Department of Reconstructive Sciences, University of Connecticut Health Center, Farmington, Connecticut.
Department of Orthopedic Surgery, University of Connecticut Health Center, Farmington, Connecticut.
J Histochem Cytochem. 2019 Jun;67(6):401-417. doi: 10.1369/0022155419836436. Epub 2019 Mar 8.
The effectiveness of autologous cell-based skeletal repair continues to be controversial in part because in vitro predictors of in vivo human bone formation by cultured human progenitor cells are not reliable. To assist in the development of in vivo assays of human osteoprogenitor potential, a fluorescence-based histology of nondecalcified mineralized tissue is presented that provides multiple criteria to distinguish human and host osteoblasts, osteocytes, and accumulated bone matrix in a mouse calvarial defect model. These include detection of an ubiquitously expressed red fluorescent protein reporter by the implanted human cells, antibodies specific to human bone sialoprotein and a human nuclear antigen, and expression of a bone/fibroblast restricted green fluorescent protein reporter in the host tissue. Using low passage bone marrow-derived stromal cells, robust human bone matrix formation was obtained. However, a striking feature is the lack of mouse bone marrow investment and osteoclasts within the human bone matrix. This deficiency may account for the accumulation of a disorganized human bone matrix that has not undergone extensive remodeling. These features, which would not be appreciated by traditional decalcified paraffin histology, indicate the human bone matrix is not undergoing active remodeling and thus the full differentiation potential of the implanted human cells within currently used mouse models is not being realized.
自体细胞基骨骼修复的效果仍然存在争议，部分原因是体外预测培养的人类前体细胞在体内形成人骨的能力不可靠。为了协助开发人类成骨前体细胞体内潜能的检测方法，本文提出了一种基于荧光的非脱钙矿化组织组织学方法，该方法提供了多种标准，可用于区分鼠颅骨缺损模型中的人源和宿主成骨细胞、骨细胞和积累的骨基质。这些标准包括检测植入的人细胞中普遍表达的红色荧光蛋白报告基因、针对人骨涎蛋白和人核抗原的抗体，以及宿主组织中骨/成纤维细胞特异性的绿色荧光蛋白报告基因的表达。使用低传代骨髓基质细胞可获得丰富的人骨基质形成。然而，一个显著的特征是缺乏人骨髓细胞在人骨基质中的投资和破骨细胞。这种缺陷可能导致未经过广泛重塑的无序人骨基质的积累。这些特征在传统的脱钙石蜡组织学中无法被识别，表明人骨基质没有进行活跃的重塑，因此目前使用的鼠模型中植入的人细胞的全部分化潜能尚未得到实现。
