scholarly journals Chondrogenic differentiation of Wharton’s Jelly mesenchymal stem cells on silk spidroin-fibroin mix scaffold supplemented with L-ascorbic acid and platelet rich plasma

2020 ◽  
Vol 10 (1) ◽  
Author(s):  
Anggraini Barlian ◽  
Hermawan Judawisastra ◽  
Ahmad Ridwan ◽  
Antonia Ratih Wahyuni ◽  
Meidiana Ebtayani Lingga
Keyword(s):  
Cell Proliferation ◽  
Compressive Strength ◽  
Ascorbic Acid ◽  
Silk Fibroin ◽  
Chondrogenic Differentiation ◽  
Platelet Rich Plasma ◽  
Blue Staining ◽  
Silk Fibroin Scaffold ◽  
Rgd Sequence ◽  
Silk Scaffolds

Abstract 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.

scholarly journals Chondrogenic differentiation of adipose-derived mesenchymal stem cells induced by L-ascorbic acid and platelet rich plasma on silk fibroin scaffold

PeerJ ◽  
2018 ◽  
Vol 6 ◽  
pp. e5809 ◽  
Author(s):  
Anggraini Barlian ◽  
Hermawan Judawisastra ◽  
Nayla M. Alfarafisa ◽  
Untung A. Wibowo ◽  
Imam Rosadi
Keyword(s):  
Tissue Engineering ◽  
Cell Proliferation ◽  
Ascorbic Acid ◽  
Silk Fibroin ◽  
Mtt Assay ◽  
Chondrogenic Differentiation ◽  
Platelet Rich Plasma ◽  
Cartilage Tissue ◽  
Induction Medium ◽  
Biomaterial Scaffold

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.

scholarly journals Effect of Platelet-Rich Plasma on Chondrogenic Differentiation in Three-Dimensional Culture

2014 ◽  
Vol 8 (1) ◽  
pp. 78-84 ◽  
Author(s):  
Steven Elder ◽  
John Thomason
Keyword(s):  
Cell Proliferation ◽  
Stromal Cells ◽  
Chondrogenic Differentiation ◽  
Transforming Growth Factor ◽  
Platelet Rich Plasma ◽  
Three Dimensional ◽  
Cartilage Regeneration ◽  
Alginate Beads ◽  
Marrow Stromal Cells ◽  
Significant Difference

Platelet-rich plasma (PRP) may have the potential to enhance articular cartilage regeneration through release of growth factors including transforming growth factor isoforms. The purpose of this study was to investigate the potential for PRP to stimulate chondrogenic differentiation in three-dimensional PRP hydrogel constructs. Allogenic PRP was prepared using a double centrifugation protocol which resulted in a platelet concentration approximately 250% above baseline. Canine marrow stromal cells were encapsulated at 6.8×106 cells/ml in either 2% sodium alginate or in a 3:1 mixture of freshly prepared PRP and 2% alginate. PRP and alginate beads were cultured in chemically defined chondrogenic medium with and without 10 ng/ml TGF-β3. PRP cultures were additionally supplemented with frozen-thawed PRP. In the absence of TGF-β3, PRP had a mild stimulatory effect on cell proliferation. PRP did not stimulate cell proliferation in the presence of TGF-β3. Cells exposed to TGF-β3 accumulated significantly more GAG/DNA than those which were not, but there was not a statistically significant difference between alginate and PRP. Total collagen content was greater in PRP than in alginate, regardless of TGF-β3. Chondrogenesis in PRP was qualitatively and spatially different than that which occurred in conventional alginate beads and was characterized by isolated centers of intense chondrogenesis. Overall the results demonstrate that PRP alone weakly promotes chondroinduction of marrow stromal cells, and the effect is greatly augmented by TGF-β3.

scholarly journals The Effect of Human Platelet-Rich Plasma and L-Ascorbic Acid on Morphology, Proliferation, and Chondrogenesis Ability towards Human Adipose-Derived Stem Cells

2019 ◽  
Vol 3 (1) ◽  
pp. 26 ◽  
Author(s):  
Imam Rosadi ◽  
Karina Karina ◽  
Iis Rosliana ◽  
Siti Sobariah ◽  
Irsyah Afini ◽  
...  
Keyword(s):  
Stem Cells ◽  
Ascorbic Acid ◽  
Alcian Blue ◽  
Human Platelet ◽  
Platelet Rich Plasma ◽  
Cartilage Tissue ◽  
Population Doubling ◽  
Blue Staining ◽  
Alcian Blue Staining ◽  
Adipose Derived Stem Cells

Background: Microtia is a congenital malformation in the external ear due to cartilage defect. Adipose-derived stem cells (ADSC) is promising cells to develop cartilage tissue engineering for microtia. In this study, we focused on proliferation and chondrogenesis of ADSC in three different media, which consist of 10% fetal bovine serum (FBS), 10% FBS with L-ascorbic acid, and 10% human platelet rich plasma (PRP). Methods: ADSC were induced to differentiate into adipocytes, chondrocyte and osteocytes. ADSC morphology, proliferation and population doubling time was compared in three different media and analysed. Observation and alcian blue staining were done every 7 days to assess chondrogenic potency of ADSC from each treatment.Results: Isolated ADSC were able to differentiate into adipocytes, osteocytes and chondrocytes. ADSC in all group have fibroblast-like morphology, but cells in 10% FBS and 10% FBS with LAA group were flattened and larger. ADSC in 10% PRP group proliferates faster than 10% FBS with and without LAA. PDT values of ADSC were 34 hours, 44 hours and 48 hours, respectively for 10% PRP, 10% FBS with LAA and 10% FBS group. Alcian blue staining revealed that ADSC in 10% FBS with LAA and 10% PRP were able to proceed to chondrogenesis when cultured time were prolong up to 21 days, but not with ADSC in 10% FBS. Conclusion: We conclude that adding 10% FBS with LAA or 10% PRP into medium culture can support proliferation and chondrogenesis of ADSC. Key words: human ADSC, PRP, L-ascorbic acid, proliferation, chondrogenesis

scholarly journals In vitro study of cartilage tissue engineering using human adipose-derived stem cells induced by platelet-rich plasma and cultured on silk fibroin scaffold

2019 ◽  
Vol 10 (1) ◽  
Author(s):  
Imam Rosadi ◽  
Karina Karina ◽  
Iis Rosliana ◽  
Siti Sobariah ◽  
Irsyah Afini ◽  
...  
Keyword(s):  
Tissue Engineering ◽  
Stem Cells ◽  
Mrna Expression ◽  
Silk Fibroin ◽  
Platelet Rich Plasma ◽  
Cartilage Tissue Engineering ◽  
Cartilage Tissue ◽  
Silk Fibroin Scaffold

Abstract 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.

scholarly journals Role of Hypoxia on Growth and Differentiation of Human Adipose Derived Stem Cells Grown on Silk Fibroin Scaffold Induced by Platelet Rich Plasma

2021 ◽  
Vol 53 (3) ◽  
pp. 415-427
Author(s):  
Anggraini Barlian ◽  
Marselina Irasonia Tan ◽  
Ergha Widya Sarjana ◽  
Noviana Vanawati
Keyword(s):  
Stem Cells ◽  
Silk Fibroin ◽  
Platelet Rich Plasma ◽  
Adipose Derived Stem Cells ◽  
Low Oxygen ◽  
Silk Fibroin Scaffold ◽  
Hypoxic Conditions ◽  
Proliferation And Differentiation ◽  
The Difference ◽  
Low Oxygen Concentration

Previous research has proven that 10% platelet-rich plasma (PRP) can enhance growth and differentiation of human adipose derived stem cells (hADSC) grown on silk fibroin scaffold into chondrocytes. A low oxygen concentration (hypoxia) condition is an important factor that potentially affects the ability of hADSC to grow and differentiate. The objective of this research was to analyze the difference in growth and differentiation capacity of hADSC grown on salt leached silk fibroin scaffold supplemented by 10% PRP under normoxic and hypoxic conditions. The growth capacity of the hADSC was determined by MTT assay and differentiation was tested using glycosaminoglycan (GAG) content analysis, while chondrocyte markers were visualized with the immunocytochemistry (ICC) method. This research observed hADSC proliferation under normoxic and hypoxic conditions for 21 days. Visualization of type 2 collagen showed that it was more abundant under hypoxia compared to normoxia.  HIF-1α was only detected in the hADSC cultured in hypoxic conditions. In conclusion, culture under hypoxic conditions increases the capacity of hADSC to grow and differentiate into chondrocytes. This is the first study that has shown that hypoxia is able to enhance the proliferation and differentiation of hADSC grown on 3D salt leached silk fibroin scaffold supplemented by 10% PRP.

scholarly journals Cell penetration and chondrogenic differentiation of human adipose derived stem cells on 3D scaffold

2021 ◽  
pp. FSO734
Author(s):  
Rizka Musdalifah Amsar ◽  
Anggraini Barlian ◽  
Hermawan Judawisastra ◽  
Untung Ari Wibowo ◽  
Karina Karina
Keyword(s):  
Ascorbic Acid ◽  
Silk Fibroin ◽  
Platelet Rich Plasma ◽  
Type Ii Collagen ◽  
Adipose Derived Stem Cells ◽  
Cell Penetration ◽  
3D Scaffold ◽  
Reverse Transcription Quantitative Pcr ◽  
Proliferation And Differentiation

The ability of cells to penetrate the scaffold and differentiate into chondrocyte is important in cartilage engineering. The aim of this research was to evaluate the use of silk fibroin 3D scaffold in facilitating the growth of stem cell and to study the role of L-ascorbic acid and platelet rich plasma (PRP) in proliferation and differentiation genes. Cell penetration and type II collagen content in the silk fibroin scaffold was analyzed by confocal microscopy. Relative expressions of CDH2, CCND1, CTNNB1 and COL2A1 were analyzed by reverse transcription-quantitative PCR (RT-qPCR). The silk fibroin 3D scaffold could facilitate cell penetration. L-ascorbic acid and PRP increased the expression of CDH2 and COL2A1 on the 21st day of treatment while PRP inhibited CTNNB1 and CCND1.

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