Fibrin with Laminin-Nidogen Reduces Fibrosis and Improves Soft Palate Regeneration Following Palatal Injury.

This study aimed to analyze the effects of fibrin constructs enhanced with laminin-nidogen, implanted in the wounded rat soft palate. Fibrin constructs with and without laminin-nidogen were implanted in 1 mm excisional wounds in the soft palate of 9-week-old rats and compared with the wounded soft palate without implantation. Collagen deposition and myofiber formation were analyzed at days 3, 7, 28 and 56 after wounding by histochemistry. In addition, immune staining was performed for a-smooth muscle actin (a-SMA), myosin heavy chain (MyHC) and paired homeobox protein 7 (Pax7). At day 56, collagen areas were smaller in both implant groups (31.25 ± 7.73% fibrin only and 21.11 ± 6.06% fibrin with laminin-nidogen)) compared to the empty wounds (38.25 ± 8.89%, p < 0.05). Moreover, the collagen area in the fibrin with laminin-nidogen group was smaller than in the fibrin only group (p ˂ 0.05). The areas of myofiber formation in the fibrin only group (31.77 ± 10.81%) and fibrin with laminin-nidogen group (43.13 ± 10.39%) were larger than in the empty wounds (28.10 ± 11.68%, p ˂ 0.05). Fibrin-based constructs with laminin-nidogen reduce fibrosis and improve muscle regeneration in the wounded soft palate. This is a promising strategy to enhance cleft soft palate repair and other severe muscle injuries.

CXCL12-CXCR4 Interplay Facilitates Palatal Osteogenesis in Mice.

Cranial neural crest cells (CNCCs), identified by expression of transcription factor Sox9, migrate to the first branchial arch and undergo proliferation and differentiation to form the cartilage and bone structures of the orofacial region, including the palatal bone. Sox9 promotes osteogenic differentiation and stimulates CXCL12-CXCR4 chemokine-receptor signaling, which elevates alkaline phosphatase (ALP)-activity in osteoblasts to initiate bone mineralization. Disintegration of the midline epithelial seam (MES) is crucial for palatal fusion. Since we earlier demonstrated chemokine-receptor mediated signaling by the MES, we hypothesized that chemokine CXCL12 is expressed by the disintegrating MES to promote the formation of an osteogenic center by CXCR4-positive osteoblasts. Disturbed migration of CNCCs by excess oxidative and inflammatory stress is associated with increased risk of cleft lip and palate (CLP). The cytoprotective heme oxygenase (HO) enzymes are powerful guardians harnessing injurious oxidative and inflammatory stressors and enhances osteogenic ALP-activity. By contrast, abrogation of HO-1 or HO-2 expression promotes pregnancy pathologies. We postulate that Sox9, CXCR4, and HO-1 are expressed in the ALP-activity positive osteogenic regions within the CNCCs-derived palatal mesenchyme. To investigate these hypotheses, we studied expression of Sox9, CXCL12, CXCR4, and HO-1 in relation to palatal osteogenesis between E15 and E16 using (immuno)histochemical staining of coronal palatal sections in wild-type (wt) mice. In addition, the effects of abrogated HO-2 expression in HO-2 KO mice and inhibited HO-1 and HO-2 activity by administrating HO-enzyme activity inhibitor SnMP at E11 in wt mice were investigated at E15 or E16 following palatal fusion. Overexpression of Sox9, CXCL12, CXCR4, and HO-1 was detected in the ALP-activity positive osteogenic regions within the palatal mesenchyme. Overexpression of Sox9 and CXCL12 by the disintegrating MES was detected. Neither palatal fusion nor MES disintegration seemed affected by either HO-2 abrogation or inhibition of HO-activity. Sox9 progenitors seem important to maintain the CXCR4-positive osteoblast pool to drive osteogenesis. Sox9 expression may facilitate MES disintegration and palatal fusion by promoting epithelial-to-mesenchymal transformation (EMT). CXCL12 expression by the MES and the palatal mesenchyme may promote osteogenic differentiation to create osteogenic centers. This study provides novel evidence that CXCL12-CXCR4 interplay facilitates palatal osteogenesis and palatal fusion in mice.

Heme Oxygenase Protects against Placental Vascular Inflammation and Abortion by the Alarmin Heme in Mice.

Both infectious as non-infectious inflammation can cause placental dysfunction and pregnancy complications. During the first trimester of human gestation, when palatogenesis takes place, intrauterine hematoma and hemorrhage are common phenomena, causing the release of large amounts of heme, a well-known alarmin. We postulated that exposure of pregnant mice to heme during palatogenesis would initiate oxidative and inflammatory stress, leading to pathological pregnancy, increasing the incidence of palatal clefting and abortion. Both heme oxygenase isoforms (HO-1 and HO-2) break down heme, thereby generating anti-oxidative and -inflammatory products. HO may thus counteract these heme-induced injurious stresses. To test this hypothesis, we administered heme to pregnant CD1 outbred mice at Day E12 by intraperitoneal injection in increasing doses: 30, 75 or 150 µmol/kg body weight (30H, 75H or 150H) in the presence or absence of HO-activity inhibitor SnMP from Day E11. Exposure to heme resulted in a dose-dependent increase in abortion. At 75H half of the fetuses where resorbed, while at 150H all fetuses were aborted. HO-activity protected against heme-induced abortion since inhibition of HO-activity aggravated heme-induced detrimental effects. The fetuses surviving heme administration demonstrated normal palatal fusion. Immunostainings at Day E16 demonstrated higher numbers of ICAM-1 positive blood vessels, macrophages and HO-1 positive cells in placenta after administration of 75H or SnMP + 30H. Summarizing, heme acts as an endogenous "alarmin" during pregnancy in a dose-dependent fashion, while HO-activity protects against heme-induced placental vascular inflammation and abortion.

Effects of Remote Ischemic Preconditioning on Heme Oxygenase-1 Expression and Cutaneous Wound Repair.

Skin wounds may lead to scar formation and impaired functionality. Remote ischemic preconditioning (RIPC) can induce the anti-inflammatory enzyme heme oxygenase-1 (HO-1) and protect against tissue injury. We aim to improve cutaneous wound repair by RIPC treatment via induction of HO-1. RIPC was applied to HO-1-luc transgenic mice and HO-1 promoter activity and mRNA expression in skin and several other organs were determined in real-time. In parallel, RIPC was applied directly or 24h prior to excisional wounding in mice to investigate the early and late protective effects of RIPC on cutaneous wound repair, respectively. HO-1 promoter activity was significantly induced on the dorsal side and locally in the kidneys following RIPC treatment. Next, we investigated the origin of this RIPC-induced HO-1 promoter activity and demonstrated increased mRNA in the ligated muscle, heart and kidneys, but not in the skin. RIPC did not change HO-1 mRNA and protein levels in the wound 7 days after cutaneous injury. Both early and late RIPC did not accelerate wound closure nor affect collagen deposition. RIPC induces HO-1 expression in several organs, but not the skin, and did not improve excisional wound repair, suggesting that the skin is insensitive to RIPC-mediated protection.

Primary and secondary cartilages of the neonatal rat: the femoral head and the mandibular condyle.

Primary and secondary cartilages differ in embryonic origin and in histological organization, and are generally considered to have a different mode of growth. However, few studies have directly compared the two types of cartilage of the same animal at the same age. Therefore, we analysed several histological and biochemical differences between secondary cartilage of the mandibular condyle and primary cartilage of the femoral head of 4-d-old rats. We evaluated the tissue organization, the level of DNA and glycosaminoglycan (GAG) synthesis, and the GAG and collagen content. The expression of collagen types I, II and III and of receptors for insulin-like growth factor (IGF)-I, fibroblast growth factor (FGF), and transforming growth factor (TGF)-beta were investigated by immunohistochemistry. The ex vivo DNA and GAG synthesis as well as the GAG content of femoral heads were much higher than that of mandibular condyles. Mandibular condyles expressed both collagen types I and II, while femoral heads expressed only type II collagen. In the mandibular condyles, receptors for IGF-I, FGF, and TGF-beta were observed mainly in the superficial layers, whereas they were found throughout the entire femoral head. In conclusion, major differences were found between both types of cartilage, which might be related to their specific functional demands.