Abstract：
Although autogenous bone implantation is considered to be the gold standard for the reconstruction of bone defects, this approach remains challenging when treating extensive bone defects (EBDs). Therefore, artificial materials (AMs) such as artificial bone and scaffolds are often used for treating EBDs. Nevertheless, complications such as material failure, foreign body reaction, and infection are common. To overcome these issues, we aimed to develop a new treatment for an EBD using scaffold-free adipose-derived stromal cells (ADSCs) to fabricate chondrogenic/osteogenic-induced constructs without AMs.
ADSCs were obtained from the subcutaneous adipose tissue of 8-week-old female Wistar rats (n = 3) and assessed to determine their potential for multilineage differentiation into adipocytes (Oil Red O staining), chondrocytes (hematoxylin and eosin, Alcian blue, and Safranin O staining), and osteoblasts (Alizarin red and von Kossa staining). Spheroids (n = 320), each containing 3.0 × 104 ADSCs, were then used to fabricate scaffold-free cell constructs using a bio-3D printer with a needle array. The spheroids and constructs were stimulated with induction medium to induce chondrogenic and osteogenic differentiation. The induced cartilage- and bone-like constructs were finally evaluated using micro-computed tomography (μCT) and histological analysis.
The collected ADSCs were capable of trilineage differentiation, and were successfully used to produce scaffold-free constructs. The fabricated constructs (n = 3) exhibited equivalent strength (load, 195.3 ± 6.1 mN; strength, 39.1 ± 1.2 kPa; and stiffness, 0.09 ± 0.01 N/mm) to that of soft tissues such as the muscles in the uninduced condition. In chondrogenic induction experiments, Alcian blue and Safranin O staining confirmed the differentiation of the constructs into cartilage, and cartilage tissue-like structures were produced. In the osteogenic induction experiment, Alizarin Red and von Kossa staining showed calcium salt deposition, and μCT images confirmed the same calcification level as that of the cortical bone.
Scaffold-free constructs consisting of ADSCs without an AM were fabricated, and cartilage- and bone-like tissues were successfully generated, demonstrating their potential for bone reconstruction.
ADSCs were obtained from the subcutaneous adipose tissue of 8-week-old female Wistar rats (n = 3) and assessed to determine their potential for multilineage differentiation into adipocytes (Oil Red O staining), chondrocytes (hematoxylin and eosin, Alcian blue, and Safranin O staining), and osteoblasts (Alizarin red and von Kossa staining). Spheroids (n = 320), each containing 3.0 × 104 ADSCs, were then used to fabricate scaffold-free cell constructs using a bio-3D printer with a needle array. The spheroids and constructs were stimulated with induction medium to induce chondrogenic and osteogenic differentiation. The induced cartilage- and bone-like constructs were finally evaluated using micro-computed tomography (μCT) and histological analysis.
The collected ADSCs were capable of trilineage differentiation, and were successfully used to produce scaffold-free constructs. The fabricated constructs (n = 3) exhibited equivalent strength (load, 195.3 ± 6.1 mN; strength, 39.1 ± 1.2 kPa; and stiffness, 0.09 ± 0.01 N/mm) to that of soft tissues such as the muscles in the uninduced condition. In chondrogenic induction experiments, Alcian blue and Safranin O staining confirmed the differentiation of the constructs into cartilage, and cartilage tissue-like structures were produced. In the osteogenic induction experiment, Alizarin Red and von Kossa staining showed calcium salt deposition, and μCT images confirmed the same calcification level as that of the cortical bone.
Scaffold-free constructs consisting of ADSCs without an AM were fabricated, and cartilage- and bone-like tissues were successfully generated, demonstrating their potential for bone reconstruction.
摘要：
尽管自体骨植入被认为是重建骨缺损的金标准，这种方法在治疗广泛的骨缺损(EBD)时仍然具有挑战性.因此,人造材料(AM)如人造骨和支架通常用于治疗EBD。然而，并发症，如材料失效，异物反应,和感染是常见的。为了克服这些问题,我们旨在开发一种新的EBD治疗方法，使用无支架的脂肪来源的基质细胞（ADSCs）来制造软骨形成/成骨诱导的无AMs的构建体。
ADSCs从8周龄雌性Wistar大鼠（n=3）的皮下脂肪组织中获得，并评估其多向分化为脂肪细胞的潜力（油红染色O），软骨细胞（苏木精和伊红，阿尔辛蓝，和SafraninO染色)，和成骨细胞（茜素红和vonKossa染色）。类球体（n=320），每个包含3.0×104个ADSCs，然后使用带有针阵列的生物3D打印机制造无支架细胞构建体。用诱导培养基刺激球体和构建体以诱导软骨形成和成骨分化。最终使用微计算机断层扫描（μCT）和组织学分析评估诱导的软骨和骨样结构。
收集的ADSCs具有三系分化能力，并成功地用于生产无支架的构建体。制造的结构(n=3)表现出等效强度(载荷，195.3±6.1mN;强度,39.1±1.2kPa;和刚度,0.09±0.01N/mm），低于未诱导状态下的肌肉等软组织。在软骨诱导实验中，Alcianblue和SafraninO染色证实了构建体分化为软骨，并产生软骨组织样结构。在成骨诱导实验中,茜素红和vonKossa染色显示钙盐沉积，和μCT图像证实钙化水平与皮质骨相同。
制作了由无AM的ADSC组成的无支架构建体，软骨和骨样组织成功生成，展示了他们重建骨骼的潜力。
ADSCs从8周龄雌性Wistar大鼠（n=3）的皮下脂肪组织中获得，并评估其多向分化为脂肪细胞的潜力（油红染色O），软骨细胞（苏木精和伊红，阿尔辛蓝，和SafraninO染色)，和成骨细胞（茜素红和vonKossa染色）。类球体（n=320），每个包含3.0×104个ADSCs，然后使用带有针阵列的生物3D打印机制造无支架细胞构建体。用诱导培养基刺激球体和构建体以诱导软骨形成和成骨分化。最终使用微计算机断层扫描（μCT）和组织学分析评估诱导的软骨和骨样结构。
收集的ADSCs具有三系分化能力，并成功地用于生产无支架的构建体。制造的结构(n=3)表现出等效强度(载荷，195.3±6.1mN;强度,39.1±1.2kPa;和刚度,0.09±0.01N/mm），低于未诱导状态下的肌肉等软组织。在软骨诱导实验中，Alcianblue和SafraninO染色证实了构建体分化为软骨，并产生软骨组织样结构。在成骨诱导实验中,茜素红和vonKossa染色显示钙盐沉积，和μCT图像证实钙化水平与皮质骨相同。
制作了由无AM的ADSC组成的无支架构建体，软骨和骨样组织成功生成，展示了他们重建骨骼的潜力。
