Diabetic humans and animals exhibit lower bone mass and healing, resulting from diminished bone formation. We have recently reported that type 1 diabetic rats have fewer bone marrow osteoprogenitor cells, and since the formation of advanced glycation end products (AGEs) in bone increases in diabetes, we explored possible mechanisms involved in AGE-induced apoptosis of rat bone marrow stromal cells (BMSCs). BMSCs isolated from 4-month-old rats were exposed to 10-400 mg/ml AGE-BSA for 16 h and apoptosis was quantified with PI/annexin V staining and flow cytometry. Signaling mechanisms were evaluated by preincubating the cells with appropriate inhibitors. The formation of reactive oxygen species (ROS) was quantified by flow cytometric analysis of DCFDA fluorescence and the expression of genes by RT-PCR analysis. AGE-BSA at a concentration of 400 mg/ml increased the apoptosis of BMSCs two-to threefold, an effect completely blocked by a pan-caspase inhibitor. BSA or high concentrations of glucose had no effect. AGE-BSA-induced BMSC apoptosis was attenuated by a p38 inhibitor but not by an NF-kB inhibitor. Treatment with AGE-BSA induced the expression of several pro-apoptotic ligands and receptors, most notably tumor necrosis factor a (TNFa), TRAIL, lymphotoxin alpha, CD40, and TNFR2. Furthermore, AGE-BSA-induced apoptosis was completely blocked by pirfenidone, an inhibitor of TNFa production/secretion. Finally, AGE-BSA increased the production of ROS in BMSCs, and its apoptogenic effect was blocked by the antioxidant N-acetylcysteine (N-acetyl-L-cysteine). Thus, AGE-BSA increases the apoptosis of rat BMSCs via the activation of caspases, involving TNFa production/ secretion, p38 MAPK signaling, and oxidative stress. We propose that increased protein glycation, such as that occurring under hyperglycemia, causes the apoptosis of BMSCs, which might significantly contribute to the development of osteopenia in diabetic animals.

The effect of different mechanical and chemical pre-treatments on the adhesion strength of hydroxyapatite (HAp) coating on a commercially pure titanium (CP-Ti) substrate was studied by means of a standard tensile test followed by microscopic and chemical analysis to determine the locus of fracture. In addition, the effects of either these pre-treatments or post-treatment by low-energy electron irradiation, which allowed tuning the wettability of the surface, on both osteoblast progenitor attachment and S. aureus bacteria attachment were investigated. A dedicated program was developed for unambiguous identification and count of stained cells. A single-phase HAp coating was formed by electrodeposition. A series of surface pre-treatments consisted of grinding down to P1000, etching in HNO₃/HF solution, grit blast, soaking in NaOH and subsequent heat treatment provided the highest adhesion strength to the HAp coating. Osteoblast progenitors derived from rats may be attached preferentially to a hydrophilic surface (post-treatment to θ = 30°), while the bacteria seemed to be less attached to hydrophobic surfaces (post-treatment to θ = 105°). However, the results were not statistically different. The bacteria seemed to be less attached to the smoother, uncoated surfaces.

Prostaglandin E(2) inhibits and EMD stimulates hGF proliferation via distinct pathways. The different sensitivities of hDFs and hGKs to PGE(2) can be explained by the levels of EP(2) expression.

Aim To examine the in vitro biokinetics of hyaluronic acid (HA) from a collagen membrane (CM) and to evaluate the in vivo effect of immersion of the CM in HA solution on its degradation in streptozotocin (STZ)‐induced diabetes conditions in a rat calvaria subcutaneous model. Background CM degradation is accelerated in uncontrolled diabetic rats. Immersion of CM in HA has been suggested to decrease their resorption rate without interfering with their tissue integration and structural degradation. However, it is unknown to what extent CM degradation may be influenced by its immersion in HA solution under a condition mimicking a medically compromised situation with an increased inflammatory level such as diabetes. Materials and Methods CMs were soaked in cross‐linked HA. Protein adsorption and the HA release were quantified by ELISA. Diabetes was induced in sixteen rats, while 16 healthy rats served as control. CM was prepared and labeled prior to implantation with Biotin. Seventeen CM were immersed in HA and 17 CM in PBS. In each animal, one test or one control disk was implanted. In order to compare the collagen content, two similar non‐implanted CM were used as baseline. Fourteen days after surgery, thirty‐two animals were sacrificed. The entire calvaria including the skin above, was chemically fixed, decalcified, and embedded in paraffin. Five‐μm‐thick sections were analyzed histologically and histomorphometrically using H&E and avidin‐peroxidase staining. Results The in vitro results demonstrated that the CM adsorbed roughly 80% of the total HA content. After 10 days, 36.3% of the initial HA remained on the CM. The in vivo results demonstrated that diabetes significantly reduced the thickness of the CM, while HA had a significant effect on keeping the membrane thickness. HA increased the residual collagen content in the diabetic group (P < 0.0001) but no such effect was observed in the healthy group. Conclusion Immersion of CM in HA prior to the implantation delays membrane degradation in uncontrolled diabetic compared with normoglycemic rats.

Elevated levels of prostaglandins such as PGE(2) in inflamed gingiva play a significant role in the tissue destruction caused by periodontitis, partly by targeting local fibroblasts. Only very few studies have shown that PGE(2) inhibits the proliferation of a gingival fibroblast (GF) cell line, and we expanded this research by using primary human GFs (hGFs) and looking into the mechanisms of the PGE(2) effect. GFs derived from healthy human gingiva were treated with PGE(2) and proliferation was assessed by measuring cell number and DNA synthesis and potential signaling pathways were investigated using selective activators or inhibitors. PGE(2) inhibited the proliferation of hGFs dose-dependently. The effect was mimicked by forskolin (adenylate cyclase stimulator) and augmented by IBMX (a cAMP-breakdown inhibitor), pointing to involvement of cAMP. Indeed, PGE(2) and forskolin induced cAMP generation in these cells. Using selective EP receptor agonists we found that the anti-proliferative effect of PGE(2) is mediated via the EP(2) receptor (which is coupled to adenylate cyclase activation). We also found that the effect of PGE(2) involved activation of Epac (exchange protein directly activated by cAMP), an intracellular cAMP sensor, and not PKA. While serum increased the amount of phospho-ERK in hGFs by approximately 300%, PGE(2) decreased it by approximately 50%. Finally, the PGE(2) effect does not require endogenous production of prostaglandins since it was not abrogated by two COX-inhibitors. In conclusion, in human gingival fibroblasts PGE(2) activates the EP(2)-cAMP-Epac pathway, reducing ERK phosphorylation and inhibiting proliferation. This effect could hamper periodontal healing and provide further insights into the pathogenesis of inflammatory periodontal disease.