Alteration of clot architecture using bone substitute biomaterials (beta-tricalcium phosphate) significantly delays the early bone healing process.

When a bone substitute biomaterial is implanted into the body, the material's surface comes into contact with circulating blood, which results in the formation of a peri-implant hematoma or blood clot. Although hematoma formation is vital for the early bone healing process, knowledge concerning the biomaterial-induced structural properties of blood clots is limited. Here, we report that implantation of beta-tricalcium phosphate (ß-TCP) in a bone defect healing model in rats resulted in significantly delayed early bone healing compared to empty controls (natural healing). In vitro studies showed that ß-TCP had a profound effect on the overall structure of hematomas, as was observed by fibrin turbidity, scanning electron microscopy (SEM), compaction assays, and fibrinolysis. Under the influence of ß-TCP, clot formation had a significantly shortened lag time and there was enhanced lateral fibrin aggregation during the clot polymerization, which resulted in clots composed of thinner fibers. Furthermore, fibrin clots that formed around ß-TCP exhibited reduced compaction and increased resistance to fibrinolysis. Together, these results provide a plausible mechanism for how implanted bone-substitute materials may impact the structural properties of the hematoma, thereby altering the early bone healing processes, such as cell infiltration, growth factor release and angiogenesis.

Leptin Overexpression in Bone Marrow Stromal Cells Promotes Periodontal Regeneration in a Rat Model of Osteoporosis.

BACKGROUND: Osteoporosis is associated with widespread periodontitis and impaired periodontal healing. However, there is a lack of information about the outcomes of regenerative approaches under the influence of osteoporosis. This study investigates the effect of leptin (LEP) overexpression on the regenerative potential of bone marrow stromal cells (BMSCs) in an osteoporotic rat periodontal fenestration defect model. METHODS: Rat BMSCs were transfected with adenoviruses harboring the human (h)LEP gene. Cell proliferation and osteogenic differentiation were evaluated. A ß-tricalcium phosphate scaffold seeded with transfected cells was implanted into nude mice to investigate ectopic osteogenesis and into an osteoporotic rat defect to study periodontal regeneration. Regenerated periodontal and bone-like tissues were analyzed by histologic methods. RESULTS: hLEP overexpression induced osteogenic differentiation of BMSCs as evidenced by the upregulation of osteogenesis-related genes such as Runt-related transcription factor 2, alkaline phosphatase (ALP), and collagen Type I, as well as increased ALP activity and enhanced mineralization. Mice implanted with hLEP-BMSC-containing scaffolds showed more extensive formation of bone-like tissue than those in other groups. Periodontal defects were also filled to a greater degree when treated with hLEP-BMSCs and contained cementum and a well-organized periodontal ligament after 10 and 28 days. CONCLUSION: hLEP overexpression in BMSCs can stimulate periodontal regeneration in osteoporotic conditions and might be a promising strategy for periodontal regeneration in patients with osteoporosis.

Prediction of pain in orthodontic patients based on preoperative pain assessment.

AIM: To investigate whether pretreatment assessment of experimental pain can predict the level of pain after archwire placement. METHODS: One hundred and twenty-one general university students seeking orthodontic treatment were enrolled in this study. A cold pressor test was performed to estimate the pain tolerance of subjects before treatment. Self-reported pain intensity was calculated using a 10 cm visual analog scale during the 7 days after treatment. The relationship between pain tolerance and orthodontic pain was analyzed using Spearman's correlation analysis. RESULTS: The maximum mean level of pain intensity occurred at 24 hours after bonding (53.31±16.13) and fell to normal levels at day 7. Spearman's correlation analysis found a moderate positive association between preoperative pain tolerance and self-reported pain after archwire placement (P<0.01). There was no significant difference in pain intensity between male and female patients at any time point (P>0.05). CONCLUSION: A simple and noninvasive preoperative sensory test (the cold pressor test) was useful in predicting the risk of developing unbearable pain in patients after archwire placement. Self-reported pain after archwire placement decreased as individual pain tolerance increased.

[Construction of leptin gene modified tissue engineered composites in vitro].

PURPOSE: To evaluate the feasibility of constructing tissue engineered composites in vitro by combining human leptin (hLEP) gene modified rat bone marrow stromal cells (BMSCs) and guided tissue regeneration collagen membrane (Bio-Gide). METHODS: BMSCs of SD rats were isolated and cultured by whole bone marrow adherent method. BMSCS were transfected with adenovirus carrying hLEP gene (Ad-hLEP-EGFP) and observed under inverted fluorescence microscope. Enzyme linked immunosorbent assay (ELISA) was used to detect the expression of hLEP. The proliferation activity of transfected cells was assessed by MTT assay. Ad-hLEP-EGFP transfected BMSCs were cultured for 24 h in combination with Bio-Gide collagen membrane, hLEP modified tissue engineered composite was observed under laser scanning confocal microscope (LSCM) and scanning electron microscope (SEM). RESULTS: Through Ad-hLEP-EGFP transfection, hLEP was overexpressed in BMSCs, which didn't affect the proliferation of cells. SEM showed hLEP modified BMSCs grew well on Bio-Gide collagen membrane and secreted extracellular matrix. LSCM suggested BMSCs could migrate to different scales of Bio-Gide collagen membrane. CONCLUSIONS: hLEP modified BMSCs can be combined with Bio-Gide collagen membrane and grow well, suggesting that hLEP modified tissue engineered composite can be successfully constructed. The composite might be suitable for periodontal tissue engineering.

Increased osteogenesis in osteoporotic bone marrow stromal cells by overexpression of leptin.

Osteoporosis leads to increased bone fractures and net bone loss, in part because of the dysfunction of bone marrow stromal cells (BMSCs). Leptin is an adipokine that plays important roles in many biological processes, including the regulation of the actions of mesenchymal stem cells. Our aim is to investigate the osteogenic effects of leptin in osteoporotic BMSCs in vitro and in vivo. The leptin gene was transferred into BMSCs isolated from osteoporotic rats by using recombinant adenoviruses. Once the gene and protein expression of leptin had been confirmed, MTT assays were performed; leptin overexpression was confirmed not to affect the viability of osteoporotic BMSCs. However, alkaline phosphatase (ALP) activity measurements, Alizarin red staining and analyses by quantitative real-time reverse transcription with the polymerase chain reaction revealed that leptin upregulated ALP activity, mineral deposition and the mRNA levels of runt-related transcription factor 2, ALP and collagen type І. Lastly, the effects of leptin on osteogenic differentiation were assessed in vivo. Cells transfected with leptin exhibited increased osteogenic differentiation and enhanced formation of bone-like structures. This study thus reveals, for the first time, that the overexpression of leptin in osteoporotic BMSCs (1) enhances their capacity to differentiate into osteoblasts and to form bone-like tissue and (2) might be a useful skeletal regenerative therapy in osteoporotic patients.

[Gene expression of latent TGF-ß binding protein-3 in the follicle of mouse first mandibular molar in mice].

PURPOSE: To observe and analyze the gene expression of latent TGF-ß binding protein-3(LTBP-3) in mice first mandibular molars during tooth eruption period. METHODS: The first mandibular molar germs of KM mice, from postnatal 1.5 to 9.5 days, were dissected. Anatomical microscope was used to observe and separate dental follicles of each time-point. After total RNA extracted from separated dental follicles of the first mandibular molars, reverse transcription-polymerase chain reaction (RT-PCR) was performed using LTBP-3 specific primers to detect its gene expression. Then densitometry analysis was performed using Gene Genius Bio-Imaging system and GeneSnap software. Expression differences between any two time points were assessed by LSD test in one-way ANOVA at α=0.05 level using SPSS 13.0 software package. RESULTS: A circle of fibrae sac like loose tissue could be seen around all the germs in anatomical microscope and could be carefully dissected from the germs. There was an aging progressive gene expression of LTBP-3 in the dental follicles of mice first mandibular molars among the detecting time-points. LSD test analysis revealed that the expression of both P7.5 and P9.5 was higher than that on the P1.5 (P<0.05 and P<0.01, respectively) and P9.5 expression level was higher than that on the P3.5 (P<0.05), while there was no difference in expression between any other two detecting days. CONCLUSIONS: LTBP-3 is confined to have an aging progressive gene expression in the follicles of mice first mandibular molars during tooth eruption period. LTBP-3 may play a role in the events of mice teeth eruption.