BACKGROUND:Scholars are stil looking for ideal bone tissue-engineered scaffolds, and three-dimensional (3D) printing technology is a novel construction method. In the meanwhile, bone extracel ular matrix is becoming a hotspot in osteogenic induction. OBJECTIVE:To construct the polycaprolactone/bone extracel ular matrix scaffold using 3D printing technology and co-culture method, and to detect its osteogenic property. METHODS:216 3D-printed polycaprolactone scaffolds were divided into group A (96 pores, n=72) and group B(48 pores, n=144). Passage 5 bone marrow mesenchymal stem cel s from Sprague-Dawley rats were seeded onto the two kinds of polycaprolactone scaffolds, and the group A was used for alizarin red staining and Masson staining, while the group B for col agen and glycosaminoglycan detection at 1, 2 and 3 weeks of incubation. Afterwards, the scaffolds at 1, 2 and 3 weeks of culture were decel ularized and labeled as groups AE1, AE2, AE3, BE1, BE2 and BE3. Then passage 5 bone marrow mesenchymal stem cel s from Sprague-Dawley rats were seeded onto each scaffold again, and the former three groups underwent alizarin red staining, and the latter three were used for calcium, alkaline phosphatase activity and DNA quantitative analysis at 1, 2 and 3 weeks of culture. RESULTS AND CONCLUSION:Masson staining, glycosaminoglycan and hydroxyproline quantitative analysis showed that the extracel ular matrix on the composite scaffold increased with time. Alkaline phosphatase activity revealed that the composite scaffold had a significantly stronger osteogenic differentiation than the normal polycaprolactone scaffold (P<0.05). Alizarin red staining and calcium quantitative analysis showed that the mineralization of the composite scaffold was more obvious than that of the normal polycaprolactone scaffold (P<0.05), but the total DNA analysis did not differ significantly between scaffolds. These results suggest that the composite scaffold with extracel ular matrix is constructed successful y using the 3D technology and co-culture method and exhibits a better osteoinductivity.%背景：骨组织工程学者们仍在寻找理想的骨组织工程支架，3D打印技术为支架的构建提供了新的方法，同时骨细胞外基质在成骨诱导中的作用也越来越受到关注。目的：采用3D细胞-支架共培养的方式获取聚己内酯-骨细胞外基质复合支架，检测其成骨性能。方法：将216枚3D打印聚己内酯支架分为A组(与96孔板匹配，n=72)、B组(与48孔板匹配，n=144)。①第1轮三维培养：将第5代SD大鼠骨髓间充质干细胞分别接种于两组支架上，接种1，2，3周，A组进行茜素红染色、Masson染色，B组进行胶原、糖胺多糖检测；②第2轮三维培养：将第1轮三维培养1，2，3周的支架进行脱细胞处理，分别记为AE1、AE2、AE3、BE1、BE2、BE3，将第5代SD大鼠骨髓间充质干细胞再次接种于6组支架上，接种1，2，3周，AE1、AE2、AE3组进行茜素红染色，BE1、BE2、BE3组进行钙定量、碱性磷酸酶活性及DNA定量分分析。结果与结论：①Masson染色及糖胺多糖、羟脯氨酸定量分析结果提示，随着培养时间延长，复合支架表面的细胞外基质成分逐渐增加；②碱性磷酸酶活性检测结果提示，聚己内酯-骨细胞外基质复合支架的细胞成骨分化程度显著高于普通聚己内酯支架(P<0.05)；③茜素红染色分析及钙定量分析结果提示，复合支架的矿化程度显著高于普通聚己内酯支架(P<0.05)，复合支架的DNA总量相对于普通样本并没有显著升高；④结果表明，通过3D细胞-支架共培养的方式可以获得带有细胞外基质的复合支架，该复合支架具备更好的成骨诱导性能。
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