Effects Of TNF-α On Cell Proliferation And Osteogenic Differentiation Of Human Periodontal Ligament Stem Cells And Mesenchymal Stem Cells And Their Effects On Bone Regeneration

Background and ObjectivesPeriodontitis is an inflammatory disorder of the supporting tissues of the teeth and one of the most common types of human infection. Alveolar bone resorption followed by loss of teeth is the most important clinical issue associated with the disease. Ideal clinical therapies should include predictable regenerative procedures to reestablish these tissues lost to the disease. To develop effective regenerative therapies, it is imperative that cellular and molecular mechanisms which regulate formation of these tissues be thoroughly understood.Experimental studies have shown that the potential of periodontal regeneration seems to be limited by the regenerative capacity of the cells involved. The regeneration of damaged periodontal tissues is mediated by various periodontal cells and is regulated by a vast array of extracellular matrix informational molecules that induce both selective and nonselective responses in different cell lineages and their precursors. Because conventional periodontal regeneration methods remain insufficient to obtain a complete and reliable periodontal regeneration, the concept of periodontal tissue engineering has been based on the generation of the conditions necessary to improve the healing of periodontal tissues. PDL contains fibroblasts, macrophages, undifferentiated mesenchymal cells, cementoblasts, osteoblasts, cell rests of Malassez and vascular and neural elements that are capable of generating and maintaining periodontal tissues. However, the presence of paravascular progenitor stem cells within PDL has long been speculated, and until recently, their isolation and propagation in culture remained elusive. In an impressive work, Seo et al. isolated a population of multipotent stem cells in human PDL (PDLSCs), providing a unique reservoir of stem cells from an accessible tissue resource. PDLSCs exhibit mesenchymal stem cells characteristics such as clonogenicity, high proliferation and the expression of the putative stem cell marker Stro-1and the perivascular cell marker CD146. In addition, in vitro-expanded PDLSCs can form collagen fibers and generate cementum/PDL-like structures. Kawanabe et al. additionally demonstrated that PDLSCs exhibit a stem cell function profile and express ABCG-2(a general stem cell marker) and Oct-4(a critical transcriptional factor for maintaining pluripotentiality), however only3.9%of all cultured PDL cells exhibit such characteristics.Inflammatory cytokines have pronounced paracrine and autocrine effects on bone cells. Several mediators have been proposed to induce periodontal disease. TNF-α is an important proinflammatory cytokine associated with tissue destruction in periodontitis.The cytokine has been described as a mediator of bone loss in osteolysis and other inflammatory bone diseases. In addition to its known bone resorptive action, TNF-α reduces bone formation by inhibiting osteoblast differentiation.Many studies showed that osteoblasts and bone marrow-derived stromal cells are highly responsible to the development of local inflammatory responses, Our current understanding of how mesenchymal stem cells behave in various environments is limited at best. Their behavior in stem cell niches is a rapidly expanding field of study but mesenchymal stem cells interaction with disease microenvironment is poorly understood. Prior studies have investigated the in vitro effects of cytokines, including those with proinflammatory activity, on mesenchymal progenitor populations to determine what influence an inflammatory reaction might have, for example, on bone formation. This question is of potential significance since tissue regeneration is often needed in areas of the body where there is significant damage and hence an inflammatory reaction.TNF-α activates NF-κ B signaling pathway and mitogen activated protein kinase (MAPK) such as p38, ERK and JNK. The NF-κ B signaling pathway is long known to play an important role in inflammation and control of the immune system. In the absence of stimuli, NF-κ B is sequestered redominantly in the cytoplasm by inhibitory I κ B proteins. Stimulation by effectors such as TNF-α, interleukin-1(IL-1), viral proteins, and double-stranded RNA, triggers I κ B phosphorylation and degradation, NF-κ B nuclear translocation, and transcriptional regulation of target genes. A negative regulation of osteoblast differentiation by NF-κ B was suggested as inhibition of NF-κ B signaling activity in osteosarcoma cells (Saos2), Less is known about the role of NF-κ B signaling in osteoblast differentiation.The aim of our present study was to determine how human periodontal ligament stem cells and a murine bone marrow-meshenchymal cells ST2might behave in an inflammatory environment. The aim is to find a new way to repair tissue in an inflammatory microenvironment like periodontitis.Materials and Methods:1. Effects of TNF-α on osteogenic differentiation of human periodontal ligament stem cells (PDLSCs) and mesenchymal stem cells (BMSCs)PDLSCs and BMSCs were cultured and isolated by magnetic Dynabeads. Cells were treated with human or murine TNF-α at concentrations of0,0.01,0.1,1,10and100ng/ml, respectively. At24,48and72h after the TNF-α treatment, the proliferation/survival of the cells was evaluated using the methylthiazolyldiphenyl-tetrazolium bromide (MTT) test;The changes in the expression levels of osteogenic transcription factors (Runx2and Osx) and bone matrix proteins (OC and ALP) during osteogenic differentiation were observed by real-time PCR; The change in ALP activity was determined using PNPP assay; In vitro mineralization of PDLSCs and BMSCs treated with TNF-a at concentrations of0,0.1,1and100ng/ml for four weeks under osteogenic induction was monitored using Alizarin red staining.2. Effect of overexpression of the NF-κ B antogonist I κ B α-mut on osteogenic differentiation of ST2cellspBABE-puro-I κ B α-mut or pBABE-puro was transfected into PT67packaging cells using LipofectamineTM2000. Stably virus-producing cell line was obtained by puromycine election. The medium from packaging cells comprising virus was added to infect ST2cells and chosen by puromycine to gain the stably overexpressing I κ B α-mut cells. MTT, Real-time PCR, PNPP and mineralization assay were adopted to determine the effect of overexpression of I κ B α-mut on cell proliferation/survival and the osteoblast related genes expression, ALP activity and the mineral ability in ST2cells respectively.3. Effects of TNF-a on bone regeneration in mice skull wound modelSkull wound model was established in BABL/C mice.The experimental groups were:group A:BMSCs implanted without TNF-α injection(n=5)group B:BMSCs implanted with intraperitoneal injection of5ug/kg of TNF-α every two days (n=5)group C:I κ B α-mut overexpressed BMSCs implanted with intraperitoneal injection of5ug/kg of TNF-α every two days (n=5)After two weeks of surgery, animals were sacrificed, H&E staining and histomorphometric analysis were performed to evaluate the effect of TNF-a on bone regeneration.Results1. Effects of TNF-α on osteogenic differentiation of PDLSCs and BMSCs(1) The stem cells were successfully obtained using magnetic Dynabeads. The stem cells were STRO-1+and Vimentin+stained. The ability of colony formation was high, and both sorts of stem cells had formed mineralized nodules when cultured in osteogenic medium.(2)Real time PCR analysis showed that the mRNA levels of the potent osteogenic transcription factors (Runx2and Osx) and some bone matrix proteins (OC and ALP) were up-regulated in cell cultures treated with TNF-a at lower concentrations (0.01and0.1ng/ml) when compared with cells treated with0ng/ml TNF-α. In contrast, the mRNA levels of Runx2, Osx, OC and ALP were dose-dependently down-regulated in cell cultures treated with TNF-a at concentrations above1ng/ml.(3)48hr treatment of the PDLSCs and BMSCs with TNF-α at lower concentrations (0.01and0.1ng/ml) enhanced ALP activity in these cells when compared with the control cells (treated with0ng/ml TNF-a). Cells treated with TNF-a at higher concentrations (10and100ng/ml) showed decreased ALP activity when compared with the control cells.(4) Long term treatment with TNF-α resulted in a dose-dependent decrease in mineral nodule formation after the4-week osteogenic induction.(5) MTT assay did not reveal any statistically significant changes in the proliferation/survival of PDLSCs and BMSCs treated with TNF-α at concentrations ranging from0.01to100ng/ml.2. Effect of overexpression of the NF-κ B antogonist I κ B α-mut on osteogenic differentiation of ST2cells(1) The NF-κ B signaling pathway was successfully inhibited via the overexpression of the NF-κ B antogonist I κ B α-mut in ST2cells. Western blot analysis showed that the protein level of I κ B α was increased significantly in ST2cells transduced with pBABE-puro-I κ B α-mut when compared with the control cells. While the nuclear protein level of p65, a subunit of NF-κ B, was slightly decreased in ST2cells stably overexpressing the exogenous I κ B α-when compared with the control cells.(2) Blockade of NF-κ B signaling via the overexpression of the NF- κ B antogonist IκBa in ST2cells only reversed the inhibitory effect observed in cell cultures treated with TNF-α at concentrations higher that1ng/ml, while had no impact on the positive changes observed in cell cultures treated with TNF-α at lower concentrations (0.01and0.1ng/ml).(3) Overexpression of I κ B a in ST2cells reversed the down-regulation of ALP activity by TNF-α treatment at concentrations of10and100ng/ml, while failed to induce significant changes in ALP activity of cells treated with TNF-α at lower concentrations (0.01and0.1ng/ml).(4) The inhibitory effect of TNF-α on in vitro mineral nodule formation in ST2cells was partially reversed by the blockade of NF-κ B signaling.(5) The blockade of NF-κ B signaling pathway via the overexpression of I κ B a in ST2cells showed a dose-dependent negative effect of TNF-a on the proliferation/survival of cells.3. Effects of TNF-α on bone regeneration in mice skull wound model(1)General observation and H&E-stained sections revealed decreased newly formed bone tissues in bone defects treated with TNF-α by intraperitoneal injection5ug/kg continuously (group B) compared with the control groups, no TNF-α injection(group A), Histomorphometric analysis confirmed different newly formed bone area in the two groups.(2)The ability of bone regeneration in group that implanted I κ B α-mut overexpressed BMSCs greatly increased in proinflammotry environment, compared with the group implanted BMSCs (group C versus group B),and newly formed bone area between the two groups was significant different.Conclusions:(1) The PDLSCs and BMSCs are successfully obtained by maganetic dynabeads, and the procedure is simple.(2)TNF-α treatment at lower concentrations and short time moderately enhances expression of osteogenic genes and ALP activity in PDLSCs and BMSCs(3)TNF-α treatment at higher concentrations and long time displays inhibitory effect on osteogenic differentiation and mineralization in PDLSCs and BMSCs(4)Continuously intraperitoneal injection of TNF-α delays bone regeneration in mice wound model. The ability of bone regeneration greatly increases when implanted overexpression of I κ B α-mut BMSCs in proinflammotry environment.(5)The binding of TNF-a to its receptor results in the activation of multiple signaling pathways, which actively interact with each other to regulate the differentiation, proliferation, survival and apoptosis of mesenchymal stem cells.