PGS:Gelatin nanofibrous scaffolds with tunable mechanical and structural properties for engineering cardiac tissues

TLDR: This study suggests that the aligned nanofibrous PGS:gelatin scaffold support cardiac cell organization, phenotype and contraction and could potentially be used to develop clinically relevant constructs for cardiac tissue engineering.

About: This article is published in Biomaterials.The article was published on 2013-09-01 and is currently open access. It has received 260 citations till now. The article focuses on the topics: Tissue engineering.

Figures

Table 1 Mechanical properties of crosslinked aligned (A) and random (R) scaffolds at dry and we

Fig. 6. Interactive effects of physical and chemical properties on CMs contraction and organization. (A) Beating rate (BPM) as a function of culture period and (B) beating pattern of CMs grown on the aligned and random scaffolds at day 7 of culture. (C) CMs alignment was quantified after 7 days of culture. (*: P < 0.05).

Fig. 2. (A) Representative images of live/dead assay and phase contrast images of CFs seeded on the aligned scaffolds at day 1 of culture (Live cells: green stain, Dead cells: red stain). (B) Quantified viability results obtained at days 1, 3 and 5 of culture. (C) DNA quantification of CFs cultured for 6 h on the aligned and random scaffolds. (D) Normalized CFs metabolic activity determined by Alamar Blue assay at days 3 and 5 of culture. (*: P < 0.05). (For interpretation of the references to color in this figure legend, the reader is referred to the web version of this article.)

Fig. 5. Representative fluorescence images of CMs immunostained for (A) Sarcomeric a-acti and random scaffolds. (For interpretation of the references to color in this figure legend, th

Fig. 3. (A) Effects of physical and chemical properties of scaffolds on the on differentiation of CFs. Low PGS content along with fiber alignment increased the number of a-SMAe positive CFs. Overlaid images of CFs cultured for 5 days on random and aligned scaffolds stained for a-SMA (red), F-actin (green) and DAPI (blue). (B) Quantification of a-SMA expressing cells cultured on the aligned and random scaffolds on days 1 and 5 of culture. (C) SirCol assay analysis, confirming that the amount of the deposited collagen per sample significantly increased on A(Gelatin) scaffold after 5 days of culture.(þand *:Compared to control and other scaffolds, respectively.(*and þ: P < 0.05)). (For interpretation of the references to color in this figure legend, the reader is referred to the web version of this article.)

Fig. 4. Effects of scaffold composition and architecture on the morphology, alignment and elongation of CFs. (A) F-actin filaments and nuclei stained CFs with phalloidin and DAPI on the random and aligned scaffolds. Main panels indicate the orientation and morphology of cells. Insets: higher magnification images showing elongated stress fibers within the cells cytoplasm increasing with lower PGS content. Quantified (B) alignment and (C) elongation of CFs on each scaffold. Cell alignment was significantly higher on A(PGS:2Gelatin) scaffold compared to A(Gelatin) scaffold (*: P < 0.05).

Frequently asked questions

Q1. What is the role of the scaffolds in the development of cardiac tissue?

Since extremely stiff substrates inhibit the contractile properties of cardiac cells, relatively elastic scaffolds (similar to native tissue tensile modulus w 54e240 kPa) could potentially provide a suitable microenvironment to mimic the native myocardial tissue [9,37,38]. 

Q2. What are the contributions mentioned in the paper "Pgs:gelatin nanofibrous scaffolds with tunable mechanical and structural properties for engineering cardiac tissues" ?

In this study, the authors utilized an electrospinning approach to fabricate elastomeric biodegradable poly ( glycerol sebacate ) ( PGS ): gelatin nanofibrous scaffolds with a wide range of chemical composition, stiffness and anisotropy. Furthermore, the authors studied attachment, proliferation, differentiation and alignment of neonatal rat cardiac fibroblast cells ( CFs ) as well as protein expression, alignment, and contractile function of cardiomyocyte ( CMs ) on PGS: gelatin scaffolds with variable amount of PGS. Overall, their study suggests that the aligned nanofibrous PGS: gelatin scaffold support cardiac cell organization, phenotype and contraction and could potentially be used to develop clinically relevant constructs for cardiac tissue engineering. 

Q3. What is the role of CMs in synchronized beating?

synchronized beating of CMs is directly coupled to the expression of gap junctions [62], immunostaining results confirmed the suitability of the aligned blended scaffolds to induce synchronized beating of the cells. 

Q4. What was the spectroscopic method used to determine the chemical composition of the scaffolds?

Fourier-transform infrared spectroscopy (FTIR) (Bruker Optics, MA, USA)was performed over a range of 500e4000 cm 1 and resolution of 2 cm 1 to verify the chemical composition of the scaffolds. 

Q5. Why do the authors expect higher proliferation rate on rigid scaffolds?

In addition, the authors expect that higher proliferation rate on rigid scaffolds (Gelatin) is mainly due to the differentiated state of the cells to myofibroblasts [50]. 

Q6. How many times were the goat anti-rabbit secondary antibody added to the samples?

The sampleswerewashed three times inDPBS, and a1:200dilution of theAlexa Fluor-598 conjugatedgoat anti-rabbit secondary antibody was added. 

Q7. What is the effect of the CMs coupling with their neighboring cells?

The ventricle myocardium posses a highly aligned and organized architecture along with contractile ability as the result of CMs coupling with their neighboring cells [61]. 

Q8. What are the main factors that affect the degradation of the scaffolds?

In addition to chemical factors, topographical cues such as pore size and alignment of the fibers are expected to affect the degradation profile of the scaffolds. 

Q9. What is the reason for the higher cell attachment on the scaffolds?

higher cell attachment on the scaffolds with higher PGS content (softer substrate) is expected to be mainly dominated by the smaller fiber size, higher surface-to-volume ratio, which ultimately enhances protein adsorption as well as selective protein secretions as a favorable substrate for cell attachment [45]. 

Q10. What is the effect of the pore size of pure gelatin on the degradation of the scaffold?

The larger pore size of pure gelatin scaffold likely accelerated the hydrolysis and the weight loss of the scaffold compared to their blended analogs due to the increased contact area and permeability of the scaffolds with DPBS. 

Q11. What is the reason for the increased strength and stiffness of random scaffolds?

according to previous results, the increased fiber size within random scaffolds reduced their strength and stiffness since the scaffolds displayed bulk-like properties making the stretching of the polymer chains more difficult [41,43]. 

Q12. What is the reason for the higher stiffness of the scaffolds?

In addition to chemical composition, the architecture of the scaffolds also influenced their mechanical properties; within both dry and wet states, aligned fibrous scaffolds exhibited notably higher stiffness and strength compared to the random ones, while elongation remained unaltered (Table 1). 

Q13. What is the effect of PGS content on cellular alignment?

according to mechanical characterization of the scaffolds (Table 1), increasing PGS content resulted in higher elasticity of the matrix and lower resistance to deformation therefore leading to the formation of thinner stress fibers within cytoskeleton of CFs. 

Q14. Why do the authors expect higher levels of a-SMA expression on pure gelatin scaffolds?

the authors anticipate that higher level of a-SMA expression along with collagen deposition on pure gelatin scaffolds compared to the blended ones, specifically 2PGS:Gelatin, is mainly due to fairly higher stiffness of the matrix. 

Q15. How many goat antimouse secondary antibodies were added to the samples?

The scaffolds were then washed three times in DPBS and a 1:200 dilution of Alexa Fluor-488 conjugated goat antimouse secondary antibody for sarcomeric a-actinin, Alexa Fluor-594 goat antimouse secondary antibody for troponin I, and Alexa Fluor-594 goat anti-rabbit secondary antibody for Cx-43 were added to samples. 

Q16. What was the tensile strength of the PGS:gelatin scaffolds?

Hydrophilic properties of the developed PGS:gelatin scaffolds (n¼ 3) were determined by water contact angle measurement using the static sessile drop technique. 

Q17. What is the significance of a-SMA expression in CFs?

in this study, the authors also quantified a-SMA expression, which is a myofibroblast specific marker, along with actin cytoskeletal organization to assess the CFs differentiation on the developed scaffolds (Fig. 3(A,B)). 

Q18. What is the importance of maintaining the balance of both cell types?

maintaining the balance of both cell types is important to ensure proper physiological properties of the engineered tissue constructs.