Recombinant human bone morphogenetic protein-2–induced ossification of the ligamentum flavum in rats and the associated global modification of histone H3

Abstract

<jats:sec> <jats:title>Object</jats:title> <jats:p>The primary object of this investigation was to study recombinant human bone morphogenetic protein–2 (rhBMP-2)–induced ossification of the ligamentum flavum and associated histone H3 modification in a rat model. In an additional set of studies the authors investigated spinal cord and behavioral changes in the same model.</jats:p></jats:sec> <jats:sec> <jats:title>Methods</jats:title> <jats:p>The authors report on 2 separate sets of studies. A total of 90 rats were used for the 2 sets of studies (45 each); in each study, a lyophilized rhBMP-2 and collagen mixture (20 μg rhBMP-2 and 200 μl collagen) was implanted in the lumbar extradural space in 18 rats; another 18 animals were used for a sham-operation control group and underwent implantation of lyophilized collagen without rhBMP-2 at the same level; an additional 9 animals were used as untreated controls. Lumbar spinal samples were harvested from the rhBMP-2 groups and the shamoperation control groups at 1 week, 3 weeks, and 9 weeks after the operation. Samples were also obtained from untreated controls at the same time points. All samples were scanned using micro-CT and then made into paraffinembedded sections. The sections from the first set of 45 rats were stained using elastica van Gieson and toluidine blue, and the expression of histone modifications (H3K9ac, H3K18ac, H3K4me3, and H3K36me3) and osteogenic transcription factors (osterix, Runx2) was detected by immunohistochemistry. In the second set of studies, hindlimb motor function was assessed at 1 week, 3 weeks, and 9 weeks after surgery. After behavioral evaluation, samples were harvested, scanned using micro-CT, and then made into paraffin-embedded sections. The sections were stained using Luxol fast blue. The expression of NeuN was also detected using immunohistochemistry.</jats:p></jats:sec> <jats:sec> <jats:title>Results</jats:title> <jats:p>Ossification was seen in the rhBMP-2 group from 1 week after insertion, and the volume of ossified mass increased at 3 and 9 weeks. There was no ossification seen in the sham-surgery and normal controls. The pathological changes of ossification involved ligament degeneration, cartilage formation, and, finally, bone replacement. Spinal cord evaluation showed a significant decrease in white matter content and number of neurons at 9 weeks after operation in the rhBMP-2–treated group (compared with findings in the sham-surgery and control groups as well as findings at the earlier time points in the rhBMP-2 group).</jats:p> <jats:p>Using immunohistochemical staining, histone modifications (H3K9ac, H3K18ac, H3K4me3, and H3K36me3) and osteogenic transcription factors (osterix, Runx2) all were found to be expressed in the fibrocartilage area of the rat ossified ligamentum flavum samples (rhBMP2 group).</jats:p></jats:sec> <jats:sec> <jats:title>Conclusions</jats:title> <jats:p>This rhBMP-2–induced OLF is a typical endochondral ossification, which is similar to clinical OLF. The compressed spinal cord around the ossification site showed signs of a chronic degenerative process. Histone H3 modifications (H3K9ac, H3K18ac, H3K4me3, and H3K36me3) may play an important role in OLF.</jats:p></jats:sec>

Notch signaling pathways in human thoracic ossification of the ligamentum flavum

“…A previous study suggested that many transcription factors, including Osterix and Runx2, play important roles in the development of OLF . We previously created a rat model using recombinant human BMP‐2‐induced OLF, and found Osterix and Runx2 expression in the fibrocartilage area of the rat ossified ligamentum flavum . Expression of Runx2 and Osterix increased similarly to that of Notch2 during the osteogenic process of ligamentum flavum cells in the present study.…”

“…38 We previously created a rat model using recombinant human BMP-2-induced OLF, and found Osterix and Runx2 expression in the fibrocartilage area of the rat ossified ligamentum flavum. 39 Expression of Runx2 and Osterix increased similarly to that of Notch2 during the osteogenic process of ligamentum flavum cells in the present study. Overexpression of Notch2 significantly increased Runx2 and Osterix levels, and Notch2 knockdown produced the opposite effect.…”

Meniscal and ligament modifications in spontaneous and post-traumatic mouse models of osteoarthritis

“…It was also reported that COL6A1 and RUNX2 may be common susceptibility genes in ligament ossification in the spine [53,54] and both of these genes have also been linked to OA [55,56]. TGFβ and BMPs are known regulators of ossification and fibrosis as well as being involved in OA development, and have also been associated with ligament ossification, including recombinant BMP2 in rat spines [57] and abnormal BMP signalling in human spines [58]. Recently, epidermal growth factor signalling was linked to OA development, comprising of ectopic chondro-osseous pathologies in the ligament and meniscus [10].…”

Evaluating Osteogenic Potential of Ligamentum Flavum Cells Cultivated in Photoresponsive Hydrogel that Incorporates Bone Morphogenetic Protein-2 for Spinal Fusion

“…The pathogenesis of spontaneous ossification of LF in vivo and the differentiation of normal and abnormal LF are still undergoing experimental research being experimentally examined. Hou et al ossified rat LF cells by embedding recombinant BMP-2 in their epidural space [ 23 ]. They found that pathological changes in ossification involved ligament degeneration, cartilage formation and, finally, bone formation.…”