Fabrication of Decellularized Cartilage-derived Matrix Scaffolds

Podsumowanie

Decellularized cartilage-derived scaffolds can be used as a scaffold to guide cartilage repair and as a means to regenerate osteochondral tissue. This paper describes the decellularization process in detail and provides suggestions to use these scaffolds in in vitro settings.

Streszczenie

Osteochondral defects lack sufficient intrinsic repair capacity to regenerate functionally sound bone and cartilage tissue. To this extent, cartilage research has focused on the development of regenerative scaffolds. This article describes the development of scaffolds that are completely derived from natural cartilage extracellular matrix, coming from an equine donor. Potential applications of the scaffolds include producing allografts for cartilage repair, serving as a scaffold for osteochondral tissue engineering, and providing in vitro models to study tissue formation. By decellularizing the tissue, the donor cells are removed, but many of the natural bioactive cues are thought to be retained. The main advantage of using such a natural scaffold in comparison to a synthetically produced scaffold is that no further functionalization of polymers is required to drive osteochondral tissue regeneration. The cartilage-derived matrix scaffolds can be used for bone and cartilage tissue regeneration in both in vivo and in vitro settings.

Wprowadzenie

Articular cartilage defects in the knee caused by traumatic events can lead to discomfort, and above all can have a large impact on the lives of the young and active population^1,2,3. Moreover, cartilage damage at a young age may lead to a more rapid onset of osteoarthritis later in life⁴. Currently, the only salvage therapy for generalized osteoarthritis of the knee is joint replacement surgery. As cartilage is a hypocellular, aneural, and avascular tissue, its regenerative capacity is severely limited. Therefore, regenerative medicine approaches are sought after to aid and stimulate the regenerative capacity of the native tissue. For this purpose, scaffolds are designed and used as either a cell-carrier or as an inductive material that incites differentiation and regeneration of tissue by the body's native cells⁵.

Decellularized scaffolds have been widely studied within regenerative medicine⁶. It has had some success, for example, in aiding the regeneration of skin⁷, abdominal structures⁸, and tendons⁹. The advantage of using decellularized scaffolds is their natural origin and their capacity to retain bioactive cues that both attract and induce cell differentiation into the appropriate lineage required for tissue repair^6,10. Moreover, since extracellular matrix (ECM) is a natural biomaterial, and decellularization prevents a potential immune response by removing cellular or genetic content, issues regarding biocompatibility and biodegradability are overcome.

Cartilage-derived matrix (CDM) scaffolds have shown great chondrogenic potential in in vitro experiments when seeded with mesenchymal stromal cells¹¹. In addition, these scaffolds have shown the potential to form bone tissue through endochondral ossification on ectopic locations in in vivo settings¹². As CDM scaffolds guide the formation of both bone and cartilage tissue, these scaffolds may hold potential for osteochondral defect repair in addition to cartilage repair.

This article describes a protocol adapted from Yang et al. (2010)¹³ for the production of decellularized CDM scaffolds from equine stifle cartilage. These scaffolds are rich in collagen type II and devoid of cells, and do not contain any glycosaminoglycans (GAGs) after decellularization. Both in vitro and in vivo experiments on (osteo)chondral defect repair can be conducted using these scaffolds.

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Protokół

For this protocol, equine stifle cartilage was obtained from horses that had died from other causes than osteoarthritis. Tissue was obtained with permission of the owners, in line with the institutional ethical regulations.

NOTE: This protocol describes the fabrication of scaffolds from decellularized equine cartilage, which can be used for applications such as in vitro tissue culture platforms or for in vivo implantation in regenerative medicine strategies. The enzymatic treatment steps must be performed in the described chronological order.

1. Harvesting of Articular Cartilage from Donor (Cadaveric) Joints

1. Ahead of the harvesting step, prepare 1 L of cartilage washing solution, consisting of sterile phosphate-buffered saline (PBS), supplemented with 100 U/mL penicillin, 100 µg/mL streptomycin, and 1% (v/v) Amphotericin B.
2. Wear gloves and a lab coat during the entire harvesting procedure, since the donor can carry pathogens.
3. Obtain an intact (cadaveric) joint for cartilage isolation.
   NOTE: At this point, the skin around the joint is still intact. Equine stifle cartilage obtained from horse was used in this protocol, but other joints from other species can also be used.
4. Remove the skin that is located around the joint area of interest by using a scalpel and a surgical tweezer. In addition, remove excessive fat and muscle tissue if necessary, without damaging the underlying joint. Optionally, transfer the prepared cadaver to a biological safety cabinet.
5. To perform an arthrotomy, i.e., a separation of the joint, first define the location where the joint articulates by performing extension and flexion of the joint.
6. Carefully remove more layers of fat, muscles, and tendons with a scalpel and surgical tweezer to open up the joint area.
7. Make an incision to reach the joint cavity.
   NOTE: The joint cavity is filled with synovial fluid, which can drip from the cavity when performing a correct incision.
8. Continue to open up the joint completely by cutting through the tendons that keep the joint together.
9. Inspect the cartilage for any macroscopic damage.
   NOTE: If the articular cartilage does not have a glossy and smooth appearance or if evident blistering, clefts, or defects are present, discard the cartilage.
10. Use a sterile scalpel to remove the cartilage from the subchondral bone. Cut all the way down to the subchondral bone to also remove the deep zone of the cartilage (Figure 1). To prevent the cartilage from drying out, regularly drip cartilage washing solution on the cartilage.
    NOTE: At this time, the size of the cartilage slices does not matter.
11. Collect the cartilage slices in 50 mL tubes containing previously prepared cartilage washing solution (Figure 2A).
12. After collecting the cartilage from all the areas of interest, snap freeze the cartilage slices in liquid nitrogen for 5 min.
13. Transfer the cartilage slices into 50 mL tubes and immediately lyophilize the cartilage slices for 24 h in a freeze dryer. Set the freeze dryer at approximately 0.090 mbar while the ice condenser is -51 °C (Figure 2B).
14. Store the cartilage slices in a dry place at room temperature until further use.
    NOTE: Up to the point of scaffold formation, the solutions and cartilage do not have to be prepared and treated in a sterile way.

2. Creating Decellularized Cartilage Particles

1. Submerse the lyophilized cartilage slices in liquid nitrogen.
2. Directly grind the samples either manually or by a milling machine. When grinding the cartilage slices by hand, use a mortar and pestle and grind the slices for approximately 45 min until they are pulverized (Figure 2C and 2D). When grinding automatically, use a milling machine at its pre-set speed for a few seconds up to a minute, until the snap-frozen cartilage slices are pulverized.
   NOTE: Pre-cool the mortar or the grinding compartment of the milling machine by adding liquid nitrogen. When using a milling machine, make sure that all particles are ground, and that no particles stay in the bottom of the grinding compartment.
3. Sieve the cartilage particles to get rid of larger parts by using a sieve with a 0.71 mm mesh size.
4. Store the particles in a dry place at room temperature.

3. Enzymatic Decellularization with Trypsin 0.25%-EDTA

1. Prepare the digestion solution, consisting of 0.25% trypsin with EDTA supplemented with 100 U/mL penicillin, 100 μg/mL streptomycin, and 1% (v/v) Amphotericin B. Store the solution at 4 °C.
   NOTE: Trypin is a protease that degrades proteins residing in the cartilage tissue. This enzymatic step will open up the dense cartilage structure.
2. Fill the 50 mL tubes with the pulverized cartilage particles up to a volume of approximately 7.5 mL per tube.
3. Incubate the cartilage particles with the prepared digestion solution at 37 °C for 24 h in total, while refreshing the digestion solution every 4 h.
   1. First, add 30 mL of the digestion solution to each 50 mL tube that contains cartilage particles.
   2. Then, resuspend the particles by using a vortex or pipet so that all particles are exposed to the enzymatic solution.
   3. Incubate the samples for 4 h at 37 °C on a roller.
   4. To refresh the digestion solution, centrifuge the tubes for 20 min at 3113 x g to cause sedimentation of the cartilage particles. Discard the supernatant.
      NOTE: The supernatant will become clearer with every trypsin incubation period.
   5. Repeat step 3.3.1–3.3.4 until the particles have been incubated with the digestion solution for 6 cycles of 4 h.
4. After the final cycle, remove the supernatant after centrifuging and wash the particles in 30 mL of cartilage washing solution twice. Centrifuge the particles for 20 min at 3113 x g between each of the washes.

4. Enzymatic Decellularization with Nuclease Treatment

1. Prepare a 10 mM Tris-HCl solution at pH 7.5 in deionized water.
2. Add 50 U/mL deoxyribonuclease and 1 U/mL ribonuclease A to the Tris-HCl buffer, to obtain the nuclease solution.
   NOTE: This step is performed to specifically degrade deoxyribonucleases and ribonucleases.
3. To remove the cartilage washing solution from the cartilage particles (step 3.4), centrifuge for 20 min at 3,113 x g, and remove the supernatant.
4. Add 30 mL of the nuclease solution to the cartilage particles and stir the particles through the solution, making sure that all particles are exposed to the nuclease solution.
5. Incubate the samples on a roller for 4 h at 37 °C.
6. Remove the nuclease solution by centrifuging the cartilage particles for 20 min at 3,113 x g and discard the supernatant.
7. Wash the samples by adding 30 mL of 10 mM Tris-HCl solution without the deoxyribonuclease and ribonuclease A to the cartilage particles. Resuspend the particles and leave the samples on a roller for 20 h at room temperature.

5. Detergent Decellularization

1. Make the detergent solution by dissolving 1% (v/v) octoxynol-1 in PBS.
2. Remove the Tris-HCl solution from the cartilage particles by centrifuging for 20 min at 3,113 x g and discarding the supernatant.
3. Add 30 mL of the prepared 1% detergent solution to the cartilage particles. Resuspend the cartilage particles gently to avoid foaming of the solution.
   NOTE: This step breaks down cellular membranes.
4. Incubate the samples on a roller for 24 h at room temperature on a roller.
5. Remove the detergent solution by centrifugation for 20 min at 3,113 x g and discard the supernatant.
6. To remove all of the remnants of the decellularization solutions, wash the cartilage particles in 6 cycles of 8 h in 30 mL of cartilage washing solution. Perform washing on a roller at room temperature.
   1. To change to the next wash, centrifuge the suspension for 20 min at 3,113 x g, discard the supernatant and add fresh cartilage washing solution.
7. Leave the last wash in the tubes and store the decellularized cartilage particles at -80 °C.

6. Creating Scaffolds from the Decellularized Particles

1. If the particles have been stored at -80 °C, thaw the closed tubes that contain the cartilage particles in warm water before creating the scaffolds.
2. Transfer the cartilage particles with a small ladle to a cylindrical mold, for example, a plastic vial with a diameter of 8 mm and a height of 2 cm.
3. When placing the cartilage particles into the plastic mold, press all the air-bubbles out to avoid cavities in the scaffold, and fill the mold until the edge.
   NOTE: It will be more difficult to take the scaffolds out of a metal mold after scaffold formation, which can lead to cracks in the scaffold.
4. Freeze the molds with cartilage particles for 10 min at -20 °C.
5. Lyophilize the cartilage scaffolds within their molds for 24 h in a freeze dryer.
6. After lyophilization, take the scaffold out of the mold (Figure 3) and cross-link them with ultraviolet (UV) light at 30 cm distance and 365 nm overnight.
7. In order to use the scaffolds for in vitro cell culture or in vivo implantation, sterilize the scaffolds with, for example, ethylene oxide (EtO) gas.
   NOTE: EtO sterilization is performed by an external party.

7. Characterization of the Decellularized Scaffolds with Histological Stainings

NOTE: To ensure complete decellularization and to visualize the remaining natural character of the cartilage, perform several histological stainings before using the scaffolds in any experiment, including hematoxylin and eosin (H&E) staining to ensure decellularization, Safranin-O staining to visualize residual GAG presence, collagen type I immunohistochemistry to differentiate between collagen content, and collagen type II immunohistochemistry to differentiate between collagen content.

1. Cut the scaffolds in thin slices of approximately 3 mm with a scalpel.
   NOTE: If the scaffolds are cut in larger or smaller sizes, the durations of the dehydration cycles need to be adapted.
2. Embed the scaffolds in a drop of 4% (w/v) alginate and induce cross-linking by adding a similar volume of 3.7% non-buffered formalin that contains 20 mM CaCl₂.
   NOTE: Alginate embedding makes the scaffold slices more rigid compared to the washing steps prior to paraffin embedding. In case the scaffolds have been cell-seeded or in vivo implanted, alginate embedding is not necessary, as the composition of the scaffolds will be resistant enough due to ECM incorporation by the cells.
3. Dehydrate the samples by placing them in a graded ethanol series, starting with one-hour cycles of 70%, 96%, 96%, 100%, and 100% ethanol, followed by two 1 h cycles of xylene, and ending with two 1 h cycles of paraffin at 60 °C.
4. After dehydration, paraffin-embed the samples in a mold and cool the samples down.
5. Cut the samples with a microtome in slices of 5 μm thick.
6. Before staining, rehydrate the samples by placing them in a returned graded ethanol series. Start with two washes of xylene for 5 min, followed by 2 washes of 100%, 95%, and 70% ethanol for 3 min per wash. Finally, wash the samples 3 times in water for 2 min.
   NOTE: In case of using alginate to process samples for paraffin embedding, make sure to wash off the alginate with 10 mM citric acid prior to rehydration of the paraffin sections.
7. Stain the samples with hematoxylin and eosin, Safranin-O, collagen I, and collagen II as previous described¹¹.

8. Characterization of the Decellularized Scaffolds with Quantitative Analyses

1. Obtain papain digests of the scaffolds as described previously¹¹.
2. Perform an assay with a fluorescence-based DNA quantification kit to measure the double-strand DNA content of the scaffolds to ensure complete decellularization. Follow the protocol provided by the manufacturer. Express the amount of DNA per dry weight of the scaffold.
3. Perform a dimethylmethylene blue assay to quantify the remainder of the GAGs within the scaffold, as described previously¹¹. Express the amount in GAG per DNA.

9. Seeding of the Decellularized Scaffolds

1. Cut sterilized scaffolds from step 6.7 into 3 mm thick slices.
2. Transfer the scaffolds in separate wells of a 6-well plate.
3. Rehydrate scaffolds with cell culture medium by pipetting 1 mL of medium on top of the scaffold and let it soak for 30 min. Use either chondrocyte or mesenchymal stem cell (MSC) expansion medium.
   Note: Use the same medium for scaffold rehydration as for cell culture of the cells that will be seeded. Chondrocyte expansion medium consists of Dulbecco's modified media (DMEM) with 10% heat-inactivated fetal bovine serum (FBS), 100 U/mL penicillin, 100 μg/mL streptomycin, and 10 ng/mL fibroblast growth factor-2 (FGF-2). MSC expansion medium consists of minimum essential medium alpha (a-MEM) with 10% heat-inactivated FBS, 0.2 mM L-ascorbic acid 2-phosphate, 100 U/mL penicillin, 100 μg/mL streptomycin, and 10 ng/mL FGF-2.
4. Prepare 3 x 10⁶ cells in 100 μL of medium, which is the required volume for each scaffold with a diameter of 8 mm and a height of 3 mm.
   NOTE: Multiple cell types from different species can be used for seeding. When using chondrocytes or mesenchymal stromal cells, isolate the cells as previously described¹¹. When using chondrocytes, make sure that they have not been expanded past the P1 passage in order to minimize the number of dedifferentiated chondrocytes. When using mesenchymal stromal cells, they must be tested on their ability for multi-lineage differentiation, as previously described¹⁴.
5. Pipet 50 μL of prepared cell suspension on top of the pre-soaked scaffold and incubate the scaffold for 1 h at 37 °C.
6. Carefully turn the scaffold upside down, pipet the remaining 50 μL of cell suspension on this side of the scaffold and incubate for 1 h at 37 °C.
7. After incubation, add 3 mL of medium to the wells, and culture the cell-seeded scaffolds at 37 °C. Handle the culture plate gently to avoid detachment of the cells.
8. Culture the scaffolds for the period of time that is required for the experiment and change the medium 2–3 times a week. Pipet slowly and as far away from the scaffold as possible.
9. After culturing of the cell-seeded scaffold, cut the scaffolds in half and process them for both histology and biochemical analyses.

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Wyniki

Decellularization of CDM scaffolds must always be confirmed using histological stainings as well as using DNA quantification to measure the amount of DNA remnants. Insufficient decellularization might lead to undesired immunological responses that influence the results in in vivo settings^15,16,17. For this specific decellularization method, DNA was below the detection range, which started at 13.6...

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Dyskusje

The ECM of articular cartilage is very dense and quite resilient to different enzymatic treatments. The multi-step decellularization protocol described in this article addresses such resistance and successfully generates decellularized matrices. To achieve that, the process spans over several days. Many decellularization processes have been proposed for different types of tissues¹⁸, and this article describes a protocol suitable for the decellularization of cartilage. In this protocol, it is, howe...

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Materiały

Name	Company	Catalog Number	Comments
Cadaveric joint			This can be obtained as rest material from the local butcher or veterinary center.
Sterile phosphate-buffered saline (PBS)
Penicillin-Streptomycin	Gibco	15140
Amphotericin B	Thermo Fischer Scientific	15290026
Liquid nitrogen
Trypsin-EDTA (0.25%), phenol red	Thermo Fischer Scientific	25200072
Tris-HCl pH 7.5
Deoxyribonuclease I from bovine pancreas	Sigma-Aldrich	DN25
Ribonuclease A from bovine pancreas	Sigma-Aldrich	R6513
Triton X-100 (octoxynol-1)	Sigma-Aldrich	X100
Papain	Sigma-Aldrich	P3125
Trisodium citrate dihydrate	Sigma-Aldrich	S4641
Alginate	Sigma-Aldrich	180947
Formalin
CaCl2
Ethanol
Xylene
Paraffin
Ethylene oxide sterilization	Synergy Health, Venlo, the Netherlands
Multipotent Stromal cells/chondrocytes from equine donors			MSCs and chondrocytes can be isolated from donor joints that are rest material, coming from the local butcher or veterinary center.
MEM alpha	Thermo Fischer Scientific	22561
L-ascorbic acid 2-phosphate	Sigma-Aldrich	A8960
DMEM	Thermo Fischer Scientific	41965
Heat inactivated bovine serum albumin	Sigma-Aldrich	10735086001
Fibroblast growth factor-2 (FGF-2)	R & D Systems	233-FB
DNA quantification kit (Quant-iT PicoGreen dsDNA Reagent)	Thermo Fischer Scientific	P7581
1,9-Dimethyl-Methylene Blue zinc chloride double salt	Sigma-Aldrich	341088
Freeze-dryer	SALMENKIPP	ALPHA 1-2 LD plus
Analytical mill	IKA	A 11 basic
mortar/pestle	Haldenwanger 55/0A
Roller plate	CAT	RM5
Centrifuge (for 50 mL tubes)	Eppendorf	5810R
Capsule (cylindric mold)	TAAB	8 mm flat
Superlite S UV	Lumatec	2001AV
Incubator
Microtome
Sieve (mesh size 0.71 mm)	VWR	34111229
Scalpel
Scalpel holder
Small laddle