Clausen Christian, Hermund Niels Ulrich, Donatsky Ole, Nielsen Henrik
Interface Biotech A/S, c/o Glostrup Hospital, Opgang 8, 4. sal. 2600 Glostrup, Denmark.
Clin Oral Implants Res. 2006 Oct;17(5):533-40. doi: 10.1111/j.1600-0501.2006.01254.x.
In this study, we have characterized bone cell cultures derived from the human maxillary alveolar ridge, which could be a potential cell source for tissue engineering of the severely resorbed maxilla. From 10 individuals, an osseous core was obtained. Without the use of collagenase, 10 explant cultures were established and the morphology of the cells (human maxilla-derived cells (hMDCs)) was studied with light microscopy (LM). Explant cultures were analyzed by flow cytometry with respect to size, granularity and surface marker expression. Fluorochrom-conjugated monoclonal antibodies (CD13, CD31, CD44, CD90 or CD73) were used. hMDCs were cultured in standard medium (SCM) or osteoinductive medium (OIM) for 21 days and analyzed for the presence of alkaline phosphatase (ALP) and calcium deposits (Von Kossa). Furthermore, osteogenic gene expression (osteocalcin [OC], ALP, collagen type 1) were analyzed by reverse transcription polymerase chain reaction (RT-PCR). LM demonstrated that hMDCs had a polygonal morphology containing a central nucleus with two to three nucleoli. Size/granularity analysis revealed differences between individuals. Immunophenotypically, these cells were positive for CD13, CD44, CD90 and CD73 while negative for CD31. Cells cultured in SCM for 21 days showed moderate ALP staining and many calcium deposits. Culturing cells in OIM for 21 days significantly increased both ALP staining and the number of calcium deposits. RT-PCR demonstrated expression of osteogenic marker genes and the ability to upregulate osteocalcin and ALP in response to osteogenic inducers. To our knowledge, it is the first time that surface marker expression has been studied on bone cells originating from this site. Cells were positive for markers characteristic for immature mesenchymal stem cells and had osteogenic differentiation capability. This study indicates that cells derived from maxillary biopsies could be a potential cell source for bone tissue engineering.
在本研究中，我们对源自人上颌牙槽嵴的骨细胞培养物进行了特性分析，其可能是严重吸收的上颌骨组织工程的潜在细胞来源。从10名个体获取了骨芯。在不使用胶原酶的情况下，建立了10个外植体培养物，并通过光学显微镜（LM）研究了细胞（人上颌来源细胞（hMDCs））的形态。通过流式细胞术分析外植体培养物的大小、颗粒度和表面标志物表达。使用了荧光染料偶联的单克隆抗体（CD13、CD31、CD44、CD90或CD73）。将hMDCs在标准培养基（SCM）或骨诱导培养基（OIM）中培养21天，并分析碱性磷酸酶（ALP）和钙沉积（冯·科萨染色）的存在情况。此外，通过逆转录聚合酶链反应（RT-PCR）分析成骨基因表达（骨钙素[OC]、ALP、I型胶原）。光学显微镜显示hMDCs呈多边形形态，含有一个带有两到三个核仁的中央细胞核。大小/颗粒度分析揭示了个体之间的差异。免疫表型分析显示，这些细胞CD13、CD44、CD90和CD73呈阳性，而CD31呈阴性。在SCM中培养21天的细胞显示出中等程度的ALP染色和许多钙沉积。在OIM中培养细胞21天显著增加了ALP染色和钙沉积的数量。RT-PCR证明了成骨标志物基因的表达以及响应成骨诱导剂上调骨钙素和ALP的能力。据我们所知，这是首次对源自该部位的骨细胞进行表面标志物表达研究。细胞对于未成熟间充质干细胞特征性的标志物呈阳性，并且具有成骨分化能力。本研究表明，源自上颌活检的细胞可能是骨组织工程的潜在细胞来源。
