Decellularized Apple-Derived Scaffolds for Bone Tissue Engineering In Vitro and In Vivo

Summary

In this study, we detail methods of decellularization, physical characterization, imaging, and in vivo implantation of plant-based biomaterials, as well as methods for cell seeding and differentiation in the scaffolds. The described methods allow the evaluation of plant-based biomaterials for bone tissue engineering applications.

Abstract

Plant-derived cellulose biomaterials have been employed in various tissue engineering applications. In vivo studies have shown the remarkable biocompatibility of scaffolds made of cellulose derived from natural sources. Additionally, these scaffolds possess structural characteristics that are relevant for multiple tissues, and they promote the invasion and proliferation of mammalian cells. Recent research using decellularized apple hypanthium tissue has demonstrated the similarity of its pore size to that of trabecular bone as well as its ability to effectively support osteogenic differentiation. The present study further examined the potential of apple-derived cellulose scaffolds for bone tissue engineering (BTE) applications and evaluated their in vitro and in vivo mechanical properties. MC3T3-E1 preosteoblasts were seeded in apple-derived cellulose scaffolds that were then assessed for their osteogenic potential and mechanical properties. Alkaline phosphatase and alizarin red S staining confirmed osteogenic differentiation in scaffolds cultured in differentiation medium. Histological examination demonstrated widespread cell invasion and mineralization across the scaffolds. Scanning electron microscopy (SEM) revealed mineral aggregates on the surface of the scaffolds, and energy-dispersive spectroscopy (EDS) confirmed the presence of phosphate and calcium elements. However, despite a significant increase in the Young's modulus following cell differentiation, it remained lower than that of healthy bone tissue. In vivo studies showed cell infiltration and deposition of extracellular matrix within the decellularized apple-derived scaffolds after 8 weeks of implantation in rat calvaria. In addition, the force required to remove the scaffolds from the bone defect was similar to the previously reported fracture load of native calvarial bone. Overall, this study confirms that apple-derived cellulose is a promising candidate for BTE applications. However, the dissimilarity between its mechanical properties and those of healthy bone tissue may restrict its application to low load-bearing scenarios. Additional structural re-engineering and optimization may be necessary to enhance the mechanical properties of apple-derived cellulose scaffolds for load-bearing applications.

Introduction

Large bone defects caused by an injury or disease often require biomaterial grafts for complete regeneration¹. Current techniques designed to improve bone tissue regeneration regularly use autologous, allogeneic, xenogeneic, or synthetic grafts². For autologous bone grafting, considered the "gold standard" grafting practice to repair large bone defects, bone is extracted from the patient. However, this grafting procedure has several drawbacks, including size and shape limitations, tissue availability, and sampling site morbidity³. Moreover, autologous grafting procedures are susceptible to surgical site infections, subsequent fractures, hematoma formation at the sampling or reconstructed site, and post-operative pain⁴. Bone tissue engineering (BTE) offers a potential alternative to conventional bone grafting methods⁵. It combines structural biomaterials and cells to build new functional bone tissue. When designing biomaterials for BTE, it is critical to combine a macroporous structure, surface chemistry that promotes cell attachment, and mechanical properties that closely resemble those of native bone⁶. Past research has indicated that the ideal pore size and elastic modulus for biomaterials utilized in BTE are approximately 100-200 µm⁷ and 0.1-20 GPa, respectively, depending on the grafting site⁸. Besides, the porosity and pore interconnectivity of the scaffolds are critical factors affecting cell migration, nutrient diffusion, and angiogenesis⁸.

BTE has shown promising results with various biomaterials developed and evaluated as alternative options to bone grafts. Some of these biomaterials are osteoinductive materials, hybrid materials, and advanced hydrogels⁸. Osteoinductive materials stimulate the development of newly formed bone structures. Hybrid materials are composed of synthetic and/or natural polymers⁸. Advanced hydrogels mimic the extracellular matrix (ECM) and are capable of delivering the necessary bioactive factors to promote bone tissue integration⁸. Hydroxyapatite is a traditional material and a common choice for BTE due to its composition and biocompatibility⁹. Bioactive glass is another type of biomaterial for BTE, which has been shown to stimulate specific cell responses to activate genes necessary for osteogenesis^10,11. Biodegradable polymers, including poly(glycolic acid) and poly(lactic acid), have also been extensively used in BTE applications¹². Finally, natural or naturally-derived polymers like chitosan, chitin, and bacterial cellulose have also demonstrated encouraging outcomes for BTE¹³. However, while both synthetic and natural polymers show potential for BTE, the development of a functional scaffold with the desired macrostructure typically necessitates extensive protocols.

Conversely, native macroscopic cellulose structures can be readily derived from diverse plants and our research group previously demonstrated the applicability of cellulose-based scaffolds derived from plants to different tissue reconstructions. Indeed, following a simple surfactant treatment, we harnessed the inherent structure of the plant material, highlighting its potential as a versatile biomaterial¹⁴. Moreover, these cellulose-based scaffolds can be used for in vitro mammalian cell culture applications¹⁴, are biocompatible, and support spontaneous subcutaneous vascularization^14,15,16,17. Both our research group and others have demonstrated that these scaffolds can be obtained from specific plants based on the intended application^{14,15,16,17,18,19,20}. For example, the vascular structure observed in plant stems and leaves exhibits a striking similarity to the structure found in animal tissues¹⁹. Additionally, cellulose scaffolds derived from plants can be readily shaped and subjected to surface biochemical modifications to achieve the desired characteristics¹⁶. In a recent study, we incorporated a salt buffer during the decellularization process, leading to enhanced cell attachment observed both in in vitro and in vivo settings¹⁶. In the same study, we demonstrated the applicability of plant-derived cellulose scaffolds in composite biomaterials by casting hydrogels onto the surface of the scaffolds. In recent studies, the functionalization of plant-derived scaffolds has been shown to enhance their effectiveness¹⁸. For example, a study conducted by Fontana et al. (2017) revealed that the adhesion of human dermal fibroblasts was supported by RGD-coated decellularized stems, whereas non-coated stems did not exhibit the same capability¹⁸. Moreover, the authors also demonstrated that modified simulated body fluid could be utilized to artificially mineralize decellularized plant stems. In more recent studies, we explored the concept of mechanosensitive osteogenesis in plant-derived cellulose scaffolds and assessed their potential for BTE^17,20. Furthermore, Lee et al. (2019) utilized plant-derived scaffolds to cultivate bone-like tissues in an in vitro setting²¹. Through comprehensive evaluations of different plant sources, the authors identified apple-derived scaffolds as the most optimal for the culture and differentiation of human induced pluripotent stem cells (hiPSCs). Furthermore, the authors proposed that the structural and mechanical attributes of the apple-derived scaffolds play a pivotal role in their suitability for the intended purpose. Being the initial plant-derived scaffolds implemented in tissue engineering applications, apple-derived scaffolds have been extensively shown to possess a strikingly similar architecture to that of human bone, notably in terms of their interconnected pores ranging from 100 to 200 µm in diameter^14,21.

In the present study, we further investigated the potential of apple-derived cellulose scaffolds for BTE and conducted an analysis of their mechanical properties both in vitro and in vivo. Although there have been studies on the potential of apple-derived scaffolds for BTE^17,20,21, their mechanical properties have been underinvestigated. Results showed wildspread invasion and osteogenic differentiation of MC3T3-E1 preosteoblasts seeded in scaffolds that were cultured in differentiation medium for 4 weeks. The Young's modulus of these scaffolds was 192.0 ± 16.6 kPa, which was significantly higher than those of the blank scaffolds (scaffolds without seeded cells) (31.6 ± 4.8 kPa) and the cell-seeded scaffolds cultured in non-differentiation medium (24.1 ± 8.8 kPa). However, it should be noted that the Young's modulus of healthy human bone tissue typically falls within the range of 0.1-2 GPa for trabecular bone and approximately 15-20 GPa for cortical bone⁸. Nevertheless, following an 8-week implantation in a rodent calvarial defect, the cell-seeded scaffolds appeared to be well integrated into the surrounding bone, as demonstrated by an average peak force of 113.6 N ± 18.2 N in push-out tests, which is similar to the previously reported fracture load of native calvarial bone²². Overall, results obtained from this study show significant promise, particularly for non-load-bearing applications. However, apple-derived cellulose scaffolds do not currently possess the necessary mechanical properties to precisely match the surrounding bone tissue at an implant site. Consequently, further development is required to unlock the full potential of these scaffolds.

Protocol

The experimental protocols were reviewed and approved by the University of Ottawa Animal Care Committee.

1. Scaffold preparation

1. Use a mandolin slicer to cut McIntosh apples (Canada Fancy) into 8 mm-thick slices. Cut the hypanthium tissue of the apple slices into 5 mm x 5 mm squares.
2. Place the square samples in 0.1% sodium dodecyl sulfate (SDS) for 2 days.
3. Wash the decellularized samples with deionized water and incubate them overnight at room temperature (RT) in 100 mM CaCl₂ to remove the remaining surfactant.
4. Sterilize the samples (i.e., scaffolds) in 70% ethanol for 30 min, wash them with deionized water, and place them in a 24-well culture plate prior to cell seeding.

2. Cell culture and scaffold seeding

1. Maintain MC3T3-E1 Subclone 4 cells in 10-cm diameter cell culture-treated dishes in cell culture conditions (37°C in a humidified atmosphere of 95% air and 5% CO₂).
2. Prepare cell culture medium made of Eagle's minimum essential medium - alpha modification (α-MEM) supplemented with 10% fetal bovine serum (FBS) and 1% penicillin/streptomycin.
3. Detach the cells from the culture dishes by trypsinization (0.05% trypsin-EDTA) once they reach 80% confluency.
4. Centrifuge the cell suspension at c.a. 200 x g for 3 min. Aspirate the supernatant and resuspend the cells in α-MEM at 2.5 x 10⁷ cells per mL.
5. Pipette a 40 µL aliquot of the cell suspension on the surface of the scaffolds, and let the cells adhere for 1 h in cell culture conditions. Subsequently, add 2 mL of culture medium to each culture well  of the culture plate.
6. Replenish the culture medium every 2-3 days for 14 days.
7. Prepare a differentiation medium by adding 50 µg/mL ascorbic acid and 4 mM sodium phosphate to the previously described cell culture medium.
8. Induce differentiation of MC3T3-E1 cells by incubating the scaffolds in differentiation medium for 4 weeks. Replenish the medium every 3-4 days. In parallel, incubate scaffolds in non-differentiation culture medium (i.e., medium without the supplements to induce differentiation) for the same duration, with the same medium change schedule to serve as a negative control.

3. Pore size measurements using confocal laser scanning microscopy

1. Wash the decellularized apple-derived scaffolds with phosphate-buffered-saline (PBS).
2. Incubate the scaffolds in 1 mL of 10% (v/v) calcofluor white stain for 25 min in the dark at RT.
3. Wash the scaffolds (n = 3) with PBS and image three randomly selected areas per scaffold with a high-speed resonant confocal laser scanning microscope (CLSM) at 10x magnification, using a DAPI channel, as follows:
   1. Laser-emission filter configuration: 405 nm (laser); 425-475 nm (emission)
   2. Adjust the laser power and detector manually to ensure optimal image acquisition. Acquire a z-stack of 20 images with a 5-µm step size.
4. Using ImageJ software, process and analyze the confocal images as follows:
   1. Use the Z-Project to Maximum Intensity function to create an image, and apply the Find Edges function to highlight the edge of the pores.
   2. Trace the pores manually using the Freehand Selection tool.
   3. Fit each pore as an ellipse, measure the length of the major axis, compile all the measurements (a total of 54 in the present study - 6 in 3 randomly selected areas for each scaffold), and calculate the average length.

4. Cell distribution analysis using confocal laser scanning microscopy

1. Wash the cell-seeded scaffolds cultured in the non-differentiation or differentiation medium three times with PBS. Fix the scaffolds with 4% paraformaldehyde for 10 min.
2. Thoroughly wash each scaffold with deionized water, permeabilize the cells with a Triton-X 100 solution for 5 min, and wash again with PBS.
3. Incubate the scaffolds in 1 mL of 1% periodic acid for 40 min and rinse with deionized water^14,16.
4. Incubate the scaffolds in 1 mL of a solution containing 100 mM sodium metabisulphite and 0.15 M hydrochloric acid, supplemented with 100 µg/mL propidium iodide. Completely immerse the scaffolds in the solution.
5. Wash the scaffolds with PBS and stain the cell nuclei by incubating the scaffolds in a 5 mg/mL DAPI solution for 10 min in the dark. Thoroughly wash again and store the scaffolds in PBS prior to imaging.
6. Image three randomly selected surfaces of three different cell-seeded scaffolds with a high-speed resonant CLSM at 10x magnification, using DAPI and TRITC channels, as follows:
   1. Laser-emission filter configuration:
      DAPI: Laser: 405 nm; Emission: 425-475 nm
      ​TRITC: Laser: 561 nm; Emission: 570-620 nm
   2. Adjust the laser power and detector manually to ensure optimal image acquisition. Acquire a z-stack of 20 images with a 5-µm step size.
7. Use ImageJ software to process the confocal images and create a maximum projection in the z-axis for image analysis using the Z-Project to Maximum Intensity function.

5. Alkaline phosphatase analysis

1. Wash the cell-seeded scaffolds cultured in the non-differentiation or differentiation medium three times with PBS. Fix the scaffolds with 10% neutral buffered formalin for 30 min. Fix blank scaffolds (scaffolds without seeded cells) to serve as a negative control.
2. Prepare a 5-bromo-4-chloro-3'-indolyphosphate and nitro-blue tetrazolium (BCIP/NBT) staining solution by dissolving one BCIP/NBT tablet in 10 mL of deionized water.
3. Wash the fixed scaffolds with a 0.05% Tween solution and stain with the BCIP/NBT solution for 20 min at RT. Wash the stained scaffolds with a 0.05% Tween solution and store them in PBS prior to imaging.
4. Image the stained scaffolds with a 12-megapixel digital camera.

6. Calcium deposition analysis

1. Wash the cell-seeded scaffolds cultured in the non-differentiation or differentiation medium three times with PBS. Fix the scaffolds with 10% neutral buffered formalin for 30 min. Fix blank scaffolds (scaffolds without seeded cells) to serve as a negative control.
2. Prepare a 2% (w/v) alizarin red S (ARS) staining solution.
3. Wash the fixed scaffolds with deionized water and stain them with the ARS solution for 1 h at RT. Wash the stained scaffolds with deionized water and store them in PBS prior to imaging.
4. Image the stained scaffolds with a 12-megapixel digital camera.

7. Mineralization analysis

1. Wash the cell-seeded scaffolds cultured in the non-differentiation or differentiation medium three times with PBS. Fix the scaffolds with 4% paraformaldehyde for 48 h. Fix blank scaffolds (scaffolds without seeded cells) to serve as a negative control.
2. Dehydrate the samples in solutions containing concentrations of ethanol increasing from 50% to 100%, as previously described²³.
3. Perform scanning electron microscopy (SEM) and energy-dispersive spectroscopy (EDS) to analyze mineral aggregates as follows:
   1. Dry the samples using a critical point dryer, following the manufacturer's protocol²⁴.
   2. Apply a 5-nm gold coat on the scaffolds using a gold sputter coater, following the manufacturer's protocol²⁵.
   3. Image the surface of the scaffolds with a scanning electron microscope set at 3 kV, at 85x magnification.
   4. Perform EDS by setting the scanning electron microscope at 15 kV. On three randomly selected areas per scaffold, acquire EDS spectra for mineral aggregate composition analysis.

8. Young's modulus measurements

1. Remove the cell-seeded scaffolds from their respective incubation medium and immediately test the samples.
2. Using a custom-built uniaxial compression apparatus equipped with a 1.5 N load cell, compress the scaffolds (n = 3 per condition) at a constant rate of 3 mm·min^-1 to a maximum compressive strain of 10% of the scaffold height.
3. Determine the Young's modulus from the slope of the linear portion of the stress-strain curves. In the present study, the modulus was determined between 9% and 10% strain.

9. Cell infiltration and mineralization analysis by histology: In vitro scaffolds

1. Wash the cell-seeded scaffolds cultured in non-differentiation or differentiation medium three times with PBS.
2. Fix the cell-seeded scaffolds with 4% paraformaldehyde for 48 h before resuspension in 70% ethanol for storage.
3. Histology:
   NOTE: In the present study, the entire histological preparation (embedding, sectioning, and staining) described in the next steps was performed by the Louise Pelletier Histology Core Facility (University of Ottawa).
   1. After dehydration and embedding in paraffin, cut the samples into 5-µm thick serial sections, starting 1 mm inside the scaffolds, and mount the sections on microscope slides.
   2. Stain the sections with hematoxylin and eosin (H&E) or von Kossa (VK) stains.
   3. Image the sections with a slide scanner microscope at 40x magnification (n = 1 scaffold in non-differentiation medium and n = 2 scaffolds in differentiation medium in the present study).
   4. Using ImageJ software, visually evaluate cell infiltration (H&E staining) and mineralization (VK staining).

10. Rat calvarial defect model

1. Get experimental protocols reviewed and approved by the local animal care committee.
2. Prepare circular (5-mm diameter and 1-mm thickness) decellularized scaffolds following Section 1 described above and using a 5-mm biopsy punch.
3. Perform bilateral craniotomy following an established protocol²⁶, as follows:
   1. Anesthetize male Sprague-Dawley rats with isoflurane, first at 3% until they are unconscious, and then at 2-3% throughout the procedure.
   2. Expose the periosteum and cranium by cutting the overlying skin using a scalpel blade. Remove the periosteum.
   3. Create bilateral defects in both parietal bones on each side of the sagittal suture using a dental drill equipped with a 5-mm diameter trephine under constant irrigation of 0.9% NaCl.
   4. Clean the surrounding bone with 0.9% NaCl to remove any bone fragments.
   5. Place the circular, decellularized scaffolds in the defects.
   6. Close the overlying skin with 4-0 sutures.
4. Give the rats unlimited access to food and water and monitor them daily.
5. After 8 weeks post-implantation, euthanize the rats by CO₂ inhalation and thoracic perforation as a secondary euthanasia measure.
6. To expose the cranium and retrieve the implants, remove the skin covering the skull using a scalpel blade.
7. Cut the skull at the frontal and occipital bones and on the side of both parietal bones using a dental drill to remove the top section of the skull completely.

11. Push-out test

1. Connect a uniaxial compression device (with a 445 N load cell) to the USB-data acquisition module.
2. Connect the data acquisition module to a computer equipped with a data acquisition software application.
3. Immediately after cranial extraction, place each sample (n = 7 implants from 4 animals in the present study) on the sample holder of the uniaxial compression device so that the dorsal side of the bone is facing up.
4. Lower the plunger at 0.5 mm/min until slightly touching the extracted implant.
5. Initiate the test by lowering the plunger through the implant until complete push-out while applying compression at a constant speed of 0.5 mm/min using the data acquisition software.
6. Record the peak force on the force vs. displacement curve using the data acquisition software.

12. Cell infiltration and mineralization analysis by histology: In vivo scaffolds

1. Fix the extracted calvaria and implants in 10% neutral buffered formalin for 72 h before resuspension in 70% ethanol for storage.
2. Histology:
   NOTE: In the present study, all histological preparation (embedding, sectioning, and staining) described in the next steps was performed by Accel Labs (Montreal, QC, Canada).
   1. Cut the samples (embedded in methyl methacrylate) into 6-µm thick sections at three different levels (top, bottom, and towards the center) and mount them on microscope slides.
   2. Stain the sections with either H&E or Masson-Goldner's trichrome (MGT).
3. Image the sections with a slide scanner microscope at 40x magnification (4 explants from 2 animals in the present study).
4. Using ImageJ software, visually evaluate cell infiltration (H&E staining) and collagen deposition (MGT staining).

Results

Pore size measurement, cell distribution, and in vitro mineralization (Figure 1 and Figure 2)
Complete removal of native cellular components of the apple tissue scaffolds was achieved after treating the scaffolds with SDS and CaCl₂ (Figure 1A). The scaffolds exhibited a highly porous structure, which was confirmed using confocal microscopy. The quantification of the images demonstrated an a...

Discussion

Several in vitro and in vivo studies have demonstrated the biocompatibility of plant-derived cellulose and its potential use in tissue engineering^{14,15,16,18,19,20}, more specifically for hosting osteogenic differentiation^20,21. The objectives of...

Materials

Name	Company	Catalog Number	Comments
4′,6-diamidino-2-phenylindole	ThermoFisher	D1306	DAPI
5-bromo-4-chloro-3'-indolyphosphate and nitro-blue tetrazolium	Sigma-Aldrich	B5655	BCIP/NBT
Alizarin red S	Sigma-Aldrich	A5533	ARS
Ascorbic acid	Sigma-Aldrich	A4403	Cell Culture
Calcium Chloride	ThermoFisher	AA12316	CaCl2
Calcofluor White	Sigma-Aldrich	18909
Dental drill			Surgical tool
Ethanol	ThermoFisher	615095000
Fetal bovine serum	Hyclone Laboratories	SH30396	FBS
Formalin	Sigma-Aldrich	HT501128	10% Formalin
Goldner's trichrome stain	Sigma-Aldrich	1.00485	GTC
Hematoxylin and eosin stain	Fisher Scientific	NC1470670	H&E
High-speed resonant confocal laser scanning microscope	Nikon	Nikon Ti-E A1-R
Hydrochloric acid	Sigma-Aldrich	258148
ImageJ software	National Institutes of Health
Irrigation saline	Baxter	JF7123	0.9% NaCl
MC3T3-E1 Subclone 4 cells	ATCC	CRL-2593	Pre-osteoblast cells
McIntosh apples			Canada Fancy grade
Methyl methacrylate	Sigma-Aldrich	M55909	Histological embedding
Minimum Essential Medium	ThermoFisher	M0894	α-MEM
Paraformaldehyde	Fisher Scientific	O4042	4%; PFA
Penicillin/Streptomycin	Hyclone Laboratories	SV30010	Cell Culture
Periodic acid	Sigma-Aldrich	375810
Phosphate buffered saline	Hyclone Laboratories	2810305	PBS; without Ca2+ and Mg2+
Propidium iodide	Invitrogen	p3566
Scanning electron microscope	JEOL	JSM-7500F FESEM	SEM and EDS
Slide scanner microscope	Zeiss	AXIOVERT 40 CFL
Sodium dodecyl sulfate	Fisher Scientific	BP166	SDS
Sodium metabisulphite	Sigma-Aldrich	31448
Sodium phosphate	ThermoFisher	BP329
Sprague-Dawley rats	Charles-River Laboratories	400	Male
Sutures	Ethicon	J494G	4-0
Trephine	ACE Surgical Supply Co	583-0182	5-mm diameter
Triton-X 100	ThermoFisher	807423
Trypsin	Hyclone Laboratories	SH30236.02	Cell Culture
Tween	Fisher Scientific	BP337
Universal compression Device	CellScale	UniVert
Von Kossa stain	Sigma-Aldrich	1.00362	Histology