本项目由加拿大自然科学与工程研究理事会（NSERC）（发现资助）和李嘉诚基金会提供资助。MLL得到了安大略省卓越中心TalentEdge计划的支持，而RJH则得到了NSERC研究生奖学金和安大略省研究生奖学金（OGS）的支持。

实验方案由渥太华大学动物护理委员会审查和批准。
1. 支架准备
<ol><li>使用曼陀林切片机将麦金托什苹果（Canada Fancy）切成 8 毫米厚的薄片。将苹果片的 hypanthium 组织切成 5 毫米 x 5 毫米的正方形。</li>
	<li>将方形样品置于 0.1% 十二烷基硫酸钠 （SDS） 中 2 天。</li>
	<li>用去离子水洗涤脱细胞样品，并在室温 （RT） 下在 100 mM CaCl2 中孵育过夜以除去剩余的表面活性剂。</li>
	<li>在70%乙醇中对样品（即支架）灭菌30分钟，用去离子水洗涤，并在细胞接种前将其置于24孔培养板中。</li>
</ol>2. 细胞培养和支架接种
<ol><li>在细胞培养条件下（在95%空气和5%CO2的湿化气氛中为37°C），在直径为10cm的细胞培养处理的培养皿中维持MC3T3-E1亚克隆4细胞。</li>
	<li>制备由 Eagle 最低必需培养基 - α 修饰 （α-MEM） 制成的细胞培养基，并补充有 10% 胎牛血清 （FBS） 和 1% 青霉素/链霉素。</li>
	<li>一旦细胞达到80%汇合度，通过胰蛋白酶消化（0.05%胰蛋白酶-EDTA）将细胞从培养皿中分离出来。</li>
	<li>将细胞悬液以约200× g 离心3分钟。吸出上清液，并以每mL2.5×107 个细胞的速度将细胞重悬于α-MEM中。</li>
	<li>将40μL等分试样的细胞悬液移液到支架表面，让细胞在细胞培养条件下粘附1小时。随后，向培养板的每个培养孔中加入 2 mL 培养基。</li>
	<li>每 2-3 天补充一次培养基，持续 14 天。</li>
	<li>通过向先前描述的细胞培养基中加入 50 μg/mL 抗坏血酸和 4 mM 磷酸钠来制备分化培养基。</li>
	<li>通过将支架在分化培养基中孵育 4 周来诱导 MC3T3-E1 细胞的分化。每 3-4 天补充一次培养基。同时，将支架在非分化培养基（即没有添加剂诱导分化的培养基）中孵育相同的持续时间，使用相同的培养基更换时间表作为阴性对照。</li>
</ol>3. 使用共聚焦激光扫描显微镜测量孔径
<ol><li>用磷酸盐缓冲盐水 （PBS） 清洗脱细胞的苹果衍生支架。</li>
	<li>将支架在 1 mL 10% （v/v） calcofluor 白色染色剂中在室温下在黑暗中孵育 25 分钟。</li>
	<li>用PBS洗涤支架（n = 3），并使用DAPI通道，用高速共振共聚焦激光扫描显微镜（CLSM）以10倍放大倍率对每个支架随机选择的三个区域进行成像，如下所示：<ol><li>激光发射滤光片配置：405 nm（激光）;425-475 nm （发射）</li>
		<li>手动调整激光功率和探测器，以确保最佳图像采集。以 5 μm 步长获取 20 张图像的 z 堆栈。</li>
	</ol></li>
	<li>使用 ImageJ 软件，按如下方式处理和分析共聚焦图像：<ol><li>使用 “Z投影至最大强度 ”功能创建图像，并应用 “查找边缘 ”功能突出显示孔隙的边缘。</li>
		<li>使用 “手绘选择 ”工具手动描摹毛孔。</li>
		<li>将每个孔拟合为椭圆，测量长轴的长度，编译所有测量值（本研究中总共 54 个 - 每个支架随机选择的 3 个区域中的 6 个），并计算平均长度。</li>
	</ol></li>
</ol>4. 使用共聚焦激光扫描显微镜进行细胞分布分析
<ol><li>用PBS洗涤在非分化或分化培养基中培养的细胞接种支架三次。用4%多聚甲醛固定支架10分钟。</li>
	<li>用去离子水彻底洗涤每个支架，用Triton-X 100溶液透化细胞5分钟，然后再次用PBS洗涤。</li>
	<li>将支架在 1 mL 1% 高碘酸中孵育 40 分钟，并用去离子水冲洗 14,16。</li>
	<li>将支架在含有 100 mM 焦亚硫酸钠和 0.15 M 盐酸的 1 mL 溶液中孵育，并补充有 100 μg/mL 碘化丙啶。将支架完全浸入溶液中。</li>
	<li>用PBS洗涤支架，并通过将支架在5mg / mL DAPI溶液中在黑暗中孵育10分钟来染色细胞核。再次彻底清洗并在成像前将支架存放在 PBS 中。</li>
	<li>使用 DAPI 和 TRITC 通道，在 10 倍放大倍率下使用高速共振 CLSM 对三种不同细胞种子支架的三个随机选择的表面进行成像，如下所示：<ol><li>激光发射滤光片配置： DAPI：激光：405 nm;发射波长：425-475 nm TRITC：激光：561 nm;发射波长：570-620 nm</li>
		<li>手动调整激光功率和探测器，以确保最佳图像采集。以 5 μm 步长获取 20 张图像的 z 堆栈。</li>
	</ol></li>
	<li>使用 ImageJ 软件处理共聚焦图像，并在 z 轴上创建最大投影，以便使用 Z 投影到最大强度 功能进行图像分析。</li>
</ol>5. 碱性磷酸酶分析
<ol><li>用PBS洗涤在非分化或分化培养基中培养的细胞接种支架三次。用10%中性缓冲福尔马林固定支架30分钟。固定空白支架（没有接种细胞的支架）作为阴性对照。</li>
	<li>通过将一片 BCIP/NBT 片剂溶解在 10 mL 去离子水中来制备 5-溴-4-氯-3'-吲哚磷酸盐和硝基蓝四唑 （BCIP/NBT） 染色溶液。</li>
	<li>用0.05%吐温溶液洗涤固定支架，并在室温下用BCIP / NBT溶液染色20分钟。</li>
	<li>用 1200 万像素数码相机对染色的支架进行成像。</li>
</ol>6. 钙沉积分析
<ol><li>用PBS洗涤在非分化或分化培养基中培养的细胞接种支架三次。用10%中性缓冲福尔马林固定支架30分钟。固定空白支架（没有接种细胞的支架）作为阴性对照。</li>
	<li>制备2%（w / v）茜素红S（ARS）染色溶液。</li>
	<li>用去离子水洗涤固定支架，并在室温下用ARS溶液染色1小时。</li>
	<li>用 1200 万像素数码相机对染色的支架进行成像。</li>
</ol>7. 矿化分析
<ol><li>用PBS洗涤在非分化或分化培养基中培养的细胞接种支架三次。用4%多聚甲醛固定支架48小时。固定空白支架（没有接种细胞的支架）作为阴性对照。</li>
	<li>如前所述，在含有乙醇浓度从50%增加到100%的溶液中对样品进行脱水23。</li>
	<li>执行扫描电子显微镜 （SEM） 和能量色散光谱 （EDS） 以分析矿物聚集体，如下所示：<ol><li>按照制造商的方案24，使用临界点干燥器干燥样品。</li>
		<li>按照制造商的方案25，使用金溅射镀膜机在支架上涂上 5 nm 金涂层。</li>
		<li>使用设置为3 kV，放大倍率为85倍的扫描电子显微镜对支架表面进行成像。</li>
		<li>通过将扫描电子显微镜设置为 15 kV 来执行 EDS。在每个支架随机选择的三个区域上，获取用于矿物聚集体成分分析的 EDS 光谱。</li>
	</ol></li>
</ol>8. 杨氏模量测量
<ol><li>从各自的孵育培养基中取出细胞接种的支架，并立即测试样品。</li>
	<li>使用配备 1.5 N 称重传感器的定制单轴压缩装置，以 3 mm·min-1 的恒定速率将支架（每种条件下 n = 3）压缩至支架高度 10% 的最大压缩应变。</li>
	<li>根据应力-应变曲线线性部分的斜率确定杨氏模量。在本研究中，模量在9%至10%应变之间确定。</li>
</ol>9. 组织学细胞浸润和矿化分析： 体外 支架
<ol><li>用PBS洗涤在非分化或分化培养基中培养的细胞接种支架三次。</li>
	<li>用4%多聚甲醛固定细胞接种的支架48小时，然后重悬于70%乙醇中储存。</li>
	<li>组织学： 注：在本研究中，后续步骤中描述的整个组织学准备（包埋、切片和染色）由 Louise Pelletier 组织学核心设施（渥太华大学）进行。<ol><li>脱水并包埋在石蜡中后，将样品切成 5 μm 厚的连续切片，从支架内 1 mm 开始，然后将切片安装在显微镜载玻片上。</li>
		<li>用苏木精和伊红 （H&E） 或 von Kossa （VK） 染色切片。</li>
		<li>用载玻片扫描仪显微镜以 40 倍放大倍率对切片进行成像（在本研究中，非分化培养基中的 n = 1 个支架，分化培养基中的 n = 2 个支架）。</li>
		<li>使用 ImageJ 软件，目视评估细胞浸润（H&E 染色）和矿化（VK 染色）。</li>
	</ol></li>
</ol>10. 大鼠颅骨缺损模型
<ol><li>获得当地动物护理委员会审查和批准的实验方案。</li>
	<li>按照上述第 1 节并使用 5 毫米活检冲头制备圆形（直径 5 毫米，厚度为 1 毫米）脱细胞支架。</li>
	<li>按照既定方案26 进行双侧开颅手术，如下所示：<ol><li>用异氟醚麻醉雄性Sprague-Dawley大鼠，首先以3%麻醉直到它们失去知觉，然后在整个过程中以2-3%麻醉。</li>
		<li>使用手术刀刀片切割覆盖的皮肤，暴露骨膜和颅骨。切除骨膜。</li>
		<li>在 0.9% NaCl 的持续冲洗下，使用配备有 5 毫米直径环钻的牙钻在矢状缝线两侧的双侧顶骨中形成双侧缺损。</li>
		<li>用 0.9% NaCl 清洁周围的骨骼以去除任何骨碎片。</li>
		<li>将圆形脱细胞支架放置在缺陷中。</li>
		<li>用 4-0 缝合线封闭覆盖的皮肤。</li>
	</ol></li>
	<li>让老鼠无限制地获取食物和水，并每天监测它们。</li>
	<li>植入后 8 周后，通过吸入 CO2 和胸穿孔对大鼠实施安乐死作为次要安乐死措施。</li>
	<li>要暴露颅骨并取回植入物，请使用手术刀刀片去除覆盖颅骨的皮肤。</li>
	<li>使用牙钻在额骨和枕骨以及两个顶骨的侧面切开颅骨，以完全切除颅骨的顶部。</li>
</ol>11. 推出测试
<ol><li>将单轴压缩设备（带有 445 N 称重传感器）连接到 USB 数据采集模块。</li>
	<li>将数据采集模块连接到配备数据采集软件应用程序的计算机。</li>
	<li>颅骨提取后，立即将每个样本（本研究中来自 4 只动物的 n = 7 个植入物）放在单轴压缩装置的样品架上，使骨骼的背侧朝上。</li>
	<li>以 0.5 毫米/分钟的速度降低柱塞，直到稍微接触拔出的植入物。</li>
	<li>通过使用数据采集软件以 0.5 mm/min 的恒定速度施加压缩，同时将柱塞降低到植入物直至完全推出，从而开始测试。</li>
	<li>使用数据采集软件在力与位移曲线上记录峰值力。</li>
</ol>12. 组织学细胞浸润和矿化分析： 体内 支架
<ol><li>将提取的颅骨和植入物固定在10%中性缓冲福尔马林中72小时，然后重悬于70%乙醇中储存。</li>
	<li>组织学： 注：在本研究中，后续步骤中描述的所有组织学制备（包埋、切片和染色）均由 Accel Labs（加拿大魁北克省蒙特利尔）进行。<ol><li>将样品（嵌入甲基丙烯酸甲酯中）切成三个不同水平（顶部，底部和朝向中心）的6μm厚切片，并将它们安装在显微镜载玻片上。</li>
		<li>用 H&E 或 Masson-Goldner 三色 （MGT） 对切片进行染色。</li>
	</ol></li>
	<li>用载玻片扫描仪显微镜以 40 倍放大倍率对切片成像（本研究中 2 只动物的 4 个外植体）。</li>
	<li>使用 ImageJ 软件，目视评估细胞浸润（H&E 染色）和胶原蛋白沉积（MGT 染色）。</li>
</ol>

分析细胞接种的脱细胞苹果衍生支架用于骨组织工程

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H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Induced+osteogenesis+in+plants+decellularized+scaffolds.">Induced osteogenesis in plants decellularized scaffolds.</a> Scientific Reports. 9 (1), 20194 (2019).</li><li>Zhao, J., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Enhanced+healing+of+rat+calvarial+defects+with+sulfated+chitosan-coated+calcium-deficient+hydroxyapatite/bone+morphogenetic+protein+2+scaffolds.">Enhanced healing of rat calvarial defects with sulfated chitosan-coated calcium-deficient hydroxyapatite/bone morphogenetic protein 2 scaffolds.</a> Tissue Engineering. Part A. 18 (1-2), 185-197 (2012).</li><li>Murtey, M. D., Ramasamy, P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=.">.</a> Sample Preparations for Scanning Electron Microscopy - Life Sciences. In: Modern Electron Microscopy in Physical and Life Sciences. , 161-186 (2016).</li><li>. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=.">.</a> tousimis Critical Point Dryers - Samdri&#174;-PVT-3D. , (2022).</li><li>. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=.">.</a> Leica EM ACE200 Vacuum Coater. , (2023).</li><li>Spicer, P. P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Evaluation+of+bone+regeneration+using+the+rat+critical+size+calvarial+defect.">Evaluation of bone regeneration using the rat critical size calvarial defect.</a> Nature Protocols. 7 (10), 1918-1929 (2012).</li><li>Leblanc Latour, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=.">.</a> Cellulose biomaterials for bone tissue engineering [dissertation]. , (2023).</li><li>Kodama, H. -. A., Amagai, Y., Sudo, H., Kasai, S., Yamamoto, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Establishment+of+a+clonal+osteogenic+cell+line+from+newborn+mouse+calvaria.">Establishment of a clonal osteogenic cell line from newborn mouse calvaria.</a> Japanese Journal of Oral Biology. 23 (4), 899-901 (1981).</li><li>Wang, D., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Isolation+and+characterization+of+MC3T3-E1+preosteoblast+subclones+with+distinct+in+vitro+and+in+vivo+differentiation/mineralization+potential.">Isolation and characterization of MC3T3-E1 preosteoblast subclones with distinct in vitro and in vivo differentiation/mineralization potential.</a> Journal of Bone and Mineral Research. 14 (6), 893-903 (1999).</li><li>Addison, W. N., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Extracellular+matrix+mineralization+in+murine+MC3T3-E1+osteoblast+cultures:+An+ultrastructural,+compositional+and+comparative+analysis+with+mouse+bone.">Extracellular matrix mineralization in murine MC3T3-E1 osteoblast cultures: An ultrastructural, compositional and comparative analysis with mouse bone.</a> Bone. 71, 244-256 (2015).</li><li>Heary, R. F., Parvathreddy, N., Sampath, S., Agarwal, N. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Elastic+modulus+in+the+selection+of+interbody+implants.">Elastic modulus in the selection of interbody implants.</a> Journal of Spine Surgery. 3 (2), 163-167 (2017).</li><li>Lawson, Z. 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利益冲突声明：M.L.L、M.T. R.J.H.、C.M.C.、I.C. 和 AP 是渥太华大学和 Spiderwort Inc. 提交的关于将植物衍生纤维素用于 BTE 申请的专利申请的发明人。M.L.L.、R.J.H.、C.M.C. 和 AP 在 Spiderwort Inc. 拥有经济利益。

几项体外和体内研究已经证明了植物来源纤维素的生物相容性及其在组织工程中的潜在用途 14,15,16,18,19,20，更具体地说是用于宿主成骨分化 20,21.本研究的目的是进一步研究苹果衍生的纤维素...

由损伤或疾病引起的大骨缺损通常需要生物材料移植物才能完全再生1.目前旨在改善骨组织再生的技术通常使用自体、同种异体、异种或合成移植物2。对于自体骨移植，被认为是修复大骨缺损的“金标准”移植实践，从患者身上提取骨骼。然而，这种移植手术有几个缺点，包括大小和形状限制、组织可用性和采样部位发病率3。此外，自体移植手术容易受到手术部位感染、随后的骨折、取样或重建部位血肿形成以及术后疼痛的影响4.骨组织工程 （BTE） 为传统骨移植方法提供了一种潜在的替代方案5.它结合了结构性生物材料和细胞来构建新的功能性骨组织。在设计用于 BTE 的生物材料时，将大孔结构、促进细胞附着的表面化学以及与天然骨非常相似的机械性能相结合至关重要6.过去的研究表明，BTE 中使用的生物材料的理想孔径和弹性模量分别约为 100-200 μm7 和 0.1-20 GPa，具体取决于接枝位点8。此外，支架的孔隙度和孔隙互连性是影响细胞迁移、营养扩散和血管生成的关键因素8。
BTE在开发和评估各种生物材料作为骨移植物的替代选择方面显示出有希望的结果。其中一些生物材料是骨诱导材料、混合材料和高级水凝胶8.骨诱导材料刺激新形成的骨骼结构的发育。混合材料由合成和/或天然聚合物组成8.先进的水凝胶模仿细胞外基质 （ECM），能够提供必要的生物活性因子来促进骨组织整合8.羟基磷灰石是一种传统材料，由于其成分和生物相容性，是 BTE 的常见选择9.生物活性玻璃是BTE的另一种生物材料，已被证明可以刺激特定的细胞反应以激活成骨所必需的基因10,11。可生物降解的聚合物，包括聚乙醇酸和聚乳酸，也已广泛用于BTE应用12。最后，壳聚糖、几丁质和细菌纤维素等天然或天然衍生的聚合物也显示出令人鼓舞的 BTE13 结果。然而，虽然合成聚合物和天然聚合物都显示出BTE的潜力，但开发具有所需宏观结构的功能支架通常需要广泛的方案。
相反，天然的宏观纤维素结构可以很容易地从不同的植物中衍生出来，我们的研究小组之前证明了来自植物的纤维素基支架对不同组织重建的适用性。事实上，经过简单的表面活性剂处理后，我们利用了植物材料的固有结构，突出了其作为多功能生物材料的潜力14。此外，这些基于纤维素的支架可用于体外哺乳动物细胞培养应用14，具有生物相容性，并支持自发皮下血管形成14,15,16,17。我们的研究小组和其他人都已经证明，这些支架可以根据预期的应用从特定植物中获得 14,15,16,17,18,19,20。例如，在植物茎和叶中观察到的维管结构与在动物组织中发现的结构表现出惊人的相似性19。此外，源自植物的纤维素支架可以很容易地成型并进行表面生化修饰，以实现所需的特性16。在最近的一项研究中，我们在脱细胞过程中掺入了盐缓冲液，从而增强了在体外和体内环境中观察到的细胞附着16。在同一项研究中，我们通过将水凝胶浇注到支架表面，证明了植物来源的纤维素支架在复合生物材料中的适用性。在最近的研究中，植物来源支架的功能化已被证明可以提高其有效性18.例如，Fontana等人（2017）进行的一项研究表明，人真皮成纤维细胞的粘附得到了RGD涂层脱细胞茎的支持，而未涂层的茎则没有表现出相同的能力18。此外，作者还证明，改良的模拟体液可用于人工矿化脱细胞的植物茎。在最近的研究中，我们探索了植物来源的纤维素支架中机械敏感成骨的概念，并评估了它们对 BTE17,20 的潜力。此外，Lee 等人（2019 年）利用植物来源的支架在体外环境中培养骨样组织21。通过对不同植物来源的综合评估，作者确定苹果衍生的支架是人类诱导多能干细胞（hiPSCs）培养和分化的最佳选择。此外，作者提出，苹果衍生支架的结构和机械属性在其是否适合预期用途方面起着关键作用。作为组织工程应用中最早实施的植物衍生支架，苹果衍生支架已被广泛证明具有与人体骨骼惊人相似的结构，特别是在其直径为 100 至 200 μm 的相互连接的孔隙方面14,21。
在本研究中，我们进一步研究了苹果衍生纤维素支架对BTE的潜力，并对其在 体外 和 体内的力学性能进行了分析。尽管已经对苹果衍生支架对 BTE17、20、21 的潜力进行了研究，但其机械性能尚未得到充分研究。结果显示，在分化培养基中培养 4 周的支架中接种的 MC3T3-E1 成骨前细胞具有野生扩散侵袭和成骨分化。这些支架的杨氏模量为192.0±16.6 kPa，显著高于空白支架（无接种细胞的支架）（31.6±4.8 kPa）和在非分化培养基中培养的细胞接种支架（24.1±8.8 kPa）。然而，应该注意的是，健康人体骨组织的杨氏模量通常在小梁骨的 0.1-2 GPa 范围内，皮质骨的 Young 模量通常在 15-20 GPa 的范围内8.然而，在啮齿动物颅骨缺损中植入 8 周后，细胞播种的支架似乎很好地融入了周围的骨骼，正如推出测试中的平均峰值力为 113.6 N ± 18.2 N 所证明的那样，这与先前报道的天然颅骨22 的骨折负荷相似。总体而言，从这项研究中获得的结果显示出巨大的前景，特别是对于非承重应用。然而，苹果衍生的纤维素支架目前不具备必要的机械性能，无法精确匹配植入部位的周围骨组织。因此，需要进一步开发以释放这些支架的全部潜力。

<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

植物来源的纤维素生物材料已被用于各种组织工程应用。 体内 研究表明，由天然来源的纤维素制成的支架具有显着的生物相容性。此外，这些支架具有与多种组织相关的结构特征，它们促进哺乳动物细胞的侵袭和增殖。最近使用脱细胞苹果蒺菌组织的研究表明，其孔径与小梁骨的孔径相似，并且能够有效支持成骨分化。本研究进一步研究了苹果衍生纤维素支架在骨组织工程（BTE）应用中的潜力，并评估了其 体外 和 体内 力学性能。将 MC3T3-E1 成骨前细胞接种在苹果来源的纤维素支架中，然后评估其成骨潜力和机械性能。碱性磷酸酶和茜素红 S 染色证实了在分化培养基中培养的支架的成骨分化。组织学检查显示整个支架上有广泛的细胞侵袭和矿化。扫描电子显微镜（SEM）显示支架表面有矿物聚集体，能量色散光谱（EDS）证实了磷酸盐和钙元素的存在。然而，尽管细胞分化后杨氏模量显着增加，但仍低于健康骨组织。 体内 研究表明，在大鼠颅骨中植入 8 周后， 脱细胞的苹果衍生支架内的细胞浸润和细胞外基质的沉积。此外，从骨缺损中移除支架所需的力与先前报道的自体颅骨骨折负荷相似。总体而言，这项研究证实了苹果衍生的纤维素是BTE应用的有前途的候选者。然而，其机械性能与健康骨组织之间的差异可能会限制其在低承重情况下的应用。可能需要进行额外的结构重新设计和优化，以增强用于承重应用的苹果衍生纤维素支架的机械性能。

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.

作者聚焦：深入了解 Apple-derived Cellulose 支架在骨组织工程中的应用 

用于体外和体内骨组织工程的脱细胞苹果衍生支架

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.

孔径测量、细胞分布和 体外 矿化（图1 和 图2） 用 SDS 和 CaCl2 处理支架后，完全去除苹果组织支架的天然细胞成分（图 1A）。支架表现出高度多孔的结构，使用共聚焦显微镜证实了这一点。图像的定量表明平均孔径为 154 μm ± 40 μm。孔径分布范围在73 μm至288 μm之间。然而，大多数孔隙在100μm至200μm?...

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

在这项研究中，我们详细介绍了植物基生物材料的脱细胞、物理表征、成像和 体内 植入方法，以及支架中细胞接种和分化的方法。所描述的方法允许评估用于骨组织工程应用的植物基生物材料。

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

研究范围的重点是评估苹果衍生的纤维素支架在骨组织工程中的潜力，并评估其在体外和体内的机械性能。所讨论的问题包括支架的成骨潜力、它们支持细胞侵袭、矿化的能力以及它们的机械性能。该研究领域的最新进展涉及利用来自各种植物的纤维素基支架进行组织重建。这些支架可以被塑造和修改，以实现所需的特性，并且已经采用了功能化技术来增强其有效性。该协议解决的研究差距是对用于骨组织工程的苹果衍生纤维素的机械性能的有限研究。虽然以前的研究已经探索了支架的潜在应用，但其机械特性尚未得到广泛研究。本研究通过评估支架的机械性能来填补这一空白，强调它们与健康骨组织相比的局限性，并强调需要进一步开发以优化其性能。这项研究的结果证实了来自苹果的纤维素支架具有促进成骨细胞前细胞粘附、成骨细胞分化和增殖的潜力，从而促进了该领域的研究进展。在研究之后，人们可以提出一些问题，例如，如何优化来自植物的纤维素支架的机械性能，以密切匹配或模仿天然骨组织的机械性能？可以采用化学改性或复合策略来增强植物来源的纤维素支架的刚度和承载能力，用于骨组织工程应用。

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

Research

JoVE Journal

Bioengineering

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

1.1K 次观看.  University of Ottawa. 研究范围的重点是评估苹果衍生的纤维素支架在骨组织工程中的潜力，并评估其在体外和体内的机械性能。所讨论的问题包括支架的成骨潜力、它们支持细胞侵袭、矿化的能力以及它们的机械性能。该研究领域的最新进展涉及利用来自各种植物的纤维素基支架进行组织重建。这些支架可以被塑造和修改，以实现所需的特性，并且已经采用了功能化技术来增强其有效性。?...

视频: 作者聚焦：深入了解 Apple-derived Cellulose 支架在骨组织工程中的应用 

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.

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.