牙髓干细胞在实验兔牙槽骨缺损修复中的作用研究

Efficacy of dental pulp stem cells in repairing experimental alveolar bone defect

目的探索牙髓干细胞(DPSC)在兔牙槽骨缺损再生修复中的作用,为临床上骨缺损的修复提供一定的理论基础。方法 (1)从新西兰幼兔(4周)前牙及磨牙牙髓组织中分离DPSC进行体外培养,测定细胞克隆形成率、抗Vimentin、抗CD44免疫组化染色,鉴定DPSC的增殖能力和多分化潜能的生物学特征。(2)16只实验新西兰兔随机分为空白组,实验组两组,每组8只,均在动物下颌无牙区牙槽骨人工制备10 mm×4 mm×4 mm骨缺损区,其中空白组骨缺损区填入浸有PBS溶液的0.25 g Bio-oss骨粉,实验组加入1×108/L的DPSC及0.25 g Bio-oss骨粉;于术后第6周同期处死动物,无菌获取牙槽骨组织,制作石蜡切片,HE染色及免疫组化检测骨涎蛋白(BSP)。组间图像平均光密度比较采用两样本t检验。结果 (1)原代培养的幼兔牙髓细胞多呈成纤维细胞样细胞,少数纺锤形或多角形,光镜下传代培养的细胞形态特点与原代培养无明显差异;幼兔牙髓细胞体外多次传代仍具有良好的增殖能力;原代和次代克隆,免疫细胞化学对未经诱导的细胞进行表面标志物Vimentin、CD44鉴定,结果均为染色阳性。(2)术后6周,空白组HE染色可见少量红细胞,未见明显新骨生成,见少量成骨细胞;BSP免疫组化成弱阳性表现,图像平均光密度0.192±0.030。实验组HE染色提示炎性渗出明显吸收,可见新生骨组织,骨缺损区周围有大量成骨细胞,骨小梁较空白组致密,BSP免疫组化成强阳性表达,图像平均光密度0.258±0.035,与空白组相比,差异有统计学意义(P<0.05)。结论 DPSC具有向成骨细胞分化的潜能,可促进牙槽骨缺损修复再生。

Objective To explore the efficacy of dental pulp stem cells(DPSC) in repairing rabbit alveolar bone defect. Methods(1) DPSC were isolated from pulp tissue of New Zealand rabbits incisors and molars and cultured in vivo. Cells were stained for Vimentin and CD44 as indicators for differentiation. Cell proliferation was evaluated;(2) 16 New Zealand rabbits were divided into control group and experimental group randomly. 10 mm×4 mm×4mm alveolar bone defect were made in animal mandibular edentulous. In the experimental group, the defect was filled with 1 × 108/L DPSC and 0.25 g Bio-oss powder and in the control group, the defect was filled with 0.25 g Bio-oss powder only. The peripheral tissue coating of alveolar bone defect was harvested for histopathologic study 6 week later. Results(1) Primary cultures of immature rabbit dental pulp cells were fibroblast-liked, spindle-shaped or polygonal, there was no significant difference in cell morphology after passaging under light microscope. Cells proliferated well in vitro; Cells expressed osteoblast marker Vimentin and CD44;(2) 6 weeks later after treatment, HE staining showed few red blood cells, no obvious new bone formation and weak expression of BSP in the control group(MOD= 0.192 ± 0.030), while inflammatory exudation was absorbed in the experimental group, with new bone formation positive staining of BSP(MOD = 0.258 ± 0.035, P < 0.05). Conclusions Dental pulp stem cells may be a potential treatment for alveolar bone defect.