Electrospinning and decellularized matrix combination

Decellularized extracellular matrix (ECM) material contains a range of proteins and other extracellular matrix (ECM) components which not only facilitates cell adhesion and proliferation, it may also drive stem cell differentiation. Electrospinning facilitates in the introduction of decellularized ECM by acting as a carrier or to provide the mechanical support the decellularized ECM. There are a few ways in which decellularized materials may be used with other materials under electrospinning context. The first is to mix the decellularized materials with the material solution and electrospinning to form fibers containing the mixture. The other method is to form fibers first using the material and mixed the scaffold with the decellularized materials. In either case, a small amount of decellularized matrix material may be used to generate a much larger volume of scaffold. Another method is to culture cells on electrospun scaffold for an extended period of time such that extracellular matrix are deposited within followed by decellularization.

Decellularized matrix from existing tissues is generally the most common source for loading with electrospun fibers. Grant et al (2019) constructed a scaffold using a blend of solubilized decellularized liver extracellular matrix (hLECM) and poly-L-lactic acid (PLLA) solution and electrospinning. THLE-3 hepatocytes were cultured on hLECM/PLLA scaffold, polymer only scaffold and polymer scaffold containing individual ECM component (Collagen I, Laminin-521 and Fibronectin) for comparison. hLECM and all the individual proteins were solubilized prior to blending with PLLA solution. The presence of electrospun PLLA fibers increases the Young's Modulus of the decellularized ECM scaffold thereby providing mechanical support for hepatocytes. All electrospun scaffolds were able to maintain hepatocyte growth and albumin production. However, only hLECM cultured on hLECM/PLLA scaffold showed albumin expression increasing over time. On day 16, upregulation of cytochrome P450s (CYP1A1, CYP1A2 and CYP3A4), a family of enzymes involved in metabolism of drugs and other toxic compounds in the liver, were shown on hepatocytes cultured on hLECM/PLLA scaffold. Collagen IV was downregulated over time on all single protein scaffolds but increases for hLECM/PLLA scaffold. This study demonstrated the role of hLECM in introducing the complex biological cue to influence the response of hepatocytes and its synergy with electrospun scaffold in a lab-based environment. Goyal et al (2017) showed the possibility of using another source of decellularized ECM. This alternative source comes from cells that were first cultured on the electrospun scaffold. Poly(desamino tyrosyl-tyrosine carbonate) (PDTEC) was co-electrospun with poly(ethylene glycol) (PEG) used as a sacrificial polymer. The PEG was removed to create large pores size within the scaffold to facilitate cell penetration. Cells were then cultured on the electrospun scaffold and fibronectin fibrils were subsequently deposited by the cells. When sufficient ECM was deposited by the cells, decellularization was carried out to remove the cells. The resultant hybrid scaffold was shown to promote cell adhesion and proliferation. The advantage of this method is that the source of decellularized matrix can be controlled. Grant et al (2018) used sacrificial, transfected cell line (5637 human urinary bladder epithelials cells) for production of the vector derived fibronectin on an electrospun poly-L-lactic acid (PLA) scaffold for liver tissue engineering before decellularizing. HepG2 liver cells were cultured on hybrid construct, pure PLLA scaffold and PLLA scaffold with non-transfected cells for comparison and validation. HepG2 liver cells cultured on hybrid construct showed gene expression of key hepatic genes altered on day 5 but the other two scaffolds only influence CYP1A2 and COL4A1 expression. Their study provides evidence on the advantage of using cells to biofunctionalize electrospun scaffolds for specific applications. The structure and integration of the first deposited ECM with the electrospun scaffold can be maintained.

It may be difficult or impossible to maintain the structural integrity of the ECM following decellularizing of the organ or tissue. However, to take advantage of the presence of decellularized ECM, one method is to incorporate it into a matrix. A few studies have been carried to investigate the advantage of electrospinning fibers with decellularized ECM mixed into its matrix. Baiguera et al (2014) incorporated rat decellularized brain ECM with gelatin for the purpose of neural tissue engineering. They showed that just with 1% w/w of decellularized rat brain extracellular matrix in the gelatin fiber mixture was able to show differentiative potential. Gibson et al (2014) used different decellularized ECM particles in the nanofiber matrix to determine its influence on stem cell differentiation. Decellularized ECM particles of bone, cartilage and fat in the fiber were found to promote osteogenesis while those of spleen and lung reduce osteogenesis. In agreement with the study by Baiguera et al (2014), a 1% w/w of bone, cartilage and fat decellularized ECM was able to illicit a response from the stem cell.

Following decellularizing, the resultant ECM is often soft. Electrospun nanofibers may be used as a structural support to maintain the form of the decellularized ECM. Hung et al (2014) suggested creating a hybrid structure comprising of a poly-L-lactide (PLLA) coil over a metal tube with decellularized mucosa wrapped onto the coil followed by electrospinning to coat fibers onto the decellularized mucosa. This way, a tracheal scaffold can be constructed with the electrospun coating acting as a porous mesh that holds the structure together.

Alizarin red staining of hASCs in monolayer culture on fibrous scaffolds containing ECM derived from the indicated tissues. In each case, the diameter of the indicated circles is 5mm. [Gibson et al. BioMed Research International, vol. 2014, Article ID 469120, 13 pages, 2014. This work is licensed under a Creative Commons Attribution 3.0 Unported License.]

One method of introducing extracellular matrix (ECM) into the graft is by first implanting the scaffold subcutaneously and using the body as the bioreactor for cell infiltration and deposition of ECM. Su et al (2022) demonstrated this method using a rabbit carotid artery model and electrospun Poly (L-lactic-co-ε-caprolactone) (PLCL) /polyurethane (PU) small diameter vascular graft. In this study, PLCL/PU graft was first implanted into the abdominal subcutaneous area for 4 weeks. Prior to implantation, a silicone tube was placed in the lumen of electrospun PLCL/PU graft to prevent deposition and occlusion of the graft. After 4 weeks of embedding, the PLCL/PU graft with the silicone tube was removed. The silicone tube was extracted from the PLCL/PU graft and the graft was subsequently stored in 75% ethanol solution to kill the cells. This PLCL/PU biotube was covered with collagen on the inner and outer surfaces. The efficacy of the biotube and pure PLCL/PU graft was tested in a rabbit carotid artery model. The PLCL/PU grafts group were all occlusive within 4 weeks while the biotubes group was able to maintain a 62.5% primary patency after 12 weeks of implantation. The biotubes after 12 weeks of implantation showed smooth muscle cells (SMCs) arranged in layers while endothelial cells (EC) were present in the luminal surface although less than the native rabbit aorta EC count. Larger count of SMCs in the biotube compared to native rabbit aorta cell count may suggest rising risk of intimal hyperplasia although longer studies would be needed to determine this.