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 properties, including moisturization, flexibility, adhesion, bioactivity, and biodegradability⁴. This study aims to develop a bioengineered wound dressing that integrates extracellular vesicles (EVs) with a core-sheath 3D-bioprinted scaffold to provide a controlled therapeutic environment and accelerate chronic wound healing.

EVs derived from stem cells aid chronic wound healing by promoting anti-inflammatory responses, cell growth, migration, and blood vessel formation⁵. Additionally, EVs can deliver bioactive molecules, including small molecule drugs, gene and protein constructs for chronic wound management⁶. Moreover, their ability to protect cargo from enzymatic degradation improves the stability and bioavailability of therapeutic agents, offering distinct advantages over conventional growth factors and small molecule drugs, which often degrade rapidly in vivo⁷. Despite these advantages, the efficient and sustained delivery of EVs to target tissues remains a significant challenge.

3D-bioprinting scaffolds can serve as a delivery platform for EVs to boost their therapeutic effects⁸. These scaffolds mimic natural cellular environments and allow for the controlled release of EVs^9,10. They also protect EVs from degradation, enhancing the stability of their microRNAs and proteins¹¹. Han et al. demonstrated that EVs can be effectively released from 3D bioprinted GelMA scaffolds. This release led to improved cell attachment and enhanced gene expression related to mechanotransduction pathways in human buccal fat pad mesenchymal stem cells (hBFP-MSCs) seeded onto the scaffolds¹². Born et al., by optimizing the concentration of the crosslinker, achieved a controlled release of the EVs. This approach has demonstrated efficacy in promoting angiogenesis and offers a promising method for the regulated delivery of EVs¹³.

Core-sheath 3D-bioprinting enables the creation of complex, multi-material structures by printing a core material encased in a sheath. The core can include cells, growth factors, or drugs, while the sheath offers mechanical support and protection or acts as a barrier. This method has applications in tissue engineering and regenerative medicine, such as developing vascular networks, mimicking natural tissue structures, and creating drug delivery systems. It allows precise control over material distribution and composition, enhancing the functionality and biological relevance of the constructs. Compared to alternative techniques, core-sheath 3D bioprinting provides precise control over material distribution and composition, improving the functionality and biological relevance of the constructs^14,15.

Engineered degradation in wound dressings offers benefits such as reduced discomfort during changes, a moist environment for healing and infection control, timely therapeutic delivery, and optimal tissue regeneration^16,17,18. Alginate (Alg) and carboxymethyl cellulose (CMCh) hydrogels are biocompatible and effective for delivering extracellular vesicles (EVs) to wounds, enhancing healing through cellular communication and inflammation reduction¹⁸. In this study, EVs were integrated into a core of Alg, while a sheath of CMCh and AlgLyase (AlgLyase) was used to enable rapid dressing degradation and EVs delivery. This core-sheath design facilitates the rapid release of EVs in response to scaffold degradation, enhancing their therapeutic efficacy and addressing the limitations of existing chronic wound treatments. The primary objective of this study is to develop a bioengineered dressing that enhances wound healing by integrating controlled EVs release with a responsively degradable scaffold, ultimately improving the treatment outcomes for chronic wounds.

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

1. Retrieve a vial of MSCs (passage #2) from liquid nitrogen storage and handle it with aseptic techniques to prevent contamination. Rapidly thaw the cells in a 37 °C water bath, ensuring to remove them as soon as a small ice fragment remains.
2. Prepare a complete medium including MSC SFM Basal Medium, supplemented with 1% (v/v) MSC SFM XenoFree supplement, 1% (v/v) GlutaMAX, and 0.01% (v/v) Gentamicin. Add 1 mL pre-warmed (37 °C) complete medium at a rate of 3 to 5 drops every 5 s, to the vial and then gently mix it. Transfer the entire contents of the MSCs vial into a 15 mL conical tube containing 4 mL pre-warmed complete medium, under aseptic conditions in a Class II biosafety cabinet.
3. Centrifuge the cells at 200 x g for 5 min at room temperature. Ensure the centrifuge is balanced properly.
4. Aspirate the supernatant and resuspend the cells in 1 mL of pre-warmed complete media. Then, transfer the cells into a T25 flask containing 4 mL of complete medium.
5. Gently tilt the flask to ensure the cells are evenly distributed. Incubate the flask at 37 °C with 5% CO₂.
6. Change the medium every 2 days, replacing it with fresh, pre-warmed, complete medium. Use gentle pipetting to avoid cell disruption. Upon reaching 70%-80% confluence, aspirate the spent medium from the T25 flask.
7. Wash the cells with 3 mL of fresh PBS to remove residuals. Ensure complete coverage of the flask during the wash.
8. Add 1 mL of 0.25% trypsin solution to the T25 flask, incubate it at 37 °C for 3-7 min, and carefully monitor the detachment under the microscope at 4x magnification.
9. Tap the flask gently if required to ensure complete cell detachment. Add pre-warmed complete medium to the flask and pipette up and down over the surface to help detach the cells. Then, transfer the cell suspension into a 15 mL centrifuge tube.
10. Centrifuge the tube at 200 x g for 5 min at room temperature. Suspend the cell pellet in a fresh, complete medium and count the cells using a Neubauer hemocytometer. Transfer the cells to a T75 flask. Ensure a seeding density of 5,000 cells/cm² for optimal growth.
11. Incubate the cells at 37 °C with 5% CO₂. After 72 h of cell incubation, collect the conditioned media from cells for EVs isolation. Use immediately after collection.

2. EVs isolation

1. Centrifuge the 13 mL of the collected conditioned media at 800 x g for 15 min to remove cellular debris. Filter the supernatant gently using a 0.22 µm syringe filter to remove large particles.
2. Add 5 mL of precipitation buffer to the filtered conditioned media and vortex thoroughly to mix. Ensure the solution is homogenous.
3. Incubate the mixture overnight at 4 °C to allow the EVs to precipitate. Centrifuge the mixture at 3,220 x g for 30 min at 20 °C. Check the balance of the tubes.
4. Carefully discard the supernatant without disturbing the pellet. Centrifuge the pellet once again at 3,220 x g for 5 s to remove residual liquid.
5. Gently pipette the EV pellet in 200 µL of PBS to avoid damage to the EVs. Measure the EVs protein concentration following the manufacturer's instructions (BCA Protein Assay Kit).
6. Perform Western blotting to detect EV-specific markers (CD63, CD81, and CD9), to verify EV phenotype¹⁸. The analysis confirmed the presence of these markers, validating the EVs identity¹⁹.
7. To minimize the risk of RNase contamination, it is recommended to use it directly without additional storage. In case it is needed, store the EVs at -80 °C for further use. Aliquot the EVs suspension into 40 µL volumes to avoid repeated freeze-thaw cycles.

3. EVs labeling with PKH-26

1. Buffer preparation
   1. Dissolve 0.238 g of HEPES in approximately 90 mL of sterile ultra-pure water in a sterile container. Use freshly prepared HEPES solution. Add 0.85 g of NaCl to the HEPES solution.
   2. Adjust the pH to 7.4 using 1 M NaOH or HCl, as required, using a calibrated pH meter. Add deionized water to bring the total volume to 100 mL. Filter the solution through a 0.22 µm filter to sterilize it.
2. Dilute 4 µL of PKH-26 dye in 1 mL of prepared buffer. Mix thoroughly by pipette to ensure homogeneity.
3. Resuspend purified EVs in 1 mL of PBS at a concentration of 700 µg/mL. Add 1 mL of the EVs suspension to 1 mL of the prepared PKH-26 solution. For the dye-only control group, add 1 mL of complete medium without EVs to 1 mL of the PKH-26 solution. All subsequent steps are performed for the control group as well to account for potential nonspecific aggregation or micelle formation.
4. Mix the EVs thoroughly by gently inverting the tube 10x-15x. Incubate the mixture for 3-5 min at room temperature, ensuring even exposure of EVs to the dye.
5. Prepare a 2 mL fresh 1% BSA solution using sterile ultrapure water and add it to the 2 mL of the resultant mixture to quench the labeling reaction. Mix gently to prevent aggregation.
6. Concentrate the PKH-26 labeled EVs according to the abovementioned protocol (steps 2.2-2.7). Resuspend the labeled EVs in 300 µL of PBS. Pipette the PKH-26 labeled EVs sample into a 30 kDa filter tube.
7. Centrifuge the sample at 3,220 x g for 30 min at 4 °C. During this step, the free PKH-26 dye and small molecules will pass through the membrane into the filtrate, while the PKH-26 labeled EVs will be retained in the retentate.
8. After the initial centrifugation, discard the filtrate and add 300 µL of PBS to the retentate. Gently resuspend the EVs in the PBS by pipetting up and down.
9. Centrifuge the sample again at 3,220 x g for 30 min at 4 °C to wash away any remaining free dye or small molecules.
10. Confirm the lack of PKH-26 in the filtrate solution by a fluorescent microscope. If any dye is detected, repeat the washing steps.
11. Collect the top solution by micropipette and remove the filter from the collection tube. Carefully invert the filter (turn it upside down).
12. Centrifuge the inverted filter at 800 x g for 5 min at 4 °C. This will help collect the retained PKH-26-EVs from the filter membrane into the new collection tube. Directly use the PKH-26-EVs for the next step.

4. 3D Bio-printing

1. Bioinks preparation
   1. Prepare a fresh 4.5% (w/v) sodium Alg solution using sterile ultra-pure water. Stir overnight at 60 °C to allow the solution to dissolve completely.
   2. Dissolve CMCh in sterile ultra-pure water to achieve a 3.8% (w/v) solution. Prepare fresh. Stir overnight at 60 °C to dissolve completely.
   3. Centrifuge the prepared bioinks at 3,220 x g for 10 min at 25°C to remove any bubbles that may interfere with the printing process.
   4. Mix the prepared 3 mL of Alg solution with the labeled EVs or the dye-only control using a syringe mixer to achieve the 0.01% (w/v) concentration of EVs. Ensure even distribution by gentle mixing.
   5. Using a syringe mixer, combine 1 mL of CMCh with a freshly prepared AlgLyase solution in sterile ultra-pure water to achieve a final concentration of 0.5 mU/mL.
2. As depicted in Figure 1A, simultaneously load the Alg/Exo bioink into the core part and the CMCh/AlgLyase bioink into the sheath part of the core/sheath syringe setup.
3. Allow the bioinks to rest for 15 min before printing.
4. 3D-bio-printer setup
   1. Using the 3D-bioprinter software, create the scaffold structure by selecting a Cylindrical Shape with a Diagonal Infill Pattern. For this purpose, set the cylinder diameter and height to 20 mm, and 1.1 mm, respectively. Configure the pore size to 1 mm.
   2. Set the core and sheath nozzle pumping speeds to 1 mm/s with a thickness of 0.25 mm per layer and set the moving speed to 6 mm/s. Print four layers with a thickness of 0.25 mm per layer at room temperature.
5. Start to print on polyester film.
6. Use the humidifier with aerosol calcium chloride (2.2%) to cross-link the scaffold during the extrusion process. Position the humidifier nozzle approximately 20 cm away from the extrusion head to ensure effective cross-linking without compromising the scaffold structure. For further-crosslinking, immerse the scaffold in calcium chloride solution (2.2%) for 10 min.
7. Rinse the scaffold 3x with sterile ultra-pure water to remove any excess calcium chloride and non-bound bioink.
8. Ensure that the scaffold is stored in a sterile environment at 4 °C to maintain the integrity and functionality of the scaffold for up to three months.

5. Tracking EVs release

1. Creation of circular cutaneous wound model
   1. Anesthetize male Akita heterozygous diabetic mice (8 weeks) by administering an intraperitoneal injection of Ketamine (70 mg/kg) and Xylazine (10 mg/kg). Confirm proper anesthetization by assessing the absence of reflex responses (e.g., toe pinch) and monitor the respiratory rate. To prevent corneal dryness during anesthesia, apply sterile veterinary ophthalmic ointment to the eyes.
   2. Prepare the dorsal skin area by first shaving it using an electric clipper. Avoid skin irritation or injury. Sterilize the shaved area using a povidone-iodine solution.
   3. Using a sterile seizer, create a 6 mm circular full-thickness cutaneous wound on the dorsal surface of each mouse.
   4. Gently place the 3D bioprinted scaffold containing PKH-labeled EVs directly onto the wound bed. Ensure the scaffold fully covers the wound surface with minimal air pockets by pressing lightly using sterile forceps. Ensure the animal is closely observed after the procedure and remains attended until it has fully regained consciousness and can maintain sternal recumbency.
2. Fluorescent imaging
   1. At 2 h, 4 h, 8 h, 24 h post-implantation, anesthetize the mice using isoflurane. For induction of anesthesia, place the mice in the chamber of the in vivo imaging system (IVIS) and expose them to 2%-3% isoflurane in oxygen. Apply ophthalmic ointment to the mice's eyes to prevent dryness. Once anesthetized, transfer the mice to the IVIS system and maintain them with 1%-2% isoflurane in oxygen delivered through the nose channels. Verify that the animals are fully anesthetized and stable before proceeding with imaging.
   2. Use the IVIS system to capture the fluorescent signals from the PKH-labeled EVs released from the scaffold. In the imaging wizard, select the Fluorescence Imaging option and activate the excitation and emission filters for the PKH dye. Adjust the camera settings, including the field of view and subject height, to optimize signal detection. Ensure consistent positioning across all imaging time points to enable accurate comparisons. Start acquiring images and save the resulting data.
   3. Quantify the fluorescent signal intensities using the IVIS software. This will allow for the tracking of EVs release over time.

Results

The in vivo release of EVs from both the Alg-EVs/CMCh and Alg-EVs/CMCh-AlgLyase scaffolds is depicted in Figure 1B,C. As anticipated, the Alg-EVs/CMCh-AlgLyase scaffold exhibited a more rapid release profile compared to Alg-EVs/CMCh, particularly at the 2 h and 4 h time points. The release of EVs from hydrogels is governed by a combination of physicochemical mechanisms, including diffusion, swelling, erosion, and degradation²⁰. By leveraging ...

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