Heterogeneity of engrafted bone-lining cells after systemic and local transplantation

The outcome of various osteoprogenitor-cell transplantation protocols was assessed using Col1a1-GFP reporter transgenic mice. The model requires the recipient mice to undergo lethal total body irradiation (TBI) followed by rescue with whole bone marrow. When the mice are rescued with total bone marrow from a Col1a1-GFP transgenic mouse, green fluorescence protein (GFP)-positive donor cells can be observed on most endosteal and trabecular bone surfaces. Although the cells express an osteoblast-restricted GFP, they fail to progress to osteocytes, do not form a mineralized matrix, and do not generate bone nodules in vitro. However when calvarial progenitor cells derived from the same transgenic mice are injected into the bone marrow space, osteogenesis by the donor cells is observed. Using different GFP colors that distinguish the donor and recipient osteoblasts, commingling of the 2 cells types is observed along the mineralizing osteoblast surface as well as within the osteocyte population of the endosteal bone. Despite the ability of the injected progenitor cells to produce bone within the injected bone, they lack the ability to form mineralized bone nodules when explanted to primary osteoblast culture. These reagents and imaging protocols will be useful in evaluating other cells having a better progenitor potential than calvarial-derived stromal cells

Sex Differences in Chondrocyte Maturation in the Mandibular Condyle from a Decreased Occlusal Loading Model

Temporomandibular joint disorders (TMDs) predominantly afflict women of childbearing age. Defects in mechanical loading-induced temporomandibular joint (TMJ) remodeling are believed to be a major etiological factor in the development of TMD. The goal of this study was to determine if there are sex differences in CD-1 and C57BL/6 mice exposed to a decreased occlusal loading TMJ remodeling model. Male and female CD-1 and C57BL/6 mice, 21 days old, were each divided into two groups. They were fed either a normal pellet diet (normal loading) or a soft diet and had their incisors trimmed out of occlusion (decreased occlusal loading) for 4 weeks. The mandibular condylar cartilage was evaluated by histology, and the subchondral bone was evaluated by micro-CT analysis. Gene expression from both was evaluated by real-time PCR analysis. In both strains and sexes of mice, decreased occlusal loading caused similar effects in the subchondral bone, decreases in bone volume and total volume compared with their normal loading controls. However, in both strains, decreased occlusal loading caused a significant decrease in the expression of collagen type II (Col2) and Sox9 only in female mice, but not in male mice, compared with their normal loading controls. Decreased occlusal loading causes decreased bone volume in both sexes and a decrease in early chondrocyte maturation exclusively in female mice

Use Of An Alpha-smooth Muscle Actin (SMAA) GFP Reporter To Identify An Osteoprogenitor Population

Identification of a reliable marker of skeletal precursor cells within calcified and soft tissues remains a major challenge for the field. To address this, we used a transgenic model in which osteoblasts can be eliminated by pharmacological treatment. Following osteoblast ablation a dramatic increase in a population of α-smooth muscle actin (α-SMA) positive cells was observed. During early recovery phase from ablation we have detected cells with the simultaneous expression of SMAA and a preosteoblastic 3.6GFP marker, indicating the potential for transition of α-SMA+ cells towards osteoprogenitor lineage. Utilizing α-SMAGFP transgene, α-SMAGFP+ positive cells were detected in the microvasculature and in the osteoprogenitor population within bone marrow stromal cells. Osteogenic and adipogenic induction stimulated expression of bone and fat markers in the α-SMAGFP+ population derived from bone marrow or adipose tissue. In adipose tissue, α-SMA+ cells were localized within the smooth muscle cell layer and in pericytes. After in vitro expansion, α-SMA+/CD45−/Sca1+ progenitors were highly enriched. Following cell sorting and transplantation of expanded pericyte/myofibroblast populations, donor-derived differentiated osteoblasts and new bone formation was detected. Our results show that cells with a pericyte/myofibroblast phenotype have the potential to differentiate into functional osteoblasts

sj-tif-1-car-10.1177_19476035231163691 – Supplemental material for FGF Ligands and Receptors in Osteochondral Tissues of the Temporomandibular Joint in Young and Aging Mice

Supplemental material, sj-tif-1-car-10.1177_19476035231163691 for FGF Ligands and Receptors in Osteochondral Tissues of the Temporomandibular Joint in Young and Aging Mice by Eliane H. Dutra, Po-Jung Chen, Zana Kalajzic, Sunil Wadhwa, Marja Hurley and Sumit Yadav in CARTILAGE</p

sj-tif-2-car-10.1177_19476035231163691 – Supplemental material for FGF Ligands and Receptors in Osteochondral Tissues of the Temporomandibular Joint in Young and Aging Mice

Supplemental material, sj-tif-2-car-10.1177_19476035231163691 for FGF Ligands and Receptors in Osteochondral Tissues of the Temporomandibular Joint in Young and Aging Mice by Eliane H. Dutra, Po-Jung Chen, Zana Kalajzic, Sunil Wadhwa, Marja Hurley and Sumit Yadav in CARTILAGE</p

The role of inhibition of osteocyte apoptosis in mediating orthodontic tooth movement and periodontal remodeling: a pilot study

Background Orthodontic tooth movement (OTM) has been shown to induce osteocyte apoptosis in alveolar bone shortly after force application. However, how osteocyte apoptosis affects orthodontic tooth movement is unknown. The goal of this study was to assess the effect of inhibition of osteocyte apoptosis on osteoclastogenesis, changes in the alveolar bone density, and the magnitude of OTM using a bisphosphonate analog (IG9402), a drug that affects osteocyte and osteoblast apoptosis but does not affect osteoclasts. Material and methods Two sets of experiments were performed. Experiment 1 was used to specifically evaluate the effect of IG9402 on osteocyte apoptosis in the alveolar bone during 24 h of OTM. For this experiment, twelve mice were divided into two groups: group 1, saline administration + OTM24-h (n=6), and group 2, IG9402 administration + OTM24-h (n=6). The contralateral unloaded sides served as the control. The goal of experiment 2 was to evaluate the role of osteocyte apoptosis on OTM magnitude and osteoclastogenesis 10 days after OTM. Twenty mice were divided into 4 groups: group 1, saline administration without OTM (n=5); group 2, IG9402 administration without OTM (n=5); group 3, saline + OTM10-day (n=6); and group 4, IG9402 + OTM10-day (n=4). For both experiments, tooth movement was achieved using Ultra Light (25g) Sentalloy Closed Coil Springs attached between the first maxillary molar and the central incisor. Linear measurements of tooth movement and alveolar bone density (BVF) were assessed by MicroCT analysis. Cell death (or apoptosis) was assessed by terminal dUTP nick-end labeling (TUNEL) assay, while osteoclast and macrophage formation were assessed by tartrate-resistant acid phosphatase (TRAP) staining and F4/80+ immunostaining. Results We found that IG9402 significantly blocked osteocyte apoptosis in alveolar bone (AB) at 24 h of OTM. At 10 days, IG9402 prevented OTM-induced loss of alveolar bone density and changed the morphology and quality of osteoclasts and macrophages, but did not significantly affect the amount of tooth movement. Conclusion Our study demonstrates that osteocyte apoptosis may play a significant role in osteoclast and macrophage formation during OTM, but does not seem to play a role in the magnitude of orthodontic tooth movement

Cellular and Matrix Response of the Mandibular Condylar Cartilage to Botulinum Toxin

<div><p>Objectives</p><p>To evaluate the cellular and matrix effects of botulinum toxin type A (Botox) on mandibular condylar cartilage (MCC) and subchondral bone.</p><p>Materials and Methods</p><p>Botox (0.3 unit) was injected into the right masseter of 5-week-old transgenic mice (Col10a1-RFPcherry) at day 1. Left side masseter was used as intra-animal control. The following bone labels were intraperitoneally injected: calcein at day 7, alizarin red at day 14 and calcein at day 21. In addition, EdU was injected 48 and 24 hours before sacrifice. Mice were sacrificed 30 days after Botox injection. Experimental and control side mandibles were dissected and examined by x-ray imaging and micro-CT. Subsequently, MCC along with the subchondral bone was sectioned and stained with tartrate resistant acid phosphatase (TRAP), EdU, TUNEL, alkaline phosphatase, toluidine blue and safranin O. In addition, we performed immunohistochemistry for pSMAD and VEGF.</p><p>Results</p><p>Bone volume fraction, tissue density and trabecular thickness were significantly decreased on the right side of the subchondral bone and mineralized cartilage (Botox was injected) when compared to the left side. There was no significant difference in the mandibular length and condylar head length; however, the condylar width was significantly decreased after Botox injection. Our histology showed decreased numbers of Col10a1 expressing cells, decreased cell proliferation and increased cell apoptosis in the subchondral bone and mandibular condylar cartilage, decreased TRAP activity and mineralization of Botox injected side cartilage and subchondral bone. Furthermore, we observed reduced proteoglycan and glycosaminoglycan distribution and decreased expression of pSMAD 1/5/8 and VEGF in the MCC of the Botox injected side in comparison to control side.</p><p>Conclusion</p><p>Injection of Botox in masseter muscle leads to decreased mineralization and matrix deposition, reduced chondrocyte proliferation and differentiation and increased cell apoptosis in the MCC and subchondral bone.</p></div

Reduced bone volume and density and reduced condyle width at the Botox injected side.

<p><b>Coronal</b> micro-CT images of condyles of control <b>(A)</b> and Botox <b>(B)</b> injected side masseter 4 weeks after unilateral Botox injection. Quantification of bone parameters: <b>C)</b> BVF—bone volume fraction, <b>D</b>) Trabecular Thickness, <b>E)</b> Trabecular Spacing, <b>F)</b> Tissue Density. Morphometric measurements <b>(G-H)</b> performed in Faxitron xray images of control and Botox injected side mandibles: <b>I)</b> Mandibular Lengh, <b>J)</b> Condyle Head Length, <b>L)</b> Condyle Width. Histograms <b>(C-F, I-L)</b> represent means ± SD for n = 13 <i>per group</i>. <i>*S</i>ignificant difference between control and Botox injected side (p < 0.05). Scale bar = 500μm.</p

Decrease in osteoclastic activity and mineralization at the Botox injected side condyle.

<p>Sagittal sections of control (left side) <b>(A)</b> and Botox (right side) <b>(B)</b> injected side condyles stained for TRAP. <b>C)</b> Quantification of TRAP positive pixels (yellow) over subchondral bone area. Sagittal sections stained for alkaline phosphatase, control <b>(D)</b> and Botox injected side <b>(E)</b>. <b>F)</b> Quantification of distance mapping. Histograms <b>(C,F)</b> represents means ± SD for n = 7 <i>per group</i>. <i>*</i> Significant difference between control and Botox injected side (p < 0.05). No significant difference between control and Botox injected side for alkaline phosphatase distance mapping <b>(F)</b>. Scale bar = 500μm.</p

Reduced bone volume and density and reduced condyle width at the Botox injected side.

<p><b>Coronal</b> micro-CT images of condyles of control <b>(A)</b> and Botox <b>(B)</b> injected side masseter 4 weeks after unilateral Botox injection. Quantification of bone parameters: <b>C)</b> BVF—bone volume fraction, <b>D</b>) Trabecular Thickness, <b>E)</b> Trabecular Spacing, <b>F)</b> Tissue Density. Morphometric measurements <b>(G-H)</b> performed in Faxitron xray images of control and Botox injected side mandibles: <b>I)</b> Mandibular Lengh, <b>J)</b> Condyle Head Length, <b>L)</b> Condyle Width. Histograms <b>(C-F, I-L)</b> represent means ± SD for n = 13 <i>per group</i>. <i>*S</i>ignificant difference between control and Botox injected side (p < 0.05). Scale bar = 500μm.</p