Rosiglitazone disrupts endosteal bone formation during distraction osteogenesis by local adipocytic infiltration

Abstract

Rosiglitazone (Rosi) is a drug in the thiazolidinedione class for treatment of type 2 diabetes mellitus (T2DM), which binds and activates PPARγ nuclear receptor in fat cells, sensitizing them to insulin. Despite proven antidiabetic efficacy, Rosi therapy may be associated with trabecular bone loss and an increased risk of fractures. To examine the potential side effects of Rosi treatment on bone formation, we delivered Rosi to mice using a combined model of distraction osteogenesis (DO) and type 2 diabetes mellitus (T2DM). DO provides a unique method to isolate the sequence of intramembranous bone formation, an important component of both fracture healing and bone homeostasis. Four groups of n = 6 mice were used to compare the effects of Rosi on bone formation and cellular composition in both diabetic (Avy/a strain) and non-diabetic mice (a/a strain). New bone formation was examined by high resolution radiographs, micro-computed tomography, and histology. Precursor cells in the distraction gap were quantitated using immunohistochemical stains for proliferating cell nuclear antigen. Committed osteoblasts and adipocytes in the gap were identified and quantitated by immunostaining for osteocalcin and FABP4/aP2, respectively. The diabetic model developed obesity, hyperglycemia, hyperinsulinemia and insulin resistance, while the control littermates remained lean, normoglycemic and insulin sensitive. Rosi treatment decreased levels of non-fasted glucose and insulin and improved insulin sensitivity in the A^vy/a mice, but had no effect in a/a mice, indicating antidiabetic efficacy of Rosi at the tested dose. Despite the diabetic, obese mice having twice the number of fat cells in their marrow than the non-diabetic mice, bone formation using DO was not adversely affected by the diabetes itself. However, Rosi treatment significantly diminished intramembranous endosteal bone formation, while increasing adipogenesis in and adjacent to the distraction gap up to 3.5- to 3.8-fold in both diabetic and non-diabetic models. This effect was independent of the anti-diabetic therapeutic response. These results raise the question of whether osteoblast precursors are inhibited in their development or actually converted to adipocytic phenotypes, possibly via marrow fat PPARγ nuclear receptor.

Introduction

To test the potential effects of type 2 diabetes mellitus (T2DM) and of the glucose regulating drug rosiglitazone (Rosi) on bone repair, we used a unique genetic model (A^vy/a) simulating T2DM with our established model for quantitating in vivo cellular contributions to de novo endosteal bone formation (ENB) both at the site of distraction osteogenesis in the tibia and in the local bone marrow.

Drugs from the thiazolidinedione (TZD) family for treatment of type 2 diabetes mellitus (T2DM) are thought to increase the risk of fracture occurrence [1], [2], [3], [4], [5]. The negative effect of TZDs on bone mass (bone homeostasis) has also been demonstrated in humans and animals [6], [7]. Studies using in vitro cell models demonstrate that TZD-activated PPARγ acts as a negative regulator of osteoblastic and as a positive regulator of adipocytic lineage[8], [9], [10]. Animal studies have shown that the protein target for TZDs, a transcription factor peroxisome proliferator activator receptor gamma (PPARγ), is expressed in both mesenchymal (MSC) and hematopoietic (HSC) stem cells found in the marrow and regulates lineage commitment toward osteoblasts and osteoclasts, respectively [10], [11], [12].

To better understand the histological basis of the rosiglitazone (Rosi) effects on in vivo bone formation, we utilized a unique model combining tibial distraction osteogenesis (DO) in diabetic A^vy/a mice.

DO, a clinical tool for regenerating bone loss, has been developed in several animal species including dogs, rabbits, rats and mice [13]. A pre-determined gap is created from a surgical osteotomy site in the tibia using a scaled ring external fixator to mechanically distract the bone ends. Depending on the species, the distance of distraction exceeds the critical gap for spontaneous fracture healing, usually 15% of the baseline bone length, while the rate of distraction is critical to produce de novo bone which eventually bridges the gap. For mice, the distraction rate is 0.075 mm twice a day for 14 days which results in a 15% lengthening. The new bone, unlike fracture healing, forms from uniform and reproducible zones of primarily intramembranous or direct bone formation that can be quantitated by radiodensity and decalcified histology[14], [15], [16], [17]. From distinct proliferation zones of precursor cells, immature osteoblasts line the surface and then are incorporated within bone trabeculae exhibiting different stages of osteoblast (OB) cell maturation (see Fig. 3A) [14], [15], [16]. The local host bone cortices geographically isolate periosteal (PNB) from endosteal (ENB) new bone formation [18], [19], [20], [21]. Since both the cambium layer of periosteum and the local marrow are known to contain osteoblastic precursor cells, it appears from these models that precursor cells for ENB are supplied from the local marrow and for PNB from the adjacent cambium layer. We have determined that ENB is more sensitive to adverse factors (e.g., aging, alcohol, TNF alpha ) and can be measured to decrease significantly prior to any changes in PNB, reflecting morphological changes in the local marrow[15], [16], [17], [21].

Since diabetic patients have exhibited bone healing problems, we hypothesized that (1) the diabetic mouse model would have relatively less ENB by DO than the non-diabetic controls. Based on in vitro work using Rosi, we also hypothesized that Rosi treatment would (2) significantly decrease the actual de novo bone formation within the distraction gap, (3) significantly alter the adjacent marrow cell composition and (4) significantly alter the orderly sequence of osteoblastic maturation histologically.