bone grafts through in vitro and in vivo test. The scaffolds were fabricated in each different ratios of SF and β-TCP (100:0, 75:25, 50:50, 25:75). The characterizations of scaffolds were conducted by FT-IR, compressive strength, porosity, and SEM. The in vitro and in vivo tests were carried out by MTT, ALP, RT-PCR, SEM, μ-CT, and histological staining. We found that the SF/β-TCP scaffolds have high mechanical strength and appropriate porosity for bone tissue engineering. The study showed that SF/β-TCP (75:25) scaffold exhibited the highest osteogenesis compared with other scaffolds. The results suggested that SF/β-TCP (75:25) scaffold can be applied as one of potential bone grafts for bone tissue engineering.

Development of poly(lactide-co-glycolide) scaffold-impregnated small intestinal submucosa with pores that stimulate extracellular matrix production in disc regeneration

The pore size and microstructure of scaffolds influences cell attachment, migration, proliferation and ingrowth, but the optimal pore size of scaffolds for disc tissue formation is not clearly understood. We developed porous poly(lactide‐co‐glycolide) (PLGA) scaffolds with various pore sizes for nucleus pulposus (NP) cell cultures and examined the effects of pore size on cell ingrowth and extracellular matrix (ECM) synthesis. High cell density in the small pores of scaffolds promotes collagen synthesis and cell migration through interconnected pores. Scaffolds with large pores exhibited slower cell proliferation and collagen synthesis. Guided by these results, we investigated a novel, biodegradable, synthetic/natural hybrid scaffold composed of PLGA and small intestinal submucosa (SIS) (PLGA–SIS) with the proper pore size for NP regeneration. We tested the morphological and physical properties of PLGA–SIS scaffolds and initial cell attachment and ECM production of NP in scaffolds. The mechanical and degradable properties of the PLGA–SIS scaffold were superior to those of SIS sponge and were similar to the properties of PLGA scaffold. NP cells grown on PLGA–SIS scaffold exhibited higher initial cell adhesion and ECM production than those grown on pure PLGA scaffold in a biological assay. In conclusion, this study suggests that a proper pore size of scaffolds is critical in NP regeneration, and that PLGA–SIS scaffolds with suitable pores might be useful as substrates for tissue‐engineered biodiscs. Copyright

Effect of demineralized bone particle/poly(lactic-co-glycolic acid) scaffolds on the attachment and proliferation of mesenchymal stem cells.

The aim of this study was to investigate the effect of demineralized bone particle/ poly(lactic-co-glycolic acid) (DBP/PLGA) scaffolds on the proliferation of mesenchymal stem cells (MSCs). DBP/PLGA hybrid scaffolds were fabricated by solvent casting/salt-leaching with DBP contents of 0, 20, 40, and 80 wt%. MSCs were seeded on the DBP/PLGA scaffolds and then evaluated by a series of analytical process: SEM, MTT, RT-PCR, and in vivo histological assay. As the DBP contents increased, the cell attachment behavior and cell viability also increased. A DBP content of 80 wt% marked the best water absorption performance and the highest cell viability. Gene expression of aggrecan on DBP/PLGA scaffolds tended to increase, whereas that on PLGA scaffolds was decreased at 1 week. However, strong expression of aggrecan was observed at 2 weeks regardless of the contents of DBP. Scaffolds showed a trend of increasing type II and I collagen at 2 weeks. The results showed that MSCs on DBP/PLGA scaffolds showed more efficient cell proliferation and tissue formation in the presence of tissue-inductive stimuli. Suitable biomaterials could be more conducive to proliferation of MSCs. These results suggest that the DBP/PLGA scaffolds are a feasible biomaterial for intervertebral disc regeneration.

Demineralized Bone Particle Impregnated Poly(l-Lactide-co-Glycolide) Scaffold for Application in Tissue-Engineered Intervertebral Discs

Abstract Demineralized bone particle (DBP) contains powerful bioactive molecules that facilitate new bone or cartilage growth. We developed hybrid scaffolds of poly(l-lactide-co-glycolide) (PLGA) with various concentrations of DBP (DBP/PLGA), of which phenotypes on intervertebral disc (IVD) cells were investigated. The hybrid scaffold has a cylindrical donut shape with two distinct parts; the inner is for the nucleus pulposus (NP) and the outer is for annulus fibrosus (AF). Rabbit NP and AF cells were seeded into the inner and outer regions of the DBP/PLGA scaffolds separately. Disc cell viability in DBP/PLGA scaffolds was superior to pure PLGA scaffold and increased with increasing DBP concentration. In vitro- and in vivo-formed tissues were characterized by RT-PCR, Safranin-O, Masson’s trichrome staining and immunohistochemi- cal staining for type-I and type-II collagen. DBP/PLGA hybrid scaffolds revealed more active expression of disc phenotypes, as characterized by protein and mRNA expression, than the PLGA control. This study provides valuable information for potential disc replacement using DBP and PLGA.

Skin regeneration using duck’s feet derived collagen and poly(vinyl alcohol) scaffold

In this study, we have designed scaffold by mixing collagen extracted from the duck’s flippers with poly(vinyl alcohol) (DC/PVA) via freeze-thawing and the as-prepared scaffold were evaluated for skin regeneration. The physical and chemical properties of the scaffolds were characterized using FTIR, SEM, compressive strength, degree of swelling, etc. The in vitro behavior (cell proliferation) was examined after cultured with fibroblasts through the 3-(4,5-dimethylthiazol-2-yl)-2,5 diphenyltetrazolium bromide (MTT) assay. The in vivo results demonstrated that the scaffolds implanted in rat induced full-thickness defect, confirmed by histological staining. Encompassing all results, the DC/PVA scaffolds can be anticipated as efficient platforms for skin regeneration facilitating required cell adhesion, growth and proliferation.

Effect of hyaluronic acid (HA) in a HA/PLGA scaffold on annulus fibrosus regeneration: In vivo tests

AbstractIntervertebral disc (IVD) degeneration is an age-related process that affects the biomechanical properties of the spine and is assumed to be one of the principal causes of low back pain. Poly(lactide-co-glycolide) (PLGA) and hyaluronic acid (HA) have been widely used as biocompatible scaffold materials for tissue regeneration. In the present study, we fabricated a microporous PLGA scaffold by the salt-leaching method and HA-loaded PLGA scaffolds by the penetrating method. As the porosity of PLGA and PLGA/HA is 90.7% and 96.5%, respectively, PLGA/HA scaffold has the higher porosity. However, the pore size of PLGA (240.7±14.0 μm) is the larger than HA/PLGA (212.0±12.5 μm), and cells from the annulus fibrosus (AF) were seeded into the scaffolds. For the in vivo study, the PLGA and HA/PLGA scaffolds were implanted in four-week-old nude mice. All scaffolds were characterized using a scanning electron microscope (SEM), and the amounts of glycosaminoglycan (GAG) and collagen in the scaffolds were determined by a spectrophotometer. Histological evaluation indicated the higher level of AF cell proliferation in the HA/PLGA scaffold than in PLGA scaffolds alone. In addition, AF cells showed the stronger production of GAG and collagen in HA/PLGA. Our results indicate that the HA/PLGA scaffold might be useful for intervertebral disc regeneration.