Optimizing Extracellular Vesicle Delivery Using a Core-Sheath 3D-Bioprinted Scaffold for Chronic Wound Management

Summary

This study presents a protocol for fabricating core-sheath 3D-bio-printed scaffolds for chronic wound healing. Extracellular vesicles are isolated from mesenchymal stem cells, and loaded into the core (alginate) with the sheath made from carboxymethyl cellulose and alginate lyase. This design allows controlled scaffold degradation and efficient EVs release.

Abstract

This study outlines a detailed protocol for the fabrication of core-sheath 3D-bioprinted scaffolds designed to enhance chronic wound healing. The protocol involves isolating extracellular vesicles (EVs) from mesenchymal stem cells (MSCs), known for their regenerative and immunomodulatory properties. These EVs are then incorporated into a unique scaffold structure. The scaffold features a core composed of alginate loaded with EVs, surrounded by a sheath made of carboxymethyl cellulose and alginate lyase. This innovative design ensures controlled scaffold degradation while promoting efficient and controlled release of EVs at the wound site. The protocol covers key steps, including the preparation and characterization of the EVs, the formulation of bio-inks for 3D bioprinting, and the optimization of printing parameters to achieve the desired core-sheath architecture. By combining structural integrity and bioactivity, the scaffold aims to address the limitations of conventional wound dressings, offering a targeted approach to accelerate tissue regeneration and reduce inflammation in chronic wounds. This method provides a reproducible and scalable strategy for developing advanced biomaterials with potential clinical applications in chronic wound management. The protocol also highlights critical considerations for achieving consistent results, ensuring adaptability for future therapeutic applications.

Introduction

Chronic wounds, often linked to excessive inflammation, require timely management to prevent serious complications like infections and tissue necrosis, which can lead to amputations. Despite advancements, current treatments remain costly, inconvenient, have side effects, and have limited efficacy, highlighting the need for more curative dressings^1,2,3. The development of a new generation of wound dressings specifically designed for chronic wounds is essential to address these challenges. Moreover, the complex nature of wound healing demands dressing materials with a range of ....

Protocol

The animal research was conducted in full accordance with the ethical standards established by the National Committee of Bioethics and the Animal Ethics Committee of the University of Nizwa. Ethical approval for this study was granted under clearance ID: VCGSR, AREC/01/2023. All animals were housed under standard laboratory conditions, ensuring optimal environmental controls, proper nutrition, and comprehensive care to safeguard their welfare throughout the study. All procedures involving animals adhered strictly to institutional policies, international animal care standards, and the ARRIVE guidelines.

1. Cell culture

Discussion

A pivotal aspect of the protocol is the core-sheath scaffold design, which is essential for achieving efficient EVs delivery. The design incorporates Alg as the core material and a combination of CMCh with Alglyase as the sheath. This setup facilitates controlled and rapid release of EVs. The core material, Alg, encapsulates the EVs, ensuring their protection and localized delivery. The sheath, composed of CMCh and Alglyase, enables the accelerated degradation of the Alg core, which is critical for the timely release of .......

Materials

Name	Company	Catalog Number	Comments
23 G Purple precision conical Nozzle	Cellink	KT0000002000	To provide precise extrusion of bioinks with minimal clogging
Alginate lyase (AlgLyase)	Sigma Aldrich	A1603-100MG	Algyase is an enzyme that degrades alginate.
Amicon Ultra Centrifugal Filter, 30 kDa MWCO	Merck	UFC9030	Used to wash PKH-26 labeled-EVs
BCA assay Kit	Thermo Scientific	10678484	To determine the protein/EVs concentration
Bioprinting System	Regemat	V1	To fabricate core-sheath scaffold
Bovine serum albumin (BSA)	sigma-aldrich	05470-5G	To stop PKH 26 reaction
Calcium chloride	Sigma Aldrich	C3306-100G	To crosslink and stabilize bioinks in tissue engineering
Centrifuge	Sigma	2-16P	Used for EVs isolation
Centrifuge 5810 R	Eppendorf	22625101	Used for cell culture
Class II Biological Safety Cabinet	Telstar	Bio II Advance	Cell culture
CryoCube F570 Series - ULT Freezer	Eppendorf	F571240035	To store EVs
fluorescent microscope	OLYMPUS	IX73P1F	Used to check the residual PKH-26 in the filtrate
Gentamicin (50 mg/mL)	Thermofisher	15750	Antibiotic for cell culture media
GlutaMAX-I CTS, (100X), liquid	Thermofisher	A12860	Cell culture media supplement
HCl	Sigma Aldrich	7647-01-0	Buffer preparation
HEPES	Carl Roth	Art. No. 6763.3	Buffer preparation
High viscous carboxymethyl cellulose (CMCh)	BDH	27929 4T	CMCh is a water-soluble cellulose derivative.
Incubator	New Brunswick	NB-170R	Cell culture
Invivo imaging	PerkinElmer	IVIS Lumina XRMS Series III	To track EVs release, in vivo
Magnet stirer	SalvisLAB	MC35	For Bioinks preparation
miRCURY Exosome Kits for Exosome Isolation	Qiagen	76743	Evs isolation
NaOh	Daejung	1310-73-2	Buffer preparation
phosphate buffered saline(PBS)	Thermo Scientific	J61196.AP	Cell culture
PKH 26	MCE	154214-55-8	Red fluorescent dye for labeling theEVs
Sodium alginate (Alg)	Sigma Aldrich	A0682-100G	Natural polysaccharide derived from brown seaweed.
Sodium chloride (NaCl)	Carl Roth	Art-Nr-P029.1	Buffer preparation
StemPro BM Mesenchymal Stem Cells	Thermofisher	A1382901	Mesenchymal stem cells
StemPro MSC SFM XenoFree	Thermofisher	A1067501	Cell culture media
Trypsin 0.25%	Thermofisher	25050014	Cell dissociation
Vortex-Mixer	Daihan Scientific	VM-10	Used to mix precipitation buffer with the conditioned media