Zana Kalajzic

Use of Type I Collagen Green Fluorescent Protein Transgenes to Identify Subpopulations of Cells at Different Stages of the Osteoblast Lineage

Green fluorescent protein (GFP)‐expressing transgenic mice were produced containing a 3.6‐kilobase (kb; pOBCol3.6GFPtpz) and a 2.3‐kb (pOBCol2.3GFPemd) rat type I collagen (Col1a1) promoter fragment. The 3.6‐kb promoter directed strong expression of GFP messenger RNA (mRNA) to bone and isolated tail tendon and lower expression in nonosseous tissues. The 2.3‐kb promoter expressed the GFP mRNA in the bone and tail tendon with no detectable mRNA elsewhere. The pattern of fluorescence was evaluated in differentiating calvarial cell (mouse calvarial osteoblast cell [mCOB]) and in marrow stromal cell (MSC) cultures derived from the transgenic mice. The pOBCol3.6GFPtpz‐positive cells first appeared in spindle‐shaped cells before nodule formation and continued to show a strong signal in cells associated with bone nodules. pOBCol2.3GFPemd fluorescence first appeared in nodules undergoing mineralization. Histological analysis showed weaker pOBCol3.6GFPtpz‐positive fibroblastic cells in the periosteal layer and strongly positive osteoblastic cells lining endosteal and trabecular surfaces. In contrast, a pOBCol2.3GFPemd signal was limited to osteoblasts and osteocytes without detectable signal in periosteal fibroblasts. These findings suggest that Col1a1GFP transgenes are marking different subpopulations of cells during differentiation of skeletal osteoprogenitors. With the use of other promoters and color isomers of GFP, it should be possible to develop experimental protocols that can reflect the heterogeneity of cell differentiation in intact bone. In primary culture, this approach will afford isolation of subpopulations of these cells for molecular and cellular analysis.

Use of an alpha-smooth muscle actin 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 alpha-smooth muscle actin (alpha-SMA) positive cells was observed. During early recovery phase from ablation we have detected cells with the simultaneous expression of alpha-SMA and a preosteoblastic 3.6GFP marker, indicating the potential for transition of alpha-SMA+ cells towards osteoprogenitor lineage. Utilizing alpha-SMAGFP transgene, alpha-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 alpha-SMAGFP+ population derived from bone marrow or adipose tissue. In adipose tissue, alpha-SMA+ cells were localized within the smooth muscle cell layer and in pericytes. After in vitro expansion, alpha-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.

Directing the expression of a green fluorescent protein transgene in differentiated osteoblasts: comparison between rat type I collagen and rat osteocalcin promoters

The osteocalcin (OC) and a 2.3 kb fragment of the collagen promoter (Col2.3) have been used to restrict transgenic expression of a variety of proteins to bone. Transgenic mice carrying a green fluorescent protein (GFP) gene driven by each promoter were generated. Strong GFP expression was detected in OC-GFP mice in a few osteoblastic cells lining the endosteal bone surface and in scattered osteocytes within the bone matrix in long bones from 1-day-old to 6-month-old transgenic animals. Similar findings were noted in the forming tooth in which only individual odontoblasts expressed GFP without detectable expression from the dental pulp. This limited pattern of OC-GFP-positive cells contrasts with the uniform expression in the Col2.3GFP mice in which large proportion of osteoblasts, odontoblasts, and osteocytes strongly expressed the transgene. To assess transgene expression during in vitro differentiation, marrow stromal cell and neonatal calvarial osteoblast cultures were analyzed. The activity of both transgenes was restricted to mineralized nodules but the number of positive cells was lower in the OC-GFP-derived cultures. The different temporal and spatial pattern of each transgene in vivo and in vitro reveals potential advantages and disadvantages of these two transgene models.

Histological Analysis of GFP Expression in Murine Bone

The power for appreciating complex cellular interactions during embryonic development using green fluorescent protein (GFP) as a visual histological marker has not been applied to adult tissues due to loss of GFP signal during paraffin embedding and a high autofluorescent background, particularly in section of bone and bone marrow. Here we demonstrate that the GFP signal is well preserved in frozen sections of adult decalcified bone. Using a tape-transfer system that preserves histological relationships, GFP expression can be related to standard histological stains used in bone biology research. The choice of a dual-filter cube and a strong GFP signal makes it possible to readily distinguish at least four different GFP colors that are distinctly different from the autofluorescent background. An additional advantage of the frozen sections is better preservation of immunological epitopes that allow colocalization of an immunostained section with an endogenous GFP and a strong lacZ signal emanating from a β-gal marker gene. We present an approach for recording multiple images from the same histological section that allows colocalization of a GFP signal with subsequent stains and procedures that destroy GFP. Examples that illustrate the flexibility for dual imaging of various fluorescent signals are described in this study. The same imaging approach can serve as a vehicle for archiving, retrieving, and sharing histological images among research groups.

Overexpression of Dlx5 in chicken calvarial cells accelerates osteoblastic differentiation

Our laboratory and others have shown that a homeodomain protein binding site plays an important role in transcription of the Col1a1 gene in osteoblasts. This suggests that homeodomain proteins have an important role in osteoblast differentiation. We have investigated the role of Dlx5 in osteoblastic differentiation. In situ hybridization studies indicated that Dlx5 is expressed in chick calvarial osteoblasts (cCOB) in vivo. Northern blot analysis indicated that Dlx5 expression in cultured cCOBs is induced concurrently with osteoblastic markers. To study the effect of overexpression of Dlx5 on osteoblast differentiation, we infected primary osteoblast cultures from 15‐day‐old embryonal chicken calvaria with replication competent retroviral vectors [RCASBP(A)] expressing Dlx5 or control replication competent avian splice acceptor brianhightiter polymerase subtype A [RCASBP(A)]. Expression of Col1a1, osteopontin, alkaline phosphatase, and osteocalcin messenger RNA (mRNA) occurred sooner and at higher levels in cultures infected with RCASBP(A)DLX5 than in RCASBP(A)‐infected cultures. Mineralization of Dlx5‐expressing cultures was evident by days 12‐14, and RCAS‐infected control osteoblasts did not begin to mineralize until day 17. Dlx5 also stimulated osteoblastic differentiation of calvarial cells that do not normally undergo osteoblastic differentiation in vitro. Our results suggest that Dlx5 plays an important role in inducing calvarial osteoblast differentiation.

Stage specific inhibition of osteoblast lineage differentiation by FGF2 and noggin

Fibroblast growth factor 2 (FGF2) and noggin are two unrelated ligands of two distinctly different signaling pathways that have a similar inhibitory effect on osteoblast differentiation. Because of their differences, we postulated that they probably acted at a different stage within the osteoprogenitor differentiation pathway. This study was performed on primary murine bone cell cultures under conditions where alkaline phosphatase (AP) and type I collagen expression (Col1a1) were observed by day 7 (preosteoblast stage), followed by bone syaloprotein (BSP) at day 11 (early osteoblast) and osteocalcin (OC) by day 15–18 (mature osteoblast stage). FGF2 completely inhibited expression of AP and the mRNA transcript for Col1a1, while noggin showed only a partial inhibition of these markers of preosteoblast differentiation. However, the markers of differentiated osteoblasts (BSP and OC) were completely inhibited in both the FGF2 and noggin treated cultures, suggesting that noggin acts at later point in the osteoprogenitor differentiation pathway than FGF2. To further verify that the inhibition was occurring at a different stage of osteoblasts development, primary cultures derived from transgenic mice harboring segments of the collagen promoter driving green fluorescent protein (GFP) that activate at different levels of osteoblast differentiation were analyzed. Consistent with the endogenous markers, pOBCol3.6GFP and pOBCOL2.3GFP transgene activity was completely inhibited by continuous addition of FGF2, while noggin showed partial inhibition of pOBCol3.6GFP and complete inhibition of the pOBCol2.3GFP transgene. Upon removal of either agent, endogenous and GFP markers of osteoblast differentiation reappeared although at a different temporal pattern. This work demonstrates that FGF2 and noggin can reversibly modulate osteoblast lineage differentiation at different maturational stages. These agents may be useful to enrich for and maintain a population of osteoprogenitor cells at a defined stage of differentiation. J. Cell. Biochem. 88: 1168–1176, 2003.

Col1a1-GFP Transgene Expression in Developing Incisors

Previous studies have shown that terminal differentiation of odontoblasts is accompanied by dramatic increases in type I collagen synthesis. Recently transgenic mice in which green fluorescent protein (GFP) expression is under the control of the rat 3.6 (pOBCol3.6GFPtpz) and 2.3 (pOBCol2.3GFPemd) Col1a1 promoter fragments were generated. Our analysis of these GFP-expressing transgenic mice shows that the 2.3-kb promoter fragment directs strong expression of GFP only to bones and teeth, whereas the 3.6-kb fragment of promoter directs strong expression of GFP in bone and tooth, as well as in other type I collagen producing tissues. Our observations of incisors in these transgenic mice show high levels of GFP expression in functional odontoblasts and in differentiated osteoblasts. These observations show that expression of GFP reporter genes closely follow the patterns of expression of f 1(I) collagen in various tissues including odontoblasts.

Murine TMJ Loading Causes Increased Proliferation and Chondrocyte Maturation

The purpose of this study was to examine the effects of forced mouth opening on murine mandibular condylar head remodeling. We hypothesized that forced mouth opening would cause an anabolic response in the mandibular condylar cartilage. Six-week-old female C57BL/6 mice were divided into 3 groups: (1) control, (2) 0.25 N, and (3) 0.50 N of forced mouth opening. Gene expression, micro-CT, and proliferation were analyzed. 0.5 N of forced mouth opening caused a significant increase in mRNA expression of Pthrp, Sox9, and Collagen2a1, a significant increase in proliferation, and a significant increase in trabecular spacing in the subchondral bone, whereas 0.25 N of forced mouth opening did not cause any significant changes in any of the parameters examined. Forced mouth opening causes an increase in the expression of chondrocyte maturation markers and an increase in subchondral trabecular spacing.

Analysis of Microarchitectural Changes in a Mouse Temporomandibular Joint Osteoarthritis Model

OBJECTIVE Little is known about the natural progression of the disease process of temporomandibular joint (TMJ) osteoarthritis (OA), which affects approximately 1% of the US population. The goal of this study was to examine the early microarchitectural and molecular changes in the condylar cartilage and subchondral bone in biglycan/fibromodulin (Bgn/Fmod) double-deficient mice, which develop TMJ-OA at 6 months. METHODS TMJs from 3-month-old (n=44) and 9-month-old (n=52) wild-type (WT n=46) and Bgn/Fmod (n=50) double-deficient mice were evaluated. Micro-CT analysis of the subchondral bone (n=24), transmission electron microscopy for condylar cartilage fibril diameters (n=26), and real-time PCR analysis for gene expression for bone and cartilage maturation markers (n=45) was performed. RESULTS A statistically significant increase in collagen fibril diameter of the condylar cartilage and a decrease in expression of Parathyroid related protein in the mandibular condylar head were observed in the 3-month Bgn/Fmod double-deficient mice compared to WT controls. The 9-month Bgn/Fmod double-deficient mouse demonstrated an increase in bone volume and total volume in subchondral bone, and an increase in the expression of Collagen Type X and Aggrecan in the mandibular condylar head compared to the WT controls. CONCLUSION We found that changes in the microarchitecture of the condylar cartilage preceded changes in the subchondral bone during OA in the TMJ in Bgn/Fmod double-deficient mice.

Effect of cyclical forces on the periodontal ligament and alveolar bone remodeling during orthodontic tooth movement

OBJECTIVE To investigate the effect of externally applied cyclical (vibratory) forces on the rate of tooth movement, the structural integrity of the periodontal ligament, and alveolar bone remodeling. METHODS Twenty-six female Sprague-Dawley rats (7 weeks old) were divided into four groups: CTRL (unloaded), VBO (molars receiving a vibratory stimulus only), TMO (molars receiving an orthodontic spring only), and TMO+VB (molars receiving an orthodontic spring and the additional vibratory stimulus). In TMO and TMO+VB groups, the rat first molars were moved mesially for 2 weeks using Nickel-Titanium coil spring delivering 25 g of force. In VBO and TMO+VB groups, cyclical forces at 0.4 N and 30 Hz were applied occlusally twice a week for 10 minutes. Microfocus X-ray computed tomography analysis and tooth movement measurements were performed on the dissected rat maxillae. Tartrate-resistant acid phosphatase staining and collagen fiber assessment were performed on histological sections. RESULTS Cyclical forces significantly inhibited the amount of tooth movement. Histological analysis showed marked disorganization of the collagen fibril structure of the periodontal ligament during tooth movement. Tooth movement caused a significant increase in osteoclast parameters on the compression side of alveolar bone and a significant decrease in bone volume fraction in the molar region compared to controls. CONCLUSIONS Tooth movement was significantly inhibited by application of cyclical forces.