Funding for this project was provided by the Natural Sciences and Engineering Research Council of Canada (NSERC) (Discovery Grant) and by the Li Ka Shing Foundation. M.L.L. received support from the Ontario Centers of Excellence TalentEdge program, and R.J.H. was supported by an NSERC postgraduate scholarship and an Ontario Graduate Scholarship (OGS).

The experimental protocols were reviewed and approved by the University of Ottawa Animal Care Committee.
1. Scaffold preparation
<ol>
	<li>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.</li>
	<li>Place the square samples in 0.1% sodium dodecyl sulfate (SDS) for 2 days.</li>
	<li>Wash the decellularized samples with deionized water and incubate them overnight at room temperature (RT) in 100 mM CaCl2&#160;to remove the remaining surfactant.</li>
	<li>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.</li>
</ol>
2. Cell culture and scaffold seeding
<ol>
	<li>Maintain MC3T3-E1 Subclone 4 cells in 10-cm diameter cell culture-treated dishes in cell culture conditions (37&#176;C in a humidified atmosphere of 95% air and 5% CO2).</li>
	<li>Prepare cell culture medium made of Eagle&#39;s minimum essential medium - alpha modification&#160;(&#945;-MEM) supplemented with 10% fetal bovine serum (FBS) and 1% penicillin/streptomycin.</li>
	<li>Detach the cells from the culture dishes by trypsinization (0.05% trypsin-EDTA) once they reach 80% confluency.</li>
	<li>Centrifuge the cell suspension at c.a. 200 x g for 3 min. Aspirate the supernatant and resuspend the cells in &#945;-MEM at 2.5 x 107 cells per mL.</li>
	<li>Pipette a 40 &#181;L 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 &#160;of the culture plate.</li>
	<li>Replenish the culture medium every 2-3 days for 14 days.</li>
	<li>Prepare a differentiation medium by adding 50 &#181;g/mL ascorbic acid and 4 mM sodium phosphate to the previously described cell culture medium.</li>
	<li>Induce differentiation of MC3T3-E1 cells by incubating the scaffolds in&#160;differentiation medium for 4 weeks. Replenish the medium every 3-4 days. In parallel, incubate scaffolds in&#160;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.</li>
</ol>
3. Pore size measurements using confocal laser scanning microscopy
<ol>
	<li>Wash the decellularized apple-derived scaffolds with phosphate-buffered-saline (PBS).</li>
	<li>Incubate the scaffolds in 1 mL of 10% (v/v) calcofluor white stain for 25 min in the dark at RT.</li>
	<li>Wash the&#160;scaffolds&#160;(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:
	<ol>
		<li>Laser-emission filter configuration: 405 nm (laser); 425-475 nm&#160;(emission)</li>
		<li>Adjust the laser power and detector manually to ensure optimal image acquisition. Acquire a z-stack of 20 images with a 5-&#181;m step size.</li>
	</ol>
	</li>
	<li>Using ImageJ software, process and analyze the confocal images as follows:
	<ol>
		<li>Use&#160;the Z-Project to Maximum Intensity function to&#160;create an image, and apply the Find Edges function to highlight the edge of the pores.</li>
		<li>Trace the pores manually using the Freehand Selection tool.</li>
		<li>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.</li>
	</ol>
	</li>
</ol>
4. Cell distribution analysis using confocal laser scanning microscopy
<ol>
	<li>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.</li>
	<li>Thoroughly wash each scaffold with deionized water, permeabilize the cells with a Triton-X 100 solution for 5 min, and wash again with PBS.</li>
	<li>Incubate the scaffolds in 1 mL of 1% periodic acid for 40 min and rinse with deionized water14,16.</li>
	<li>Incubate the scaffolds in 1 mL of a solution containing 100 mM sodium metabisulphite and 0.15 M hydrochloric acid, supplemented with 100 &#181;g/mL propidium iodide. Completely immerse the scaffolds in the solution.</li>
	<li>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.</li>
	<li>Image three randomly selected surfaces of three different cell-seeded scaffolds with a high-speed resonant CLSM at 10x magnification, using DAPI and TRITC&#160;channels, as follows:
	<ol>
		<li>Laser-emission filter configuration: 
		DAPI: Laser: 405 nm; Emission: 425-475 nm 
		&#8203;TRITC: Laser: 561 nm; Emission: 570-620 nm</li>
		<li>Adjust the laser power and detector manually to ensure optimal image acquisition. Acquire a z-stack of 20 images with a 5-&#181;m step size.</li>
	</ol>
	</li>
	<li>Use ImageJ software to process the confocal images and create a maximum projection in the z-axis for image analysis&#160;using the Z-Project to Maximum Intensity function.</li>
</ol>
5. Alkaline phosphatase analysis
<ol>
	<li>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.</li>
	<li>Prepare a 5-bromo-4-chloro-3&#39;-indolyphosphate and nitro-blue tetrazolium (BCIP/NBT) staining solution by dissolving one BCIP/NBT tablet in 10 mL of deionized water.</li>
	<li>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.</li>
	<li>Image the stained scaffolds with a 12-megapixel digital camera.</li>
</ol>
6. Calcium deposition analysis
<ol>
	<li>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.</li>
	<li>Prepare a 2% (w/v) alizarin red S (ARS) staining solution.</li>
	<li>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.</li>
	<li>Image the stained scaffolds with a 12-megapixel digital camera.</li>
</ol>
7. Mineralization analysis
<ol>
	<li>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)&#160;to serve as a negative control.</li>
	<li>Dehydrate the samples in solutions containing concentrations of ethanol increasing from 50% to 100%, as previously described23.</li>
	<li>Perform scanning electron microscopy (SEM) and energy-dispersive spectroscopy (EDS) to analyze mineral aggregates as follows:
	<ol>
		<li>Dry the samples using a critical point dryer, following the manufacturer&#39;s protocol24.</li>
		<li>Apply a 5-nm gold coat on the scaffolds using a gold sputter coater, following the manufacturer&#39;s protocol25.</li>
		<li>Image the surface of the scaffolds with a scanning electron microscope set at 3 kV, at 85x magnification.</li>
		<li>Perform EDS by setting the scanning electron microscope at 15 kV. On three randomly selected areas per scaffold, acquire EDS&#160;spectra for mineral aggregate composition analysis.</li>
	</ol>
	</li>
</ol>
8. Young&#39;s modulus measurements
<ol>
	<li>Remove the cell-seeded scaffolds from their respective incubation medium and immediately test the samples.</li>
	<li>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&#183;min-1 to a maximum compressive strain of 10% of the scaffold height.</li>
	<li>Determine the Young&#39;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.</li>
</ol>
9. Cell infiltration and mineralization analysis by histology: In vitro scaffolds
<ol>
	<li>Wash the cell-seeded scaffolds cultured in non-differentiation or differentiation medium three times with PBS.</li>
	<li>Fix the cell-seeded scaffolds with 4% paraformaldehyde for 48 h before resuspension in 70% ethanol for storage.</li>
	<li>Histology: 
	NOTE: In the present study, the entire&#160;histological preparation (embedding, sectioning, and staining) described in the next steps was performed by the Louise Pelletier Histology Core Facility (University of Ottawa).
	<ol>
		<li>After dehydration and embedding in paraffin, cut the samples into 5-&#181;m thick serial sections, starting 1 mm inside the scaffolds, and mount the sections on microscope slides.</li>
		<li>Stain the sections with hematoxylin and eosin (H&#38;E) or von Kossa (VK) stains.</li>
		<li>Image the sections with a slide scanner microscope at&#160;40x magnification&#160;(n = 1 scaffold in&#160;non-differentiation medium and n = 2 scaffolds in differentiation medium in the present study).</li>
		<li>Using ImageJ software, visually evaluate cell infiltration (H&#38;E staining) and mineralization (VK staining).</li>
	</ol>
	</li>
</ol>
10. Rat calvarial defect model
<ol>
	<li>Get experimental protocols reviewed and approved by the local animal care committee.</li>
	<li>Prepare circular (5-mm diameter and 1-mm thickness) decellularized scaffolds following Section 1 described above and using a 5-mm biopsy punch.</li>
	<li>Perform bilateral craniotomy following an established protocol26, as follows:
	<ol>
		<li>Anesthetize male Sprague-Dawley rats with isoflurane, first at 3% until they are unconscious, and then at 2-3% throughout the procedure.</li>
		<li>Expose the periosteum and cranium by cutting the overlying skin using a scalpel blade. Remove the periosteum.</li>
		<li>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.</li>
		<li>Clean the surrounding bone with 0.9% NaCl to remove any bone fragments.</li>
		<li>Place the circular, decellularized scaffolds in the defects.</li>
		<li>Close the overlying skin with 4-0 sutures.</li>
	</ol>
	</li>
	<li>Give the rats unlimited access to food and water and monitor them daily.</li>
	<li>After 8 weeks post-implantation, euthanize the rats by CO2 inhalation and thoracic perforation as a secondary euthanasia measure.</li>
	<li>To expose the cranium and retrieve the implants, remove the skin covering the skull using a scalpel blade.</li>
	<li>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.</li>
</ol>
11. Push-out test
<ol>
	<li>Connect a uniaxial compression device (with a 445 N load cell) to the USB-data acquisition module.</li>
	<li>Connect the data acquisition module to a computer equipped with a data acquisition software application.</li>
	<li>Immediately after cranial extraction, place each&#160;sample&#160;(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.</li>
	<li>Lower the plunger at 0.5 mm/min until slightly touching the extracted implant.</li>
	<li>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.</li>
	<li>Record the peak force on the force vs. displacement curve using the data acquisition software.</li>
</ol>
12. Cell infiltration and mineralization analysis by histology: In vivo scaffolds
<ol>
	<li>Fix the extracted calvaria and implants in 10% neutral buffered formalin for 72 h before resuspension in 70% ethanol for storage.</li>
	<li>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).
	<ol>
		<li>Cut the samples (embedded in methyl methacrylate) into 6-&#181;m thick sections at three different levels (top, bottom, and towards the center) and mount them on microscope slides.</li>
		<li>Stain the sections with either H&#38;E or Masson-Goldner&#39;s trichrome (MGT).</li>
	</ol>
	</li>
	<li>Image the sections with a slide scanner microscope at 40x magnification (4 explants from 2 animals in the present study).</li>
	<li>Using ImageJ software, visually evaluate cell infiltration (H&#38;E staining) and collagen deposition (MGT staining).</li>
</ol>

Analyzing Cell-Seeded Decellularized Apple-Derived Scaffolds for Bone Tissue Engineering

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Conflict of interest statement: M.L.L, M.T. R.J.H., C.M.C., I.C. and A.P. are inventors on patent applications filed by the University of Ottawa and Spiderwort Inc. concerning the use of plant-derived cellulose for BTE applications.&#160;M.L.L., R.J.H., C.M.C., and A.P. have financial interests in Spiderwort Inc.

Several&#160;in vitro and in vivo studies have demonstrated the biocompatibility of plant-derived cellulose and its potential use in tissue engineering14,15,16,18,19,20, more specifically for hosting osteogenic differentiation20,21. The objectives of...

Large bone defects caused by&#160;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 &#34;gold standard&#34; 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.&#160;Bone tissue engineering&#160;(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 &#181;m7 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 osteogenesis10,11. 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 vascularization14,15,16,17. Both our research group and others have demonstrated that these scaffolds can be obtained from specific plants based on the intended application14,15,16,17,18,19,20. 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&#160;BTE17,20. 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&#160;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 &#181;m in diameter14,21.
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&#160;of apple-derived scaffolds for&#160;BTE17,20,21, their mechanical properties have been underinvestigated. Results showed wildspread invasion and osteogenic differentiation of MC3T3-E1 preosteoblasts seeded in scaffolds that were&#160;cultured in differentiation medium for 4 weeks. The Young&#39;s modulus of these scaffolds was 192.0 &#177; 16.6 kPa, which was&#160;significantly higher than those of the blank scaffolds (scaffolds without seeded cells) (31.6 &#177; 4.8 kPa) and&#160;the cell-seeded scaffolds cultured in&#160;non-differentiation medium (24.1 &#177; 8.8 kPa). However, it should be noted that the Young&#39;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 &#177; 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.

<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<tbody>
		<tr>
			<td>4&#8242;,6-diamidino-2-phenylindole</td>
			<td>ThermoFisher</td>
			<td>D1306</td>
			<td>DAPI</td>
		</tr>
		<tr>
			<td>5-bromo-4-chloro-3&#39;-indolyphosphate and nitro-blue tetrazolium</td>
			<td>Sigma-Aldrich</td>
			<td>B5655</td>
			<td>BCIP/NBT</td>
		</tr>
		<tr>
			<td>Alizarin red S</td>
			<td>Sigma-Aldrich</td>
			<td>A5533</td>
			<td>ARS</td>
		</tr>
		<tr>
			<td>Ascorbic acid</td>
			<td>Sigma-Aldrich</td>
			<td>A4403</td>
			<td>Cell Culture</td>
		</tr>
		<tr>
			<td>Calcium Chloride</td>
			<td>ThermoFisher</td>
			<td>AA12316</td>
			<td>CaCl2</td>
		</tr>
		<tr>
			<td>Calcofluor White</td>
			<td>Sigma-Aldrich</td>
			<td>18909</td>
			<td></td>
		</tr>
		<tr>
			<td>Dental drill</td>
			<td></td>
			<td></td>
			<td>Surgical tool</td>
		</tr>
		<tr>
			<td>Ethanol</td>
			<td>ThermoFisher</td>
			<td>615095000</td>
			<td></td>
		</tr>
		<tr>
			<td>Fetal bovine serum</td>
			<td>Hyclone Laboratories</td>
			<td>SH30396</td>
			<td>FBS</td>
		</tr>
		<tr>
			<td>Formalin</td>
			<td>Sigma-Aldrich</td>
			<td>HT501128</td>
			<td>10% Formalin</td>
		</tr>
		<tr>
			<td>Goldner&#39;s trichrome stain</td>
			<td>Sigma-Aldrich</td>
			<td>1.00485</td>
			<td>GTC</td>
		</tr>
		<tr>
			<td>Hematoxylin and eosin stain</td>
			<td>Fisher Scientific</td>
			<td>NC1470670</td>
			<td>H&#38;E</td>
		</tr>
		<tr>
			<td>High-speed resonant confocal laser scanning microscope</td>
			<td>Nikon</td>
			<td>Nikon Ti-E A1-R</td>
			<td></td>
		</tr>
		<tr>
			<td>Hydrochloric acid</td>
			<td>Sigma-Aldrich</td>
			<td>258148</td>
			<td></td>
		</tr>
		<tr>
			<td>ImageJ software</td>
			<td>National Institutes of Health</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Irrigation saline</td>
			<td>Baxter</td>
			<td>JF7123</td>
			<td>0.9% NaCl</td>
		</tr>
		<tr>
			<td>MC3T3-E1 Subclone 4 cells</td>
			<td>ATCC</td>
			<td>CRL-2593</td>
			<td>Pre-osteoblast cells</td>
		</tr>
		<tr>
			<td>McIntosh apples</td>
			<td></td>
			<td></td>
			<td>Canada Fancy grade</td>
		</tr>
		<tr>
			<td>Methyl methacrylate</td>
			<td>Sigma-Aldrich</td>
			<td>M55909</td>
			<td>Histological embedding</td>
		</tr>
		<tr>
			<td>Minimum Essential Medium</td>
			<td>ThermoFisher</td>
			<td>M0894</td>
			<td>&#945;-MEM</td>
		</tr>
		<tr>
			<td>Paraformaldehyde</td>
			<td>Fisher Scientific</td>
			<td>O4042</td>
			<td>4%; PFA</td>
		</tr>
		<tr>
			<td>Penicillin/Streptomycin</td>
			<td>Hyclone Laboratories</td>
			<td>SV30010</td>
			<td>Cell Culture</td>
		</tr>
		<tr>
			<td>Periodic acid</td>
			<td>Sigma-Aldrich</td>
			<td>375810</td>
			<td></td>
		</tr>
		<tr>
			<td>Phosphate buffered saline</td>
			<td>Hyclone Laboratories</td>
			<td>2810305</td>
			<td>PBS; without Ca2+ and Mg2+</td>
		</tr>
		<tr>
			<td>Propidium iodide</td>
			<td>Invitrogen</td>
			<td>p3566</td>
			<td></td>
		</tr>
		<tr>
			<td>Scanning electron microscope</td>
			<td>JEOL</td>
			<td>JSM-7500F FESEM</td>
			<td>SEM and EDS</td>
		</tr>
		<tr>
			<td>Slide scanner microscope</td>
			<td>Zeiss</td>
			<td>AXIOVERT 40 CFL</td>
			<td></td>
		</tr>
		<tr>
			<td>Sodium dodecyl sulfate</td>
			<td>Fisher Scientific</td>
			<td>BP166</td>
			<td>SDS</td>
		</tr>
		<tr>
			<td>Sodium metabisulphite</td>
			<td>Sigma-Aldrich</td>
			<td>31448</td>
			<td></td>
		</tr>
		<tr>
			<td>Sodium phosphate</td>
			<td>ThermoFisher</td>
			<td>BP329</td>
			<td></td>
		</tr>
		<tr>
			<td>Sprague-Dawley rats</td>
			<td>Charles-River Laboratories</td>
			<td>400</td>
			<td>Male</td>
		</tr>
		<tr>
			<td>Sutures</td>
			<td>Ethicon</td>
			<td>J494G</td>
			<td>4-0</td>
		</tr>
		<tr>
			<td>Trephine</td>
			<td>ACE Surgical Supply Co</td>
			<td>583-0182</td>
			<td>5-mm diameter</td>
		</tr>
		<tr>
			<td>Triton-X 100</td>
			<td>ThermoFisher</td>
			<td>807423</td>
			<td></td>
		</tr>
		<tr>
			<td>Trypsin</td>
			<td>Hyclone Laboratories</td>
			<td>SH30236.02</td>
			<td>Cell Culture</td>
		</tr>
		<tr>
			<td>Tween</td>
			<td>Fisher Scientific</td>
			<td>BP337</td>
			<td></td>
		</tr>
		<tr>
			<td>Universal compression Device</td>
			<td>CellScale</td>
			<td>UniVert</td>
			<td></td>
		</tr>
		<tr>
			<td>Von Kossa stain</td>
			<td>Sigma-Aldrich</td>
			<td>1.00362</td>
			<td>Histology</td>
		</tr>
	</tbody>
</table>

decellularized apple-derived scaffolds for bone tissue engineering in vitro and in vivo

Plant-derived&#160;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&#160;its ability to effectively support osteogenic differentiation. The present study further&#160;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&#160;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&#160;energy-dispersive spectroscopy (EDS)&#160;confirmed the presence of phosphate and calcium elements. However, despite a significant increase in the Young&#39;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&#160;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.

Author Spotlight: Insights into the Use of Apple-Derived Cellulose Scaffolds for Bone Tissue Engineering 

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

The scope of the research is focused on evaluating the potential of apple-derived cellulose scaffold for bone tissue engineering and assessing their mechanical properties, both in vitro and in vivo. The question addressed include the osteogenic potentials of the scaffold, their ability to support cell invasion, mineralization, and their mechanical performance. The most recent development in the field of research involved the utilization of cellulose-based scaffold derived from various plants for tissue reconstruction.These scaffolds can be shaped and modified to achieve desired characteristics and functionalization techniques have been employed to enhance their effectiveness. The research gap addressed by this protocol is the limited investigation of the mechanical properties of apple-derived cellulose for bone tissue engineering. While previous studies have explored potential application of the scaffolds, their mechanical characteristics have not been extensively studied.The present study fills the gap by evaluating the mechanical properties of the scaffolds, highlighting their limitation compared to healthy bone tissue, and emphasizing the need for further development to optimize their performance. The findings of this study contribute to the advancements of research in the field by confirming that cellulose scaffold derived from apples have the potential to promote adhesion, osteoblastic differentiation, and proliferation of pre-osteoblastic cells. Following the study, one can ask questions such as, how can the mechanical properties of cellulose scaffolds that are from plants be optimized to closely match or mimic those of natural bone tissue?Chemical modifications or composite strategy can be employed to enhance the stiffness and load-bearing capacity of plant-derived cellulose scaffolds for bone tissue engineering applications.

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...

Watch this Scientific Journal Video about Insights into the Use of Apple-Derived Cellulose Scaffolds for Bone Tissue Engineering at JoVE.com

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.

Plant-derived&#160;cellulose biomaterials have been employed in various tissue engineering applications. In vivo studies have shown the remarkable ...

decellularized-apple-derived-scaffolds-for-bone-tissue-engineering

Research

JoVE Journal

Bioengineering

Preparation and Cell Seeding of Decellularized Apple-Derived Scaffolds

Pore Size Measurement and Cell Distribution Analysis of Decellularized Apple-Derived Scaffolds by Confocal Laser Scanning Microscopy

Alkaline Phosphatase Activity, Calcium Deposition, and Young&#39;s Modulus Measurements in Decellularized Apple-Derived Scaffolds

1.1K Views.  University of Ottawa.  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.

Analyzing Cell-Seeded Decellularized Apple-Derived Scaffolds for Bone Tissue Engineering

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