Hong Seung-Hyun, Jiang Xi, Chen Li, Josh Pujan, Shin Dong-Guk, Rowe David
Department of Computer Science and Engineering, University of Connecticut, Storrs, Connecticut, USA.
Department of Reconstructive Sciences, Biomaterials and Skeletal Development School of Dental Medicine, University of Connecticut Health Center, Farmington, Connecticut.
J Tissue Sci Eng. 2012 Dec 24;Suppl 1:004. doi: 10.4172/2157-7552.S1-004.
Dynamic and cellular histomorphometry of trabeculae is the most biologically relevant way of assessing steady state bone health. Traditional measurement involves manual visual feature identification by a trained and qualified professional. Inherent with this methodology is the time and cost expenditure, as well as the subjectivity that naturally arises under human visual inspection. In this work, we propose a rapidly deployable, automated, and objective method for dynamic histomorphometry. We demonstrate that our method is highly effective in assessing cellular activities in distal femur and vertebra of mice which are injected with calcein and alizarin complexone 7 and 2 days prior to sacrifice. The mineralized bone tissues of mice are cryosectioned using a tape transfer protocol. A sequential workflow is implemented in which endogenous fluorescent signals (bone mineral, green and red mineralization lines), tartrate resistant acid phosphatase identified by ELF-97 and alkaline phosphatase identified by Fast Red are captured as individual tiled images of the section for each fluorescent color. All the images are then submitted to an image analysis pipeline that automates identification of the mineralized regions of bone and selection of a region of interest. The TRAP and AP stained images are aligned to the mineralized image using strategically placed fluorescent registration beads. Fluorescent signals are identified and are related to the trabecular surface within the ROI. Subsequently, the pipelined method computes static measurements, dynamic measurements, and cellular activities of osteoclast and osteoblast related to the trabecular surface. Our method has been applied to the distal femurs and vertebrae of 8 and 16 week old male and female C57Bl/6J mice. The histomorphometric results reveal a significantly greater bone turnover rate in female in contrast to male irrespective of age, validating similar outcomes reported by other studies.
小梁的动态和细胞组织形态计量学是评估稳态骨骼健康最具生物学相关性的方法。传统测量方法需要由训练有素且资质合格的专业人员进行手动视觉特征识别。这种方法存在时间和成本消耗问题，以及在人工视觉检查中自然产生的主观性。在这项工作中，我们提出了一种可快速部署、自动化且客观的动态组织形态计量学方法。我们证明，在处死前7天和2天分别注射钙黄绿素和茜素络合物的小鼠的股骨远端和椎骨中，我们的方法在评估细胞活性方面非常有效。使用胶带转移方案对小鼠的矿化骨组织进行冷冻切片。实施了一个顺序工作流程，其中内源性荧光信号（骨矿物质、绿色和红色矿化线）、由ELF-97识别的抗酒石酸酸性磷酸酶以及由固红识别的碱性磷酸酶被捕获为每个荧光颜色的切片的单独平铺图像。然后将所有图像提交到一个图像分析管道，该管道可自动识别骨的矿化区域并选择感兴趣区域。使用精心放置的荧光配准珠将TRAP和AP染色图像与矿化图像对齐。识别荧光信号并将其与ROI内的小梁表面相关联。随后，流水线方法计算与小梁表面相关的破骨细胞和成骨细胞的静态测量、动态测量和细胞活性。我们的方法已应用于8周龄和16周龄雄性和雌性C57Bl/6J小鼠的股骨远端和椎骨。组织形态计量学结果显示，无论年龄大小，雌性小鼠的骨转换率均显著高于雄性，这验证了其他研究报告的类似结果。
