Ethanol treatment of nanoPGA/PCL composite scaffolds enhances human chondrocyte development in the cellular microenvironment of tissue-engineered auricle constructs

Abstract

A major obstacle for tissue engineering ear-shaped cartilage is poorly developed tissue comprising cell-scaffold constructs. To address this issue, bioresorbable scaffolds of poly-ε-caprolactone (PCL) and polyglycolic acid nanofibers (nanoPGA) were evaluated using an ethanol treatment step before auricular chondrocyte scaffold seeding, an approach considered to enhance scaffold hydrophilicity and cartilage regeneration. Auricular chondrocytes were isolated from canine ears and human surgical samples discarded during otoplasty, including microtia reconstruction. Canine chondrocytes were seeded onto PCL and nanoPGA sheets either with or without ethanol treatment to examine cellular adhesion in vitro. Human chondrocytes were seeded onto three-dimensional bioresorbable composite scaffolds (PCL with surface coverage of nanoPGA) either with or without ethanol treatment and then implanted into athymic mice for 10 and 20 weeks. On construct retrieval, scanning electron microscopy showed canine auricular chondrocytes seeded onto ethanol-treated scaffolds in vitro developed extended cell processes contacting scaffold surfaces, a result suggesting cell-scaffold adhesion and a favorable microenvironment compared to the same cells with limited processes over untreated scaffolds. Adhesion of canine chondrocytes was statistically significantly greater (p ≤ 0.05) for ethanol-treated compared to untreated scaffold sheets. After implantation for 10 weeks, constructs of human auricular chondrocytes seeded onto ethanol-treated scaffolds were covered with glossy cartilage while constructs consisting of the same cells seeded onto untreated scaffolds revealed sparse connective tissue and cartilage regeneration. Following 10 weeks of implantation, RT-qPCR analyses of chondrocytes grown on ethanol-treated scaffolds showed greater expression levels for several cartilage-related genes compared to cells developed on untreated scaffolds with statistically significantly increased SRY-box transcription factor 5 (SOX5) and decreased interleukin-1α (inflammation-related) expression levels (p ≤ 0.05). Ethanol treatment of scaffolds led to increased cartilage production for 20- compared to 10-week constructs. While hydrophilicity of scaffolds was not assessed directly in the present findings, a possible factor supporting the summary data is that hydrophilicity may be enhanced for ethanol-treated nanoPGA/PCL scaffolds, an effect leading to improvement of chondrocyte adhesion, the cellular microenvironment and cartilage regeneration in tissue-engineered auricle constructs.

Electrospun Poly(lactic acid) and Silk Fibroin Based Nanofibrous Scaffold for Meniscus Tissue Engineering

“…In the meantime, the properties such as smaller fiber diameter, more amino groups, and more hydrophilicity of electrospun PLA/SF fibrous scaffolds also have influenced the interaction between cells and scaffolds [14,32,42,57]. Treatment of scaffolds with 70% ethanol can increase the cell viability [65]. Alcohol treatment can reduce the toxicity of the residued electrospining solvents after fabricating because it effectively eliminates bacteria and viruses, and it can dissolve the chloroform and formic acid easily [66][67][68].…”

Combining bioengineered human skin with bioprinted cartilage for ear reconstruction

“…Through the use of tissue-engineered skin to cover a biofabricated autologous auricular graft, the need to harvest costal cartilage as well as the dependence on the cutaneous cover could be overcome. Strategies to generate auricular grafts range from combinations of autologous auricular chondrocytes with various scaffold materials, hydrogels, to cells alone (21)(22)(23)(24).…”

“…Since the Vacanti mouse ( 25 ) and the use of chondrocyte-seeded polyglycolic acid (PGA) meshes, several groups have aimed to produce auricular cartilage grafts in vivo ( 21 , 23 , 26 ). The polycaprolactone (PCL) and polylactic acid (PLA) reinforced PGA meshes bear the benefit that they can be manufactured relatively stiff to sustain in vivo forces acting upon them and therefore, resist deformation to a higher degree.…”

The significance of nanofiber polyglycolic acid for promoting tissue repair in a rat subcutaneous implantation model

“…8 In general, as fiber diameters decrease, the surface area of these nanofibers increases exponentially, reducing the overall amount of PGA, and shortening the degradation time of the scaffold material in the body. 5,9,10 In addition, at optimum spacing of these nanofibers, cells are more likely to migrate and proliferate in these spaces. Although the complexity of cellular responses to biomaterials needs to be examined to promote tissue repair, the detailed mechanism has not been elucidated.…”

“…To overcome these problems, we investigated a technique to form PGA into nanofibers (nanofiber‐based PGA, nano‐PGA) with diameters and thickness similar to those of natural extracellular matrix 8 . In general, as fiber diameters decrease, the surface area of these nanofibers increases exponentially, reducing the overall amount of PGA, and shortening the degradation time of the scaffold material in the body 5,9,10 . In addition, at optimum spacing of these nanofibers, cells are more likely to migrate and proliferate in these spaces.…”