Zhihe Zhao

Expression of Osteoclastogenesis Inducers in a Tissue Model of Periodontal Ligament under Compression

There is increasing interest in the development of new in vitro tissue models. In this study, a tissue model of periodontal ligament (PDL) was established by 3-D-culturing human PDL cells in a thin sheet of porous poly (lactic-co-glycolic acid) scaffold. Growth of the model was evidenced by MTT assay and various microscopies. After being subjected to static compression of 5 ~ 35 g/cm2 for 6 hrs, the RANKL mRNA expression was significantly up-regulated by force ≥ 25 g/cm2 in the model. After being subjected to static compression of 25 g/cm2 for 6 ~ 72 hrs, the mRNA expression of PTHrP, IL-11, IL-8, and FGF-2, potential osteoclastogenesis inducers, was significantly up-regulated in the model, which was further verified by the compression of human PDL in vivo. However, when human gingival fibroblasts were substituted for PDL cells in the model, almost no osteoclastogenesis inducers were up-regulated by compression. This tissue model can serve as an effective tool for the study of PDL mechanoresponse. Abbreviations: periodontal ligament, PDL; periodontal ligament cells, PDLCs; poly (lactic-co-glycolic acid), PLGA; orthodontic tooth movement, OTM; extracellular matrix, ECM.

Tensile strain induces integrin β1 and ILK expression higher and faster in 3D cultured rat skeletal myoblasts than in 2D cultures

According to previous research, integrin beta1 and ILK play an important role in the extracellular matrix (ECM)-integrin-cytoskeleton pathway for mechanotransduction. The aim of this study was to investigate strain induced integrin beta1 and ILK expression in three-dimensional (3D) and in two-dimensional (2D) cultured rat skeletal myoblasts. Sprague-Dawley (SD) rat skeletal myoblasts were isolated and seeded on the PLGA-collagen composite scaffolds. The 3D cultured and the conventionally 2D monolayer cultured myoblasts were loaded 2000 microstrain tensile strain at 0.5Hz for 2h, 4h, 8h, 12h and 24h, respectively with the self-made four-point bending system. The expressions of integrin beta1 and ILK mRNA were measured by RT-PCR and the different changes between the 3D and 2D cultures were compared. The mRNA expression levels of both integrin beta1 and ILK were up regulated after mechanical loading (P<0.05), meanwhile, it was higher and peaked faster in 3D cultures than in the 2D cultures. It can be concluded that the ECM-integrin-cytoskeleton pathway responds to tensile strain by elevated expression of integrin beta1 and ILK, and the response is stronger in 3D cultures than in conventional 2D monolayer cultures.

Validation of β-Actin Used as Endogenous Control for Gene Expression Analysis in Mechanobiology Studies: Amendments†

We write in response to the letter by Liu et al. [1] commenting on our article, ‘‘Mesenchymal Stem Cells Regulate Angiogenesis According to Their Mechanical Environment’’ [2]. The study by Liu et al. demonstrates that the commonly used endogeneous reference gene (ERG), b-actin, is upregulated by mechanical loading, indicating a potential bias in the determined target gene expression when normalizing to b-actin, such as in our report on unchanged vascular endothelial growth factor (VEGF) and hypoxia-inducible factors (HIF)-1a mRNA levels in mechanically loaded mesenchymal stem cells (MSCs).

Establishment of a three‐dimensional culture and mechanical loading system for skeletal myoblasts

Establishment of a three‐dimensional (3‐D) culture and mechanical loading system which simulates the in vivo environment is critical in cytomechanical studies. The present article attempts to do this by integrating porous PLGA scaffolds with a four‐point bending strain unit. Three types of PLGA scaffolds with three average pore sizes were synthesized, i.e., type I (60–88 μm), type II (88–100 μm) and type III (100–125 μm). To establish the 3‐D mechanical loading system, PLGA membrane was integrated with conventional force‐loading plates and the third passage skeletal myoblasts from neonatal Sprague–Dawley (SD) rats were seeded. Small PLGA membranes were put in 24‐well plates followed by cell implantation and MTT assay was performed on days 1, 2, 4, 6 and 8 to compare biocompatibility of the three types of scaffolds. After 3 days’ culture, many more cells had grown in type II than in type I or type III under fluorescence microscopy. In the MTT assay, OD of type II was significantly higher (P < 0.05) than the other two, especially at the early stage. As type II proved to be the best among the three, it was used as the scaffold in the preliminary mechanical loading study and 4000 μstrain cyclic uniaxial strain was imposed. The system worked well and it was found that short to median time of stretching enhances while prolonged time of stretching inhibits cell proliferative activity of the 3‐D cultured skeletal myoblasts(P < 0.05). It is concluded that the combination of PLGA scaffolds with a four‐point bending strain unit provides a satisfactory 3‐D mechanical loading system.

NF-κB responds to mechanical strains in osteoblast-like cells, and lighter strains create an NF-κB response more readily

This study was to examine the early responses of nuclear factor kappa B (NF‐κB) to mechanical strains in MG‐63. MG‐63 cells were subjected to cyclic uniaxial compressive or tensile strain, produced by a four‐point bending system, at 1000 μstrain or 4000 μstrain for 5 min, 15 min, 30 min and 1 h, respectively. Control cells received the same treatment with no mechanical stress loading. Expression of NF‐κB (p60) was measured by Western blotting. NF‐κB responded rapidly to mechanical stimuli in MG‐63 cells. NF‐κB was activated by cyclic uniaxial stretch at 1000 μstrain while it was restrained under a compressive strain environment at 1000 μstrain (P < 0.001). The effects reversed for tension and compression at 4000 μstrain (P < 0.001). Furthermore, strains at 1000 μstrain affected NF‐κB expression much easier than those at 4000 μstrain. This indicates that there may be different responding mechanisms or mechanotransduction pathways for different mechanical stimuli.

The effect of varying healing times on orthodontic mini-implant stability: a microscopic computerized tomographic and biomechanical analysis

OBJECTIVEnThe aim of this study was to evaluate the effect of different healing times on the stability of titanium mini-implants used for orthodontic anchorage.nnnSTUDY DESIGNnEight male beagles were used and randomized into 4 groups according to different healing times (1, 3, 5, and 7 weeks); each group had 2 beagles. Sixty-four mini-implants were inserted bilaterally in the maxilla and mandible of the beagles. Microscopic computerized tomography (μCT) and pull-out test were used for morphometric and biomechanical analysis, respectively.nnnRESULTSnAll μCT parameters and F(max) (maximum pull-out force) increased with the prolongation of healing time. One week after insertion, all 4 measurements, namely osseointegration, trabecular bone volume density, intersection surface, and F(max), were lower in the maxilla group than in the mandible group (P < .05). Between the span of 1 and 3 weeks after insertion, a more obvious rising tendency of the 4 values was observed in the maxilla group than in the mandible group. Five and 7 weeks after insertion, the maxilla group expressed higher values of the 4 measurements than the mandible group (P < .05).nnnCONCLUSIONSnAlthough insertion in the mandible could provide higher primary stability for mini-implants, with the prolongation of healing time, insertion in the maxilla achieves higher osseointegration. The results indicated that insertion in maxilla has a more positive effect on the stability of mini-implants than insertion in the mandible.

Quantitative research using computed tomographic scanning of beagle jaws for determination of safe zones for micro-screw implantation.

OBJECTIVESnThe aim of this study was to provide an anatomical map for micro-screw placement in a safe location between the posterior dental roots of the beagle jaw.nnnSTUDY DESIGNnThree dimensional (3D) reconstructed images of 12 adult male beagles and jaw ground sections of another 6 beagles were examined. We measured sagittal, vertical and horizontal parameters in the intraradicular and interradicular regions of posterior teeth.nnnRESULTSnThe data showed that the safe zones were located in the intraradicular spaces of P2, P3, P4, M1, M2 and the interradicular spaces between P4 and M1, M1 and M2 in the mandible; in the maxilla, they were located in the intraradicular spaces of P3, P2, M1 and the interradicular space between P3 and M1.nnnCONCLUSIONSnThe present study provides a map of safe areas for the application of micro-screws in beagles.

A quantitative anatomical study on posterior mandibular interradicular safe zones for miniscrew implantation in the beagle.

With the increasing expansion of miniscrew anchorage use in orthodontic treatment, more and more studies have been and will be carried out on the biochemistry, biomechanics and side effects of miniscrews in vivo. In such studies, a beagle has been the most commonly used animal model and its mandibular interradicular zones have been the greatest focus of interest. However, interradicular miniscrews risk failure by being loosened due to collision with adjacent roots. Therefore, it is necessary for the surgeon to be familiar with the anatomy of a beagles mandible, especially that of the interradicular zones. This study has been performed to investigate the beagles mandibular interradicular safe zones for miniscrew implantation to provide an anatomical guide for this type of study. Twenty-four beagle corpses were collected. Their mandible specimens were ground parallel to the respective buccal alveolar surface using a model trimmer until a horizontal plane was obtained, which was then sectioned on the line passing each tooths central groove. In the image of this plane, cut at 2, 4, 6, 8 and 10 mm beneath the top of the alveolar crest, the mesiodistal width between the roots of P2 and P3, P3 and P4, P4 and M1 and the mesial and distal roots of M1 were measured, respectively. Zones of mesiodistal width measurement larger than 3.2 mm were found between P4 and M1, below the 8mm cut and between the mesial and distal roots of M1, below the 4 mm cut. In addition, between P2 and P3, below the 8mm cut and between P3 and P4, below the 10 mm cut, the mesiodistal width measurement was larger than 2.2 mm. The mandibular interradicular safe zones for miniscrew implantation in the dog were located between the mesial and distal roots of M1 and between the roots of P4 and M1, where there was enough mesiodistal width. Alveolar bone was relatively narrow between P2 and P3, P3 and P4, where care must be taken during implanting.

The mechanism of myoblast deformation in response to cyclic strain – A cytomechanical study

Mechanical strain is one of the important epigenetic factors that cause deformation and differentiation of skeletal muscles. This research was designed to investigate how myoblast deformation occurs after cyclic strain loading. Myoblasts were passaged three times and harvested; various cyclic strains (2.5 kPa, 5 kPa and 10 kPa) were then loaded using a pulsatile mechanical system. The adaptive response of the myoblasts was observed at different time points (0.5 h, 1 h, 6 h and 12 h) post‐loading. At the early stage of cyclic strain loading (<1 h), almost no visible morphological changes were observed in the myoblasts. The actin cytoskeleton showed a disordered arrangement and a weak fluorescence expression; there was little expression of talin. At 6 h and 12 h post‐loading, the myoblasts changed their orientation to parallel (in the 2.5 kPa and 5 kPa groups) or perpendicular (in the 10 kPa group) to the direction of strain. Fluorescence expression of both the actin cytoskeleton and talin was significantly increased. The results suggest that cyclic strain has at least two ways to regulate adaptation of myoblasts: (1) by directly affecting actin cytoskeleton at an early stage post‐loading to cause depolymerization; and (2) by later chemical signals transmitted from the extracellular side to intracellular side to initiate repolymerization.