Evaluation of osteogenic cell differentiation in response to bone morphogenetic protein or demineralized bone matrix in a critical sized defect model using GFP reporter mice

Abstract

We evaluated the osteoprogenitor response to rhBMP-2 and DBM in a transgenic mouse critical sized defect. The mice expressed Col3.6GFPtopaz (a pre-osteoblastic marker), Col2.3GFPemerald (an osteoblastic marker) and a-smooth muscle actin (a-SMACherry, a pericyte/myofibroblast marker). We assessed defect healing at various time points using radiographs, frozen, and conventional histologic analyses. GFP signal in regions of interest corresponding to the areas of new bone formation was quantified using a novel computer assisted algorithm. All defects treated with rhBMP-2 healed. In contrast, the majority of the defects in the DBM (27/30) and control (28/30) groups did not heal. Quantitation of pre-osteoblasts demonstrated a maximal response (% GFPþ cells/ TV) in the Col3.6GFPtopaz mice at day 7 (7.2% AE 6.0, p < 0.05 compared to days 14, 21, 28, and 56). The maximal response of the Col2.3GFP cells was seen at days 14 (8.04% AE 5.0) and 21 (8.31% AE 4.32), p < 0.05. In contrast, DBM and control groups showed a limited osteogenic response at all time points. In conclusion, we demonstrated that the BMP and DBM induce vastly different osteogenic responses which should influence their clinical application as bone graft substitutes. ß

Combination therapy with BMP-2 and a systemic RANKL inhibitor enhances bone healing in a mouse critical-sized femoral defect

“…However, the supraphysiologic doses of rhBMP used in clinical practice and the kinetics of BMP release from the carrier [2] are associated with inconsistent clinical results and several complications [3,4,5]. Previous animal studies using a mouse critical-sized femoral defect model in our laboratory have demonstrated that treatment with rhBMP-2 leads to healing of the defect, with the bony bridge gradually becoming thinner at later time points (28 and 56 days after treatment) [6]. It has been hypothesized that this thin cortical bone repair associated with rhBMP-2 treatment is secondary to the rapid release of the protein from the collagen sponge [6,7], leading to osteoclast stimulation and subsequent bone resorption.…”

“…Previous animal studies using a mouse critical-sized femoral defect model in our laboratory have demonstrated that treatment with rhBMP-2 leads to healing of the defect, with the bony bridge gradually becoming thinner at later time points (28 and 56 days after treatment) [6]. It has been hypothesized that this thin cortical bone repair associated with rhBMP-2 treatment is secondary to the rapid release of the protein from the collagen sponge [6,7], leading to osteoclast stimulation and subsequent bone resorption. Another potential option for BMP protein delivery is ex vivo regional gene therapy [8].…”

“…This may be due to the fact that lentiviral gene therapy is characterized by a sustained production of BMP-2 (up to 12 weeks with a lentiviral vector) which leads to a prolonged osteoinductive signal and thus increased new bone formation compared to rhBMP-2 [11]. However, BMP not only induces bone formation, but also stimulates both osteoclastogenesis and premature bone resorption [2,6,12–14]. Thus, in order to optimize the biologic healing potential of BMP-2 it may be necessary to inhibit this osteoclast activation.…”

Visual reporters for study of the osteoblast lineage

“…Col2.3-GFP in particular has been widely used in lineage tracking studies to identify osteoblasts derived from various cell populations during development, adulthood, or following injury [19, 21, 63–65]. They are also useful to track differentiation following transplantation of cells.…”

Remineralization of demineralized bone matrix in critical size cranial defects in rats: A 6‐month follow‐up study

“…Alaee used an elegant mouse model of multiple osteogeneic reporter genes and observed that although DBM had osteoinductive properties, its effect was rather limited. 8 When DBM is used as a bone filler for critical size defects, its effects are even less convincing. In rat models, for example, DBM showed significant, but limited bone formation capacity at 8 weeks.…”