Localization and Quantification of Proliferating Cells During Rat Fracture Repair: Detection of Proliferating Cell Nuclear Antigen by Immunohistochemistry

Abstract

Bilateral femurs of 12-week-old female Sprague-Dawley rats were fractured, and the fractured femurs were harvested 36 h, 3, 7, 10, and 14 days after the fracture. Localization of cell proliferation in the fracture calluses was investigated using immunohistochemistry with antiproliferating cell nuclear antigen (PCNA) monoclonal antibodies. Thirty-six hours after the fracture, many PCNA-positive cells were observed in the whole callus. The change was not limited to mesenchymal cells at the fracture site where the inflammatory reaction had occurred, but extended in the periosteum along almost the entire femoral diaphysis where intramembranous ossification was initiated. On day 3, periosteal cells or premature osteoblasts in the newly formed trabecular bone during intramembranous ossification still displayed intense staining. On day 7, many premature chondrocytes and proliferating chondrocytes were PCNA positive. Endochondral ossification appeared on days 10 and 14, and the premature osteoblasts and endothelial cells in the endochondral ossification front were stained with anti-PCNA antibodies. Quantification of PCNA-positive cells was carried out using an image analysis computer system, obtaining a PCNA score for each cellular event. The highest score was observed in the periosteum early after the fracture near the fracture site. Immunohistochemistry using anti-PCNA antibodies showed that the distribution of proliferating cells and the degree of cell proliferation varied according to the time lag after the fracture, suggesting the existence of local regulatory factors such as growth factors, and that significant cell proliferation was observed at the beginning of each cellular event. (J Bone Miner Res 1997;12:96-102)

Placental growth factor mediates mesenchymal cell development, cartilage turnover, and bone remodeling during fracture repair

“…In addition, the periosteum is considered to be another major source of mesenchymal progenitor cells. The periosteum exhibits a strong mitogenic reaction early after fracture, and its removal can delay the healing process (23,24). As observed on H&E-stained sections (data not shown), the periosteum adjacent to the fracture site showed extensive thickening in WT mice at PFD3 but was only minimally enlarged in PlGF -/-calluses.…”

Smoking delays chondrogenesis in a mouse model of closed tibial fracture healing

“…Proliferation of mesenchymal stem cells, chondrocytes, and osteoblasts at the site of fracture repair has been localized and quantified in prior studies with immunohistochemical staining for proliferating cell nuclear antigen (PCNA). [16][17][18][19] After formation of the fracture callus, mechanical stability is restored through endochondral and intramembranous ossification. Endochondral ossification is believed to follow the same pattern as embryonic chondrogenesis and osteogenesis at growth plates.…”

“…In animal models of fracture healing, the predominant cells early in chondrogenesis are premature and proliferative chondrocytes in the chondroid callus, which peak at approximately 8-9 days after fracture. 14,16,18 At 14 days, the predominant cells in the chondroid callus are hypertrophic chondrocytes that are not actively proliferating, but rather preparing the cartilage matrix for subsequent calcification. 14 In the current study, proliferation was high at day 7 in both groups, but the cells of the smoking group showed a more flattened morphology indicative of immature cells.…”

Effects of cigarette smoking on early medial collateral ligament healing in a mouse model

“…Proliferation of precursor cells has been shown to be a major factor in the healing response in a number of wound healing studies, [30][31][32] but it has never been quantified in healing murine ligament or tendon. In a mouse model of the effects of smoking on tibial fracture healing used in our laboratory, the majority of cells in the cartilaginous callus stained with PCNA at day 7 after fracture in both smokers and controls.…”