Role Of HIF-1 Alpha In BMSCs Osteogenic Differentiation Induced By Cycling Stretch

Background and objectiveClassical histological change of orthodontic tooth movement is that: the external force generates two different strains in the periodontal ligament, compression and tension. At the compression site, the force that is generated by the root against the alveolar bone induces bone resorption. The periodontal ligament space is narrowing, the blood flow is slowing, collagen fibers and matrix are degrading and absorpting, osteoclasts differentiate and alveolar bone tissue is lost. At the tension site, the periodontal ligament space is widening, the blood flow is increasing, collagen fibers and matrix are proliferating, osteoblasts differentiate and new alveolar bone tissue is formed. Mechanical stress plays a crucial role in bone formation and absorption during orthodontic tooth movement via the osteoblast and osteoclast processes. This type of tooth movement is considered as a biological response to the physiologic equilibrium, when the dentofacial complex interfered by an externally applied force. The osteoblast activity plays a central role in regulation of the bone remodeling process and orthodontic tooth movement. However, the role of orthodontic force in the osteogenic differentiation of bone mesenchymal stem cells (BMSCs) remains to be determined. Notably, alveolar bone formation occurs via the differentiation of osteoblast precursor cells from primitive BMSCs, maturation of osteoblasts, and matrix formation, followed by its mineralization. However, osteoblasts are incompetent in proliferation and differentiation in vivo, and they are primarily derived from BMSCs that are preserved after the completion of development. BMSCs can differentiate into osteoblasts and induce osteoclasts generation via deposition and absorption of alveolar bone. Alternatively, BMSCs can solve periodontal bone tissue for reconstruction. It is, therefore, important to investigate the mechanism and regulation of osteogenic differentiation in BMSCs in order to better understand orthodontic tooth movement and the orthodontic-periodontal-bone tissue reconstruction.Some studies have indicated that the biomechanical signals are important for regulating phenotypic differentiation of BMSCs, and may play a complementary or coordinating role in cytokines. Cells may develop corresponding biological changes in response to a variety of physiological stresses. Unfortunately, to reveal the impact of mechanical factors in vivo is often challenging due to the complexity of the in vivo environment. In particular, to distinguish between the roles of a single factor from a combined effect is difficult for studies on the biological behavior of cells. Therefore, the effects on cell stimulation system mainly depends on the in vitro methods, among which tensile stretch is a major factor in determining both strain and function of the cell. Under different tensile stress, the overall framework of cytoskeletal rearrangement occurs, leading to deformation of cells. Changing of the cell shape can initiate a signal, which in turn can be transmitted into cell nuclear, and produce various effects. Notably, the tension that is applied to the in vitro cultures cells can provide a more physiological stimulation that better recapitulates the growth state of cells in vivo.BMSCs are isolated from the bone marrow, and hypoxic in nature (1-7% O2). Hypoxia-inducible factor 1 alpha (HIF-1 alpha) is a specific regulator of hypoxia responses in all cells, and is also the common pathway of information transmission under hypoxia-inducible condition. HIF-1 alpha plays a key role in the adaption to hypoxia. Hypoxic microenvironment can be caused by orthodontic mechanical forces between alveolar bone and the root, and the local hypoxic microenvironment is one of the main factors starting bone remodeling. However, it is unknown how HIF-1 alpha regulates the osteogenic differentiation induced by cycling stretch. Existing work shows that: the study on the role of HIF-1 alpha in the osteogenic differentiation subjected to cycling stretch is still far from adequate, and it is helpful for clinical orthodontists using different means and methods to induce or regulate bone remodeling. It also provides a theoretical basis for clinical intervention, and has important guiding significance. This is just the start of the international new research and focus area, and also the main problem to be solved during the optimize orthodontic tooth movement in clinic.In the present study, the experimental tooth movement model was firstly built in rat lateral maxillary, and the expression HIF-1 alpha in the force area was measured in vivo. Primary rat bone mesenchymal stem cells were isolated cultured and identified, and different cycling stretch was applied on cells. According to the relationship between stimulation and BMSCs osteogenic differentiation, the ideal condition was determined. Stable BMSCs with RNA interference of HIF-1 alpha were constructed by lentiviral virus, and were induced by the ideal stretch, to investigate the role of BMSCs osteogenic differentiation subjected to cycling stretch, and to explore the relationship among force, HIF-1 alpha and BMSCs osteogenic differentiation. This paper attempts to clarify the important role of mechanical factors in BMSCs osteogenic differentiation, to reveal the role of HIF-1 alpha in the BMSCs osteogenic differentiation subjected to cycling stretch in vitro, and to provide a theoretical basis for orthodontic treatment.Main methods and results1. Observe the expression change of HIF-1 alpha in periodontal tissue after orthodontic tooth movement:A titanium-nickel closed-coil spring was applied to the upper incisors with a ligature wire and the occlusal surface of the rat right maxillary first molar with a hook. The coil spring was kept constant and recorded for Od, Id, 3d, 7d, 14d, 28d. When the experiment finished, the maxillae were removed. The specimens were fixed and decalcified. After being dehydrated in ascending grades of alcohol, cleared in xylene, and paraffin-embedded, serial sections were cut parasagittally by a microtome. HE staining was applied to observe histological change of periodontal tissue and immunohistochemical staining was carried out with anti- HIF-1 alpha and anti-VEGF rabbit polyclonal antibody to examine the expression of HIF-1 alpha and VEGF.In this study, we found that in the untreated control group, HIF-1 alpha was not expressed obviously in the rat periodontal tissues, mostly located aruound the alveolar bone or cementum, rarely in the middle of periodontal tissues. VEGF, the down-stream gene of HIF-1 alpha, was also expressed at a low level. Application of orthodontic loading simultaneously induced a significant increase of periodontal ligament cells positive for HIF-1 alpha, and the strength expression changed throughout the entire orthodontic periodontal tissue remodeling process. Strong direct HIF-1 alpha expression was observed in cementoblast of the periodontium at the compressing site near the root surface, also in the middle of periodontal tissues. The expression of VEGF was less than HIF-1 alpha, but still increasing in a time-dependent manner. HIF-1 alpha participated in the periodontal tissue remodeling during orthodontic tooth movement, and probably plays an important part in this process.2. Identification of isolated rat bone mesenchymal stem cells:Rat bone meshenchymal stem cells were obtained when cultured in vitro and amplified in a short period with high quantity and purity, and they had strong proliferative ability. The formation of cells was observed with light microscope. The proliferation ability was tested by MTT and the live and dead cells were labeled by LIVE/DEAD viability kit. The colony-forming ability of BMSCs was estimated in crystal violet dye. Under some specific conditions, BMSCs could differentiate into osteoblasts by ARS staining and ALP staining, and also into adipocyte by oil red O staining. Isolated cells were identificated as rat bone mesenchymal stem cells through a series of cellular functional experiments.3. Determine the ideal condition of cycling stretch to stimulate osteogenic differentiation of BMSCs:In this study, we applied cycling stretch stimulation to BMSCs and cells were divided into 16 groups, including control group and experimental groups according to 1%, 5%, and 15% elongation for 0.5h, 2h, 6h, 8h, and 12h, respectively, with a 1 Hz sinusoidal curve. The quantitative data analysis of ALP activity indicated that 6hr- 5% stretch was the ideal condition to stimulate osteogenic differentiation of BMSCs.4. Observe the expression of HIF-1 alpha mRNA and protein after application of cycling stretch on BMSCs:There was very low level of HIF-1 alpha protein in unloaded cells, but the cycling stretch induced a time-dependent increase in the expression of HIF-1 α mRNA level. The western blot results showed similar trend in protein levels. It indicated that HIF-1 alpha could make a response to mechanical stress.5. Construction and identification of stable BMSCs with RNA interference of HIF-1 alpha:Firstly, single-stranded DNA oligo containing interference sequences was compounded, and then double-stranded DNA oligo was pairing annealed produced. Next, it was directly connected into digested RNA interference lentiviral vector by its restriction sites at both ends. The ligation product was then connected into the prepared bacterial competent cells. Monoclonal colonies were firstly identified by PCR, and then positive colonies were identified by PCR and sequencing. The correct clone was RNA interfering lentiviral vector successfully built. Then 293T cells were co-transfected by lentiviral expression vector and packaging mix, virus was packaged, the virus stock solution was collected and concentrated, and the concentration was measured. The pure and endotoxin-free lentiviral shRNA vectors and the secondary packaging of the original plasmid were extracted. The constructed lentiviral shRNA vectors and the secondary packaging of the original plasmid were co-transfected into 239T cells by HG transgene reagent, and the enhancing buffer was added after 10-12h. Fresh medium was changed after 8h, and cell supernatant containing enriched lentiviral particles was collected after 48h. High titer lentiviral was collected after concentrated, and the virus titer was measured and calibrated in 293T cells. Eventually, rat BMSCs were infected by packed lentiviral virus and negative control lentiviral virus. After infected successfully, interference effects were evaluated by Western Blotting, and the stable strains were obtained and successfully identified.6. Observe the role of HIF-1 alpha in the cycling stretch-induced BMSCs osteogenic differentiation:The expression of HIF-1 alpha mRNA and protein level in uninfected cells, HIF-1 alpha-shRNA cells, and NC cells was measured by qPCR and western blotting, respectively. Cells in three groups were applied by 5%, 1Hz cycling stretch for 6hs. The changes of ALP activity, OCN, OPN, BSP, Runx2 and Osterix mRNA and protein level were measured to assess the osteogenic differentiation of BMSCs. An increase in the specific activity of cellular ALP has frequently been used as an index for osteogenic differentiation in untreated rat BMSCs. The ALP activity of BMSCs infected with HIF-1 alpha-shRNA markedly increased (P<0.05), and it remained unchanged in NC group (P>0.05). The osteogenic genes assessed included OCN, OPN, BSP, Runx2 and Osterix. There was no statistic difference in mRNA and protein expressions of all five osteogenic genes between uninfected cells and NC group (P>0.05), but up-regulated mRNA and protein expressions of all five osteogenic genes were observed in HIF-1 alpha-shRNA BMSCs (P<0.05).The expressions of VEGF and TWIST mRNA and protein level, the down-stream genes of HIF-1 alpha, were also found in untreated rat BMSCs. There is stastical decrease in BMSCs infected with HIF-1 alpha-shRNA (P<0.05), but no obvious change in control group (P>0.05). Osteogenic genes that may be related to changes in HIF-1 alpha downstream genesVEGF and TWIST, but the specific mechanisms still need further verification.Conclusions1. HIF-1 alpha is highly expressed by mechanical stimuli during tooth movement. HIF-1 alpha participated in the periodontal tissue remodeling during orthodontic tooth movement, and probably plays a key role in this process.2. The cycling strain initially increased the osteogenic differentiation, and the ideal condition is 1Hz-5% stretch for 6hrs. The HIF-1 alpha mRNA and protein expression also increased dramatically after force application in a time-dependent manner. These observations suggest that HIF-1 alpha could make a response to mechanical stress. HIF-1 alpha plays a rival role in remodeling of periodontal tissue in vitro.3. It is suggested that the absence of HIF-1 alpha plays a rival role in the differentiation process from bone mesenchymal stem cells to osteoblast-like cells induced by cycling strech.4. This study will contribute to a better understanding of mechanism of bone remodeling during tooth movement which may set the basis for clinical work.Originality and significanceThis research firstly confirms that HIF-1 alpha plays an important part in the periodontal tissue remodeling during orthodontic tooth movement in vitro, and reveals the rival role of HIF-1 alpha in the osteogenic differentiation process induced by cycling stretch. The study will explore a better understanding and discussion of mechanism of periodontal tissue remodeling during tooth movement which may set the basis for clinical work and provides an important significance in the orthodontic tooth movement mechanism.