Lee F Y, Choi Y W, Behrens F F, DeFouw D O, Einhorn T A
Department of Orthopaedic Surgery, New Jersey Medical School, Newark, USA.
J Orthop Res. 1998 Jan;16(1):144-50. doi: 10.1002/jor.1100160124.
This investigation tested the hypothesis that the removal of chondrocytes during endochondral fracture healing involves an ordered process of programmed cell death. To accomplish this, unilateral closed fractures were created in the femora of 36 Sprague-Dawley rats. The rats were killed in groups of four on days 1, 3, 7, 14, 21, 28, 42, 49, and 56 after fracture. The femora were embedded in paraffin and tested for expression of specific markers of fragmented DNA with use of a terminal deoxyuridyl transferase-mediated deoxyuridine triphosphate-biotin nick end labeling (TUNEL) technique. To determine the potential for transdifferentiation of chondrocytes to osteoblasts, calluses were also hybridized to detect expression of osteocalcin mRNA. Cell proliferation was assessed by an immunohistochemical detection method for proliferating cell nuclear antigen. A separate group of four rats was killed on day 28 to represent the later stage of the endochondral ossification, and the calluses were examined for cellular morphology with transmission electron microscopy. The results showed a coordination in both time and space of the activities of cellular proliferation and programmed cell death. Cell proliferation was most active in the earlier phases of fracture healing (days 1 through 14), although TUNEL expression was apparent in hypertrophic chondrocytes on day 14 after fracture and persisted until day 28. In the later stages of fracture healing (days 14 through 28), proliferating cell nuclear antigen was no longer synthesized in hard callus (intramembranous bone) and cell removal was the dominant activity in soft callus chondrocytes. Expression of osteocalcin mRNA was detected in osteoblasts but not in hypertrophic chondrocytes or in any other nonosteoblastic cell type. These findings support the hypothesis that the removal of chondrocytes during endochondral fracture healing is part of an ordered transition of tissue types in which the cellular mechanisms are genetically programmed to involve proliferation, maturation, and apoptotic cell death.
在软骨内骨折愈合过程中软骨细胞的清除涉及程序性细胞死亡的有序过程。为实现这一目标，在36只Sprague-Dawley大鼠的股骨上制造单侧闭合性骨折。在骨折后的第1、3、7、14、21、28、42、49和56天，将大鼠按每组4只进行处死。将股骨嵌入石蜡中，使用末端脱氧尿苷酰转移酶介导的脱氧尿苷三磷酸生物素缺口末端标记（TUNEL）技术检测断裂DNA的特定标志物的表达。为了确定软骨细胞向成骨细胞转分化的可能性，还对骨痂进行杂交以检测骨钙素mRNA的表达。通过免疫组织化学检测方法检测增殖细胞核抗原以评估细胞增殖。在第28天处死另一组4只大鼠以代表软骨内骨化的后期阶段，并通过透射电子显微镜检查骨痂的细胞形态。结果显示细胞增殖和程序性细胞死亡活动在时间和空间上具有协调性。细胞增殖在骨折愈合的早期阶段（第1天至第14天）最为活跃，尽管TUNEL表达在骨折后第14天的肥大软骨细胞中明显可见，并持续至第28天。在骨折愈合的后期阶段（第14天至第28天），增殖细胞核抗原不再在硬骨痂（膜内骨）中合成，细胞清除是软骨干细胞中的主要活动。在成骨细胞中检测到骨钙素mRNA的表达，但在肥大软骨细胞或任何其他非成骨细胞类型中未检测到。这些发现支持以下假设：在软骨内骨折愈合过程中软骨细胞的清除是组织类型有序转变的一部分，其中细胞机制在基因上被编程为涉及增殖、成熟和凋亡性细胞死亡。
