Saito Shizu, Hamai Ryo, Shiwaku Yukari, Hasegawa Tomoka, Sakai Susumu, Tsuchiya Kaori, Sai Yuko, Iwama Ryosuke, Amizuka Norio, Takahashi Tetsu, Suzuki Osamu
Division of Oral and Maxillofacial Surgery, Tohoku University Graduate School of Dentistry, Sendai 980-8575, Japan; Division of Craniofacial Function Engineering, Tohoku University Graduate School of Dentistry, Sendai 980-8575, Japan.
Division of Craniofacial Function Engineering, Tohoku University Graduate School of Dentistry, Sendai 980-8575, Japan.
Acta Biomater. 2021 Jul 15;129:309-322. doi: 10.1016/j.actbio.2021.05.017. Epub 2021 May 24.
This study hypothesized that distant octacalcium phosphate (OCP) scaffolds may enhance osteocyte differentiation in newly formed bone matrices. The results obtained were compared with those of Ca-deficient hydroxyapatite (OCP hydrolyzate, referred to as HL hereafter). Granular OCP and HL, 300-500 µm in diameter, were implanted in critical-sized rat calvarial defects for eight weeks and subjected to histology, immunohistochemistry, histomorphometry, and transmission electron microscopy (TEM). Early osteocyte differentiation from an osteoblastic cell line (IDG-SW3) was examined using materials without contacting the surfaces for 10 days. The material properties and the medium composition were analyzed through selected area electron diffraction (SAED) using TEM observation and curve fitting of Fourier transform infrared (FT-IR) spectroscopy. The number of positive cells of an osteocyte earlier differentiation marker podoplanin (PDPN) in bone matrices, along the direction of bone formation, was significantly higher in OCP than that in HL. The ultrastructure around the OCP surfaces observed by TEM showed the infiltration of some cells, including osteocytes adjacent to the OCP surface layers. The OCP structure remained unchanged by SAED analysis. Nanoparticle deposition and hydrolysis on OCP surfaces were detected by TEM and FT-IR, respectively, during early osteocyte differentiation in vitro. The medium saturation degree varied in accord with ionic dissolution, resulting in possible hydroxyapatite formation on OCP but not on HL. These results suggested that OCP stimulates early osteocyte differentiation in the bone matrix from a distance through its metastable chemical properties. STATEMENT OF SIGNIFICANCE: This study demonstrated that octacalcium phosphate (OCP) implanted in critical-sized rat calvaria bone defects is capable of enhancing the early differentiation of osteocytes embedded in newly formed bone matrices, even when the surface OCP is separated from the osteocytes. This prominent bioactive property of OCP was demonstrated by comparing the in vivo and in vitro performances with a control material, Ca-deficient hydroxyapatite (OCP hydrolyzate). The findings were elucidated by histomorphometry, which analyzed the differentiation of osteocytes along the parallel direction of new bone growth by osteoblasts. Therefore, OCP should stimulate osteocyte differentiation through ionic dissolution even in vivo owing to its metastable chemical properties, as previously reported in an in vitro study (Acta Biomater 69:362, 2018).
本研究假设，远处的磷酸八钙（OCP）支架可增强新形成骨基质中骨细胞的分化。将所得结果与缺钙羟基磷灰石（OCP水解产物，以下简称HL）的结果进行比较。将直径为300 - 500 µm的颗粒状OCP和HL植入大鼠颅骨临界尺寸缺损处8周，并进行组织学、免疫组织化学、组织形态计量学和透射电子显微镜（TEM）检查。使用未接触表面10天的材料检测来自成骨细胞系（IDG - SW3）的早期骨细胞分化。通过TEM观察的选区电子衍射（SAED）和傅里叶变换红外（FT - IR）光谱的曲线拟合分析材料特性和培养基成分。在骨基质中，沿骨形成方向，早期骨细胞分化标志物血小板内皮细胞黏附分子（PDPN）的阳性细胞数量在OCP组显著高于HL组。TEM观察到的OCP表面周围超微结构显示有一些细胞浸润，包括与OCP表层相邻的骨细胞。SAED分析显示OCP结构未改变。在体外早期骨细胞分化过程中，分别通过TEM和FT - IR检测到OCP表面的纳米颗粒沉积和水解。培养基饱和度随离子溶解而变化，导致OCP表面可能形成羟基磷灰石，而HL表面则不会。这些结果表明，OCP通过其亚稳化学性质从远处刺激骨基质中的早期骨细胞分化。重要性声明：本研究表明，植入大鼠颅骨临界尺寸骨缺损处的磷酸八钙（OCP）能够增强新形成骨基质中骨细胞的早期分化，即使表面的OCP与骨细胞分离。通过与对照材料缺钙羟基磷灰石（OCP水解产物）比较体内和体外性能，证明了OCP这种突出的生物活性特性。通过组织形态计量学阐明了研究结果，该方法分析了成骨细胞沿新骨生长平行方向的骨细胞分化情况。因此，正如先前在一项体外研究中报道的那样（《生物材料学报》69:362，2018），由于其亚稳化学性质，OCP即使在体内也应通过离子溶解刺激骨细胞分化。
