Stability of dental implants with sandblasted and acid-etched (SLA) and modified (SLActive) surfaces during the osseointegration period

Background. The surface properties of implants are effective factors for increasing the osseointegration and activity of osteoprogenitor cells. This study compared the stability of dental implants with sandblasted and acid-etched (SLA) and modified surfaces (SLActive) using the resonance frequency analysis (RFA). Methods. In a split-mouth design, 50 dental implants with either SLA surface properties (n=25) or modified (SLActive) surface properties (n=25) were placed in the mandibles of 12 patients with a bilateral posterior edentulous area. Implant stability was measured using RFA (Osstell) at implant placement time and every week for 1, 2, and 3 months before the conventional loading time. Results. One week following the implantation, implant stability increased from 70 to 77.67 for SLA and from 71.67 to 79 for SLActive (P<0.05). Stability improved each week except in the 4th week in SLActive surface measurements. No significant differences were observed between the groups at 2 and 3 months (P>0.05). Conclusions. For both implant surfaces, increased stability was observed over time, with no significant differences between the groups.

Wnt Pathway Inhibitor Delays Fracture Healing by Targeting Ephrin B1 (EFNB1) in Osteoprogenitor Cells

Our study assessed the role of Wnt signaling inhibitor (SM04690) in fracture healing and the underlying mechanism. Sprague Dawley (SD) rats were used to establish a fracture model which was then separated into SM04690 group which received SM04690 (50 mg/kg) by intraperitoneal injection once a day for one week, and control group which received saline once a day. After rats were sacrificed, the fractured femurs were harvested to measure femoral strength by stress testing, bone density and volume by CT. Femurs were sliced for immunohistochemical staining. Mesenchymal stem cells (MSCs), endothelial cells, osteoprogenitor cells and osteoblasts were detected by flow cytometer and EFNB1 expression was detected by immunoblotting and PCR. In addition, MSCs were treated with SM04690 (5 uM), followed by detection of EFNB1 expression. SM04690 treatment significantly inhibited EFNB1 expression and reduced bone volume and callus volume as well as decreased ultimate load of bones. Immunohistochemical staining and flow cytometry analysis showed no difference of osteoclast numbers at the fracture site between two groups, but proportion of osteoclasts in the cartilage tissue of SM04690 group was significantly decreased. In addition, the number of osteoblasts, osteoprogenitor cells and endothelial cells was significantly decreased after treatment. Under the conditions favoring osteogenic differentiation, the production of minerals by osteogenic cells was significantly decreased along with upregulated TAZ phosphorylation and downregulated Osterix in SM04690 group. In conclusion, SM04690 delays fracture healing by inhibiting EpRunB1 in osteoprogenitor cells.

WNT16 elevation induced cell senescence of osteoblasts in ankylosing spondylitis

Background WNT16 is critical for bone homeostasis, but the effect of WNT16 in ankylosing spondylitis (AS) is still unknown. Here, we investigated whether WNT16 influences bone formation and pathophysiological changes of AS in an in vitro model. Methods The bone tissue from the facet joints was obtained from seven disease control and seven AS patients. Primary osteoprogenitor cells of the facet joints were isolated using an outgrowth method. Isolated osteoprogenitor cells from both control and AS tissues were analyzed by microarray, RT-qPCR, immunoblotting, and immunohistochemistry. The bone-forming activity of osteoprogenitor cells was assessed by various in vitro assays. β-galactosidase staining and senescence-associated secretory phenotype (SASP) using RT-qPCR were used to assess cell senescence. Results In microarray analysis, WNT16 expression was significantly elevated in AS osteoprogenitor cells compared to the control. We also validated that WNT16 expression was elevated in AS-osteoprogenitor cells and human AS-bone tissues. WNT16 treatment inhibited bone formation in AS-osteoprogenitor cells but not in the control. Intriguingly, AS-osteoprogenitor cells were stained markedly with β-galactosidase for cell senescence in WNT16 treatment. Furthermore, in an H2O2 stress-induced premature senescence condition, WNT16 treatment increased cell senescence in AS-osteoprogenitor cells and WNT16 treatment under the H2O2 stress condition showed an increase in p21 protein and SASP mRNA expression. The WNT16-induced SASP expression in AS-osteoprogenitor cells was reduced in WNT16 knockdown cultures. Conclusion WNT16 is highly expressed in AS and WNT16 treatment facilitated cell senescence in AS-osteoprogenitor cells during osteoblast differentiation accompanied by suppression of bone formation. The identified role of WNT16 in AS could influence bone loss in AS patients.

Epo/EpoR signaling in osteoprogenitor cells is essential for bone homeostasis and Epo-induced bone loss

High erythropoietin (Epo) levels are detrimental to bone health in adult organisms. Adult mice receiving high doses of Epo lose bone mass due to suppressed bone formation and increased bone resorption. In humans, high serum Epo levels are linked to fractures in elderly men. Our earlier studies indicated that Epo modulates osteoblast activity; however, direct evidence that Epo acts via its receptor (EpoR) on osteoblasts in vivo is still missing. Here, we created mice lacking EpoR in osteoprogenitor cells to specifically address this gap. Deletion of EpoR in osteoprogenitors (EpoR:Osx-cre, cKO) starting at 5 weeks of age did not alter red blood cell parameters but increased vertebral bone volume by 25% in 12-week-old female mice. This was associated with low bone turnover. Histological (osteoblast number, bone formation rate) and serum (P1NP, osteocalcin) bone formation parameters were all reduced, as were the number of osteoclasts and TRAP serum level. Differentiation of osteoblast precursors isolated from cKO versus control mice resulted in lower expression of osteoblast marker genes including Runx2, Alp, and Col1a1 on day 21, whereas the mineralization capacity was similar. Moreover, the RANKL/OPG ratio, which determines the osteoclast-supporting potential of osteoblasts, was substantially decreased by 50%. Similarly, coculturing cKO osteoblasts with control or cKO osteoclast precursors produced significantly fewer osteoclasts than coculture with control osteoblasts. Finally, exposing female mice to Epo pumps (10 U·d−1) for 4 weeks resulted in trabecular bone loss (−25%) and increased osteoclast numbers (1.7-fold) in control mice only, not in cKO mice. Our data show that EpoR in osteoprogenitors is essential in regulating osteoblast function and osteoblast-mediated osteoclastogenesis via the RANKL/OPG axis. Thus, osteogenic Epo/EpoR signaling controls bone mass maintenance and contributes to Epo-induced bone loss.

Altered Secretome of Diabetic Monocytes Could Negatively Influence Fracture Healing—An In Vitro Study

Diabetes mellitus is a main risk factor for delayed fracture healing and fracture non-unions. Successful fracture healing requires stimuli from different immune cells, known to be affected in diabetics. Especially, application of mononuclear cells has been proposed to promote wound and fracture healing. Thus, aim was to investigate the effect of pre-/diabetic conditions on mononuclear cell functions essential to promote osteoprogenitor cell function. We here show that pre-/diabetic conditions suppress the expression of chemokines, e.g., CCL2 and CCL8 in osteoprogenitor cells. The associated MCP-1 and MCP-2 were significantly reduced in serum of diabetics. Both MCPs chemoattract mononuclear THP-1 cells. Migration of these cells is suppressed under hyperglycemic conditions, proposing that less mononuclear cells invade the site of fracture in diabetics. Further, we show that the composition of cytokines secreted by mononuclear cells strongly differ between diabetics and controls. Similar is seen in THP-1 cells cultured under hyperinsulinemia or hyperglycemia. The altered secretome reduces the positive effect of the THP-1 cell conditioned medium on migration of osteoprogenitor cells. In summary, our data support that factors secreted by mononuclear cells may support fracture healing by promoting migration of osteoprogenitor cells but suggest that this effect might be reduced in diabetics.

Stat3 loss in mesenchymal progenitors causes Job syndrome–like skeletal defects by reducing Wnt/β-catenin signaling

Job syndrome is a rare genetic disorder caused by STAT3 mutations and primarily characterized by immune dysfunction along with comorbid skeleton developmental abnormalities including osteopenia, recurrent fracture of long bones, and scoliosis. So far, there is no definitive cure for the skeletal defects in Job syndrome, and treatments are limited to management of clinical symptoms only. Here, we have investigated the molecular mechanism whereby Stat3 regulates skeletal development and osteoblast differentiation. We showed that removing Stat3 function in the developing limb mesenchyme or osteoprogenitor cells in mice resulted in shortened and bow limbs with multiple fractures in long bones that resembled the skeleton symptoms in the Job Syndrome. However, Stat3 loss did not alter chondrocyte differentiation and hypertrophy in embryonic development, while osteoblast differentiation was severely reduced. Genome-wide transcriptome analyses as well as biochemical and histological studies showed that Stat3 loss resulted in down-regulation of Wnt/β-catenin signaling. Restoration of Wnt/β-catenin signaling by injecting BIO, a small molecule inhibitor of GSK3, or crossing with a Lrp5 gain of function (GOF) allele, rescued the bone reduction phenotypes due to Stat3 loss to a great extent. These studies uncover the essential functions of Stat3 in maintaining Wnt/β-catenin signaling in early mesenchymal or osteoprogenitor cells and provide evidence that bone defects in the Job Syndrome are likely caused by Wnt/β-catenin signaling reduction due to reduced STAT3 activities in bone development. Enhancing Wnt/β-catenin signaling could be a therapeutic approach to reduce bone symptoms of Job syndrome patients.

Accelerating Socket Repair via WNT3A Curtails Alveolar Ridge Resorption

Tooth extraction triggers alveolar ridge resorption, and when this resorption is extensive, it can complicate subsequent reconstructive procedures that use dental implants. Clinical data demonstrate that the most significant dimensional changes in the ridge occur soon after tooth extraction. Here, we sought to understand whether a correlation existed between the rate at which an extraction socket heals and the extent of alveolar ridge resorption. Maxillary molars were extracted from young and osteoporotic rodents, and quantitative micro–computed tomographic imaging, histology, and immunohistochemistry were used to simultaneously follow socket repair and alveolar ridge resorption. Extraction sockets rapidly filled with new bone via the proliferation and differentiation of Wnt-responsive osteoprogenitor cells and their progeny. At the same time that new bone was being deposited in the socket, tartrate-resistant acid phosphatase–expressing osteoclasts were resorbing the ridge. Significantly faster socket repair in young animals was associated with significantly more Wnt-responsive osteoprogenitor cells and their progeny as compared with osteoporotic animals. Delivery of WNT3A to the extraction sockets of osteoporotic animals restored the number of Wnt-responsive cells and their progeny back to levels seen in young healthy animals and accelerated socket repair in osteoporotic animals back to rates seen in the young. In cases where the extraction socket was treated with WNT3A, alveolar ridge resorption was significantly reduced. These data demonstrate a causal link between enhancing socket repair via WNT3A and preserving alveolar ridge dimensions following tooth extraction.

Notch-Wnt signal crosstalk regulates proliferation and differentiation of osteoprogenitor cells during intramembranous bone healing

Adult bone regeneration is orchestrated by the precise actions of osteoprogenitor cells (OPCs). However, the mechanisms by which OPC proliferation and differentiation are linked and thereby regulated are yet to be defined. Here, we present evidence that during intramembranous bone formation OPC proliferation is controlled by Notch signaling, while differentiation is initiated by activation of canonical Wnt signaling. The temporospatial separation of Notch and Wnt signal activation during the early stages of bone regeneration suggests crosstalk between the two pathways. In vitro and in vivo manipulation of the two essential pathways demonstrate that Wnt activation leads to initiation of osteogenic differentiation and at the same time inhibits Notch signaling, which results in termination of the proliferative phase. Here, we establish canonical Wnt signaling as a key regulator that facilitates the crosstalk between OPC proliferation and differentiation during intramembranous, primary bone healing.