Dynamic loading stimulates mandibular condyle remodeling.

BACKGROUND: We and others have reported that low-magnitude high-frequency dynamic loading has an osteogenic effect on alveolar bone. Since chondrocytes and osteoblasts originate from the same progenitor cells, we reasoned that dynamic loading may stimulate a similar response in chondrocytes. A stimulating effect could be beneficial for patients with damaged condylar cartilage or mandibular deficiency. METHODS: Studies were conducted on growing Sprague-Dawley rats divided into three groups: control, static load, and dynamic load. The dynamic load group received a dynamic load on the lower right molars 5 minutes per day with a 0.3 g acceleration and peak strain of 30 µÎµ registered by accelerometer and strain gauge. The static load group received an equivalent magnitude of static force (30 µÎµ). The control group did not receive any treatment. Samples were collected at days 0, 28, and 56 for reverse transcriptase polymerase chain reaction analysis, microcomputed tomography, and histology and fluorescent microscopy analysis. RESULTS: Our experiments showed that dynamic loading had a striking effect on condylar cartilage, increasing the proliferation and differentiation of mesenchymal cells into chondrocytes, and promoting chondrocyte maturation. This effect was accompanied by increased endochondral bone formation resulting in lengthening of the condylar process. CONCLUSIONS: Low-magnitude, high-frequency dynamic loading can have a positive effect on condylar cartilage and endochondral bone formation in vivo. This effect has the potential to be used as a treatment for regenerating condylar cartilage and to enhance the effect of orthopedic appliances on mandibular growth.

Therapeutic effect of localized vibration on alveolar bone of osteoporotic rats.

OBJECTIVES: Vibration, in the form of high frequency acceleration (HFA), stimulates alveolar bone formation under physiologic conditions and during healing after dental extractions. It is not known if HFA has an anabolic effect on osteoporotic alveolar bone. Our objective is to determine if HFA has a regenerative effect on osteoporotic alveolar bone. METHODS AND MATERIALS: Adult female Sprague-Dawley rats were divided into five groups: 1) Ovariectomized Group (OVX), 2) Sham-OVX Group that received surgery without ovariectomy, 3) OVX-HFA Group that was ovariectomized and treated daily with HFA, 4) OVX+Static Force Group that was ovariectomized and received the same force as HFA, but without vibration, and 5) Control Group that did not receive any treatment. All animals were fed a low mineral diet for 3 months. Osteoporosis was confirmed by micro-CT of the fifth lumbar vertebra and femoral head. HFA was applied to the maxillary first molar for 5 minutes/day for 28 and 56 days. Maxillae were collected for micro-CT, histology, fluorescent microscopy, protein and RNA analysis, and three-point bending mechanical testing. RESULTS: Micro-CT analysis revealed significant alveolar bone osteoporosis in the OVX group. Vibration restored the quality and quantity of alveolar bone to levels similar to the Sham-OVX group. Animals exposed to HFA demonstrated higher osteoblast activity and lower osteoclast activity. Osteogenic transcription factors (RUNX2, Foxo1, Osterix and Wnt signaling factors) were upregulated following vibration, while RANKL/RANK and Sclerostin were downregulated. HFA did not affect serum TRAcP-5b or CTx-1 levels. The osteogenic effect was highest at the point of HFA application and extended along the hemimaxillae this effect did not cross to the contra-lateral side. CONCLUSIONS: Local application of vibration generated gradients of increased anabolic metabolism and decreased catabolic metabolism in alveolar bone of osteoporotic rats. Our findings suggest that HFA could be a predictable treatment for diminished alveolar bone levels in osteoporosis patients.

Vibration paradox in orthodontics: Anabolic and catabolic effects.

Vibration in the form of High Frequency Acceleration (HFA) is anabolic on the craniofacial skeleton in the absence of inflammation. Orthodontic forces trigger an inflammation-dependent catabolic cascade that is crucial for tooth movement. It is unknown what effect HFA has on alveolar bone if applied during orthodontic treatment. The objectives of this study are to examine the effect of HFA on the rate of tooth movement and alveolar bone, and determine the mechanism by which HFA affects tooth movement. Adult Sprague Dawley rats were divided to control, orthodontic force alone (OTM), and different experimental groups that received the same orthodontic forces and different HFA regimens. Orthodontic tooth movement was assessed when HFA parameters, frequency, acceleration, duration of exposure, and direct or indirect application were varied. We found that HFA treatment significantly enhanced the inflammation-dependent catabolic cascade during orthodontic tooth movement. HFA treatment increased inflammatory mediators and osteoclastogenesis, and decreased alveolar bone density during orthodontic tooth movement. Each of the HFA variables produced significant changes in the rate of tooth movement and the effect was PDL-dependent. This is the first report that HFA enhances inflammation-dependent catabolic cascades in bone. The clinical implications of our study are highly significant, as HFA can be utilized to enhance the rate of orthodontic tooth movement during the catabolic phase of treatment and subsequently be utilized to enhance retention during the anabolic remodeling phase after orthodontic forces are removed.

F-spondin regulates chondrocyte terminal differentiation and endochondral bone formation.

This study examines the role of F-spondin, an extracellular matrix protein of osteoarthritic cartilage, during chondrocyte maturation in embryonic growth plate cartilage. In chick tibia, F-spondin expression localized to the hypertrophic and calcified zones of the growth plate. Functional studies using tibial organ cultures indicated that F-spondin inhibited (∼35%, p = 0.02), and antibodies to F-spondin increased (∼30%, p < 0.1) longitudinal limb growth relative to untreated controls. In cell cultures, induction of chondrocyte maturation, by retinoic acid (RA) or transforming growth factor (TGF)-ß treatment led to a significant upregulation of F-spondin (p < 0.05). F-spondin transfection increased mineral deposition, alkaline phosphatase (AP) and matrix metalloproteinase (MMP)-13 mRNA levels (p < 0.05), and AP activity following RA stimulation, compared to mock transfected controls. Using AP as a differentiation marker we then investigated the mechanism of F-spondin promaturation effects. Blocking endogenous F-spondin via its thrombospondin (TSR) domain inhibited RA induced AP activity 40% compared to controls (p < 0.05). The stimulatory effect of F-spondin on AP expression was also inhibited following depletion of TGF-ß from culture supernatants. Our findings indicate that F-spondin is expressed in embryonic cartilage, where it has the capacity to enhance chondrocyte terminal differentiation and mineralization via interactions in its TSR domain and TGF-ß dependent pathways.

Characterization of polymeric solutions as injectable vehicles for hydroxyapatite microspheres.

A polymeric solution and a reinforcement phase can work as an injectable material to fill up bone defects. However, the properties of the solution should be suitable to enable the transport of that extra phase. Additionally, the use of biocompatible materials is a requirement for tissue regeneration. Thus, we intended to optimize a biocompatible polymeric solution able to carry hydroxyapatite microspheres into bone defects using an orthopedic injectable device. To achieve that goal, polymers usually regarded as biocompatible were selected, namely sodium carboxymethylcellulose, hydroxypropylmethylcellulose, and Na-alginate (ALG). The rheological properties of the polymeric solutions at different concentrations were assessed by viscosimetry before and after moist heat sterilization. In order to correlate rheological properties with injectability, solutions were tested using an orthopedic device applied for minimal invasive surgeries. Among the three polymers, ALG solutions presented the most suitable properties for our goal and a non-sterile ALG 6% solution was successfully used to perform preliminary injection tests of hydroxyapatite microspheres. Sterile ALG 7.25% solution was found to closely match non-sterile ALG 6% properties and it was selected as the optimal vehicle. Finally, sterile ALG 7.25% physical stability was studied at different temperatures over a 3-month period. It was observed that its rheological properties presented minor changes when stored at 25 degrees C or at 4 degrees C.

An improved collagen scaffold for skeletal regeneration.

Bone repair and regeneration is one of the most extensively studied areas in the field of tissue engineering. All of the current tissue engineering approaches to create bone focus on intramembranous ossification, ignoring the other mechanism of bone formation, endochondral ossification. We propose to create a transient cartilage template in vitro, which could serve as an intermediate for bone formation by the endochondral mechanism once implanted in vivo. The goals of the study are (1) to prepare and characterize type I collagen sponges as a scaffold for the cartilage template, and (2) to establish a method of culturing chondrocytes in type I collagen sponges and induce cell maturation. Collagen sponges were generated from a 1% solution of type I collagen using a freeze/dry technique followed by UV light crosslinking. Chondrocytes isolated from two locations in chick embryo sterna were cultured in these sponges and treated with retinoic acid to induce chondrocyte maturation and extracellular matrix deposition. Material strength testing as well as microscopic and biochemical analyzes were conducted to evaluate the properties of sponges and cell behavior during the culture period. We found that our collagen sponges presented improved stiffness and supported chondrocyte attachment and proliferation. Cells underwent maturation, depositing an abundant extracellular matrix throughout the scaffold, expressing high levels of type X collagen, type I collagen and alkaline phosphatase. These results demonstrate that we have created a transient cartilage template with potential to direct endochondral bone formation after implantation.

Engineering endochondral bone: in vivo studies.

The use of biomaterials to replace lost bone has been a common practice for decades. More recently, the demands for bone repair and regeneration have pushed research into the use of cultured cells and growth factors in association with these materials. Here we report a novel approach to engineer new bone using a transient cartilage scaffold to induce endochondral ossification. Chondrocyte/chitosan scaffolds (both a transient cartilage scaffold-experimental-and a permanent cartilage scaffold-control) were prepared and implanted subcutaneously in nude mice. Bone formation was evaluated over a period of 5 months. Mineralization was assessed by Faxitron, micro computed tomography, backscatter electrons, and Fourier transform infrared spectroscopy analyses. Histological analysis provided further information on tissue changes in and around the implanted scaffolds. The deposition of ectopic bone was detected in the surface of the experimental implants as early as 1 month after implantation. After 3 months, bone trabeculae and bone marrow cavities were formed inside the scaffolds. The bone deposited was similar to the bone of the mice vertebra. Interestingly, no bone formation was observed in control implants. In conclusion, an engineered transient cartilage template carries all the signals necessary to induce endochondral bone formation in vivo.

Engineering endochondral bone: in vitro studies.

Chitosan scaffolds have been shown to possess biological and mechanical properties suitable for tissue engineering and clinical applications. In the present work, chitosan sponges were evaluated regarding their ability to support cartilage cell proliferation and maturation, which are the first steps in endochondral bone formation. Chitosan sponges were seeded with chondrocytes isolated from chicken embryo sterna. Chondrocyte/chitosan constructs were cultured for 20 days, and treated with retinoic acid (RA) to induce chondrocyte maturation and matrix synthesis. At different time points, samples were collected for microscopic, histological, biochemical, and mechanical analyses. Results show chondrocyte attachment, proliferation, and abundant matrix synthesis, completely obliterating the pores of the sponges. RA treatment caused chondrocyte hypertrophy, characterized by the presence of type X collagen in the extracellular matrix and increased alkaline phosphatase activity. In addition, hypertrophy markedly changed the mechanical properties of the chondrocyte/chitosan constructs. In conclusion, we have developed chitosan sponges with adequate pore structure and mechanical properties to serve as a support for hypertrophic chondrocytes. In parallel studies, we have evaluated the ability of this mature cartilage scaffold to induce endochondral ossification.

Injectability of a bone filler system based on hydroxyapatite microspheres and a vehicle with in situ gel-forming ability.

The aim of this study was to test the injectability of a bone filler system based on the combination of ceramic microspheres with a gel-like vehicle, for noninvasive surgery. Porous hydroxyapatite microspheres with a uniform size and an average diameter of 535 +/- 38 mum were prepared, and their compression strength and friability were tested. The sodium-alginate solution with a concentration of 7.25% (w/v) was used as the vehicle. To promote its in situ gelation, calcium carbonate and D-gluconic-delta-lactone were added to the solution. Microspheres were mixed with the vehicle at different percentages (20-40 wt %). Gelation times in the range of 8-20 min, were obtained, depending on the formulation. Mixtures of HAp microspheres with alginate solution at 7.25% originating a gel in 11 min present an adequate handling time to perform an injection. Their injectability was evaluated using an injection device commonly employed in vertebroplasty surgical procedures, coupled to a texturometer in compression mode. Using an extrusion rate of 0.1 mm/s, the force required to extrude any of the mixtures tested was lower than 100 N. For an extrusion rate of 1 mm/s mixtures with 40 wt % of microspheres were very difficult to inject. Mixtures with 35 wt % of microspheres presented the best compromise between injectability and compression strength of the gelled system. MicroCT analysis revealed a homogeneous distribution of the microspheres inside the vehicle, as well as full interconnection of the intra-microspheres spaces. The compression strength for the gelled systems ranged from 80 kPa (gel itself) to 600 kPa (composite with 40 wt % of microspheres).