Regulation of gingival fibroblast phenotype by periodontal ligament cells in vitro.

OBJECTIVES: Stem cell transplantation has shown modest effects on periodontal tissue regeneration, and it is still unclear how regenerative effects utilizing this modality are mediated. A greater understanding of the basic interactions between implanted and host cells is needed to improve future strategies. The aims of this study were to investigate the effects of periodontal ligament (PDL) cells on expression of periodontal markers and alkaline phosphatase (ALP) activity of gingival fibroblasts (GF). MATERIALS AND METHODS: Primary human PDL cells were co-cultured with primary GF cultures either by direct co-culture with subsequent FACS sorting or indirect co-culture using transwell cultures and PDL cell conditioned medium. Expression of periodontal markers, asporin, nestin, and periostin, was assessed by qPCR and immunofluorescence staining. Alkaline phosphatase (ALP) expression was assessed by qPCR, histochemical staining, and activity assessed by para-nitrophenol enzymatic assay. Single cultures of PDL cells and GF were used as controls. The role of Wnt signaling on ALP activity was assessed via Dkk1-mediated inhibition. RESULTS: PDL cells significantly upregulated expression of PDL markers in GF with both direct and indirect co-culture methods when compared to controls (6.05 vs. 0.73 and 59.48 vs. 17.55 fold change of asporin expression). PDL/GF cell co-cultures significantly increased ALP activity in GF when compared with single GF cultures. Similar results were obtained when using conditioned medium isolated from PDL cell cultures. Dkk1 caused dose-dependent reduction in ALP activity of GF cultured in PDL cell conditioned medium. CONCLUSIONS: PDL cells stimulate expression of periodontal markers and osteogenic capacity of gingival fibroblasts via paracrine signaling which can be partially inhibited with addition of the Wnt antagonist, Dkk1.Further studies are required to identify specific secreted factors responsible for this activity.

Gingival fibroblasts prevent BMP-mediated osteoblastic differentiation.

OBJECTIVES: The inhibitory action of the superficial gingival connective tissues may limit the regenerative potential of alveolar bone in periodontal therapy or dental implant applications. The aims of this study were to investigate the hypothesis that gingival fibroblasts (GF) can inhibit bone morphogenetic protein (BMP)-induced osteoblastic differentiation, to determine their expression of BMP inhibitors, and finally to determine whether reduction of these inhibitors can relieve suppression of osteoblastic differentiation. METHODS: Gingival fibroblasts were co-cultured either directly or indirectly with calvarial osteoblasts to assess alkaline phosphatase inhibitory activity, a marker of osteoblastic differentiation. To test total BMP-inhibitory activity of rat GF, conditioned media (GFCM) were collected from cultures. ROS 17/2.8 osteoblastic cells were stimulated with BMP2, together with GFCM. Inhibitor expression was tested using RT-qPCR, Western blotting and in situ hybridization. Removal of inhibitors was carried out using immunoprecipitation beads. RESULTS: Co-culture experiments showed GF-secreted factors that inhibit BMP-stimulated ALP activity. 10 ng/ml BMP2 increased alkaline phosphatase expression in ROS cells by 41%. GFCM blocked BMP activity which was equivalent to the activity of 100 ng/ml Noggin, a well-described BMP inhibitor. Cultured gingival fibroblasts constitutively expressed BMP antagonist genes from the same subfamily, Grem1, Grem2 and Nbl1 and the Wnt inhibitor Sfrp1. Gremlin1 (6.7 × reference gene expression) had highest levels of basal expression. ISH analysis showed Gremlin1 expression was restricted to the inner half of the gingival lamina propria and the PDL. Removal of Gremlin1 protein from GFCM eliminated the inhibitory effect of GFCM on ALP activity in ROS cells. Subsequent addition of recombinant Gremlin1 restored the inhibitory activity. CONCLUSIONS: Factors secreted by gingival fibroblasts inhibit BMP-induced bone formation and a range of BMP inhibitors are constitutively expressed in gingival connective tissues. These inhibitors, particularly Gremlin1, may limit coronal alveolar bone regenerative potential during oral and periodontal surgery.

Site-specific differences in osteoblast phenotype, mechanical loading response and estrogen receptor-related gene expression.

The osteoporosis-resistant nature of skull bones implies inherent differences exist between their cellular responses and those of other osteoporosis-susceptible skeletal sites. Phenotypic differences in calvarial and femoral osteoblastic responses to induction of osteogenesis, mechanical loading, estrogen, growth factor and cytokine stimulation were investigated. Primary rat calvarial and femoral adult male osteoblasts were cultured and osteoblastic mineralisation and maturation determined using Alizarin Red staining and expression of osteogenic marker genes assessed. Expression of known mechanically-responsive genes was compared between sites following loading of scaffold-seeded cells in a bioreactor. Cell proliferation and differentiation following growth factor and estrogen stimulation were also compared. Finally expression of estrogen receptors and associated genes during osteogenic differentiation were investigated. Calvarial osteoblasts exhibited delayed maturation (45d. vs 21d.) and produced less mineralised matrix than femoral osteoblasts when osteogenically induced. PDGF-BB and FGF2 both caused a selective increase in proliferation and decrease in osteoblastic differentiation of femoral osteoblasts. Mechanical stimulation resulted in the induction of the expression of Ccl2 and Anx2a selectively in femoral osteoblasts, but remained unchanged in calvarial cells. Estrogen receptor beta expression was selectively upregulated 2-fold in calvarial osteoblasts. Most interestingly, the estrogen responsive transcriptional repressor RERG was constitutively expressed at 1000-fold greater levels in calvarial compared with femoral osteoblasts. RERG expression in calvarial osteoblasts was down regulated during osteogenic induction whereas upregulation occurred in femoral osteoblasts. Bone cells of the skull are inherently different to those of the femur, and respond differentially to a range of stimuli. These site-specific differences may have important relevance in the development of strategies to tackle metabolic bone disorders.

Technique for direct posterior reduction in irreducible atlantoaxial dislocation: multi-planar realignment of C1-2.

OBJECTIVE: Apart from the commonly seen antero-posterior subluxation of C1 over C2, the dislocation may occur in vertical, lateral or rotational plane. Desired C1-2 realignment can be achieved by corrrecting its dislocation in all planes. We describe a technique for the same. MATERIAL AND METHODS: The clinical and radiological features of 16 patients (4 ­ traumatic and 12 ­ congenital) with irreducible atlantoaxial dislocation (AAD) admitted in the last 1.5 years were studied. Specific attention was paid to vertical dislocation with lateral and rotational components, apart from anterior-posterior subluxation. They were operated through direct posterior approach. The technique using a long rod holder as lever and screw head (tulip) as fulcrum was employed to achieve C1-2 realignment in all planes. The postoperative clinical and radiological data was analyzed and compared with preoperative data. RESULTS: Patients presented with progressive myelopathy and/or progressive worsening of neck pain. Vertical dislocation was seen in 11 patients with congenital AAD in addition to the antero-posterior subluxation seen in all. Three patients with traumatic AAD and 8 with congenital AAD had additional lateral dislocation or lateral tilt. Three patients with traumatic AAD and 7 with congenital AAD showed rotational component. Postoperatively, all patients showed clinical improvement. CONCLUSIONS: The antero-posterior and vertical realignment could be achieved in all except one. Similarly, rotational and lateral components could be completely corrected in 8 out of 10 patients. The technique appears to realign the C1-2 in all planes and provides good anatomical restoration.

Operative nuances to safeguard anomalous vertebral artery without compromising the surgery for congenital atlantoaxial dislocation: untying a tough knot between vessel and bone.

OBJECT: Stabilization of the craniovertebral junction (CVJ) by using lateral masses requires extensive dissection. The vertebral artery (VA) is commonly anomalous in patients with congenital CVJ anomaly. Such a vessel is likely to be injured during dissection or screw placement. In this study the authors discuss the importance of preoperative evaluation and certain intraoperative steps that reduce the chances of injury to such vessels. METHODS: A 3D CT angiogram was obtained in 15 consecutive patients undergoing surgery for congenital atlantoaxial dislocation. The course of the VA and its relationship to the C1-2 facets was studied in these patients. The anomalous VA was exposed intraoperatively, facet surfaces were drilled in all, and the screws were placed according to the disposition of the vessel. RESULTS: A skeletal anomaly was found in all 10 patients who had an anomalous VA. Four types of variations were noted: 1) the first intersegmental artery in 5 patients (bilateral in 1); 2) fenestration of VA in 1 patient; 3) anomalous posterior inferior cerebellar artery crossing the C1-2 joint in 1 patient; and 4) medial loop of VA in 5 patients. The anomalous vessel was dissected and the facet surfaces were drilled in all. The C-1 lateral mass screw was placed under vision, taking care not to compromise the anomalous vessel, although occipital screws or sublaminar wires were used in the initial cases. A medial loop of the VA necessitated placement of transpedicular or C-2 lateral mass screws instead of pars interarticularis screws. The anomalous vessel was injured in none. CONCLUSIONS: Preoperative 3D CT angiography is a highly useful method of imaging the artery in patients with CVJ anomaly. It helps in identifying the anomalous VA or its branch and its relationship to the C1-2 facets. The normal side should be surgically treated and distracted first because this helps in opening the abnormal side, aiding in dissection. In the posterior approach the C-2 nerve root is always encountered before the anomalous vessel. The defined vascular anatomy helps in choosing the type of screw. The vessel should be mobilized so as to aid the drilling of facets and the placement of screws and spacers under vision, avoiding its injury (direct or indirect) or compression. With these steps, C1-2 (short segment) rigid fusion can be achieved despite the presence of anomalous VA.

Akt- and Erk-mediated regulation of proliferation and differentiation during PDGFRß-induced MSC self-renewal.

Understanding the mechanisms that direct mesenchymal stem cell (MSC) self-renewal fate decisions is a key to most tissue regenerative approaches. The aim of this study here was to investigate the mechanisms of action of platelet-derived growth factor receptor ß (PDGFRß) signalling on MSC proliferation and differentiation. MSC were cultured and stimulated with PDGF-BB together with inhibitors of second messenger pathways. Cell proliferation was assessed using ethynyl-2'-deoxyuridine and phosphorylation status of signalling molecules assessed by Western Blots. To assess differentiation potentials, cells were transferred to adipogenic or osteogenic media, and differentiation assessed by expression of differentiation association genes by qRT-PCR, and by long-term culture assays. Our results showed that distinct pathways with opposing actions were activated by PDGF. PI3K/Akt signalling was the main contributor to MSC proliferation in response to activation of PDGFRß. We also demonstrate a negative feedback mechanism between PI3K/Akt and PDGFR-ß expression. In addition, PI3K/Akt downstream signal cascades, mTOR and its associated proteins p70S6K and 4E-BP1 were involved. These pathways induced the expression of cyclin D1, cyclin D3 and CDK6 to promote cell cycle progression and MSC proliferation. In contrast, activation of Erk by PDGFRß signalling potently inhibited the adipocytic differentiation of MSCs by blocking PPARγ and CEBPα expression. The data suggest that PDGFRß-induced Akt and Erk pathways regulate opposing fate decisions of proliferation and differentiation to promote MSC self-renewal. Thus, activation of multiple intracellular cascades is required for successful and sustainable MSC self-renewal strategies.