Name: Insights into the Use of Apple-Derived Cellulose Scaffolds for Bone Tissue Engineering (Video) | JoVE
Uploaded: 2024-02-23T13:00:00
Description: Watch this Scientific Journal Video about Insights into the Use of Apple-Derived Cellulose Scaffolds for Bone Tissue Engineering at JoVE.com

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 t...

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

<ol>
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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.

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

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

Insights into the Use of Apple-Derived Cellulose Scaffolds for Bone Tissue Engineering (Video) | JoVE

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.