Optimizing the combined soft tissue repair and osteogenesis using double surfaces of crosslinked collagen scaffolds.

Excessive tissue damage or loss has been solved by guided tissue regeneration and guided bone regeneration theories. However, the unfavorable degradation property of the resorbable collagen scaffold brings a big challenge to support soft tissue stabilization and time-consuming osteogenesis. The combined effect for soft tissue and bone of the collagen scaffold with better degradation pattern has not been clearly proven. This study determined whether the double surfaces of crosslinked collagen scaffolds could optimize the combined soft tissue repair and osteogenesis. In this study, we applied the chemically crosslinking treatment to the commercially available collagen scaffolds. Surface characterization, mechanical property and cell proliferation in vitro were evaluated. Combined bilateral skin and bone defects were established with the smooth surface of scaffold facing the skin defect and the rough surface facing the bone defect on the calvaria of rat. Micro-CT and histological evaluation were applied to determine the scaffold degradation pattern, soft tissue repair and osteogenesis. The crosslinked collagen scaffolds showed comparably favorable surface porosity, structure intactness, superhydrophilicity and mechanical properties. Compared to the native scaffolds, the crosslinked scaffolds could optimize the combined soft tissue repair and osteogenesis by preferably prolonged degradation time. Early pro-angiogenesis facilitated soft tissue repair and osteogenesis by upregulated soft tissue matrix degradation and balanced pro-osteogenesis with limited osteoclast-mediated bone resorption. Taken together, this study offers a promising repair strategy for the combined soft tissue and bone defects. Further, the possible mechanism of controllable scaffold degradation should be conducted.

Autophagy inhibits TLR4-mediated invasiveness of oral cancer cells via the NF-κB pathway.

OBJECTIVES: Toll-like receptor 4 (TLR4) is abnormally expressed in oral cancer tissues and promotes cancer cell invasion. The purpose of this study was to clarify the mechanism by which autophagy regulates oral cancer invasion through the TLR4-NF-κB pathway. SUBJECTS AND METHODS: We examined TLR4 expression in oral cancer tissues and analysed the relationship between its expression and clinicopathological features. The invasion and migration of LPS-stimulated oral cancer cells with up- or downregulation of TLR4 expression was detected in addition to NF-κB signalling and autophagy levels. Furthermore, the role of autophagy in regulating TLR4-mediated cell invasiveness was explored by silencing the expression of key autophagy genes ATG7 and p62. RESULTS: We found that TLR4 overexpression was closely related to cervical lymphatic metastasis and poor survival. TLR4 activated the NF-κB pathway to promote the invasiveness of OSCC cells, and autophagy partly inhibited invasiveness by suppressing the NF-κB pathway. We observed that p62 translocated from the cytoplasm to the nucleus when autophagy was activated by LPS. Finally, silencing p62 further promoted LPS-mediated cell invasiveness. CONCLUSION: Toll-like receptor 4 significantly enhanced the invasiveness of OSCC cells. Autophagy may regulate cell invasiveness through the NF-κB pathway by modulating both the cytoplasmic and nuclear levels of p62.

An individual patient data meta-analysis on the effect of chemotherapy on survival in patients with craniofacial osteosarcoma.

Chemotherapy improves the survival of patients with long bone osteosarcomas. However, the benefits of chemotherapy in the treatment of craniofacial osteosarcoma (CFOS) are still controversial. We searched PubMed and EMBASE from February 1997 to December 2016 to identify studies on CFOS. The individual patient data of these studies were pooled into a meta-analysis. Univariate and multivariate survival analyses were performed. Thirteen studies with a total of 184 patients met our inclusion criteria. Positive resection margin was a poor prognostic factor for CFOS in the univariate and multivariate survival analyses. Chemotherapy improved overall survival (OS) and disease-specific survival (DSS) in patients with CFOS who had tumors in the maxilla, positive resection margins, or high-grade tumors. Patients with local tumor recurrence had better OS and DSS when treated with chemotherapy. Chemotherapy improves survival in patients with CFOS with adverse factors, such as tumors with positive margins, high-grade tumors, and recurrent tumors.

Role of Noxa in proliferation, apoptosis, and autophagy in human adenoid cystic carcinoma.

BACKGROUND: Noxa, which is subset of the Bcl-2 family of proteins, was previously reported to have considerable therapeutic potential in diverse cancers. However, its expression and role in salivary gland adenoid cystic carcinoma (ACC) have not been well studied. This study aimed to elucidate the expression and role of Noxa in salivary gland ACC. MATERIALS AND METHODS: The expression levels of NOXA and its association with overall survival in salivary gland ACC were analyzed by quantitative real-time PCR. We next examined the effects of Noxa overexpression or inhibition on colony formation, proliferation, apoptosis, and autophagy of salivary gland ACC cells. Furthermore, promoter analysis was performed to identify the potential transcriptional activator of NOXA. RESULTS: NOXA was markedly down-regulated and significantly correlated with a more aggressive phenotype and poor overall survival of salivary gland ACC. Ectopic expression of Noxa suppressed the viability and growth of ACC cells, which involved the induction of apoptosis and autophagy. Moreover, the transcriptional activity of NOXA gene could be enhanced by p53. CONCLUSION: The findings of this study indicate that Noxa, activated transcriptionally by p53, suppress the progression of ACC, whereby it regulates proliferation, apoptosis, and autophagy.

Investigation of cancer-associated fibroblasts and p62 expression in oral cancer before and after chemotherapy.

PURPOSE: The aim of this study is to investigate the expression of the autophagy protein p62 in oral squamous cell carcinoma (OSCC) cells before and after chemotherapy. We also detected cancer-associated fibroblasts (CAFs) in these OSCC samples to explore the roles of p62 and CAFs in chemotherapy. MATERIALS AND METHODS: Immunohistochemistry was used to analyze the expression of p62 and α-SMA in 26 paired OSCC samples before and after chemotherapy. The relationships between clinicopathological features, clinical outcome and the expression of these proteins were analyzed. RESULTS: Our results indicated an increased stromal α-SMA expression after chemotherapy in OSCC samples. High p62 expression of OSCC cells closely correlated with stromal α-SMA expression after chemotherapy. Furthermore, the post-chemotherapy p62 expression was associated with the prognosis for OSCC patients. CONCLUSION: These results suggest that chemotherapy may increase CAFs in OSCC. High cytoplasmic p62 expression may serve as a poor prognostic marker for OSCC patients.

Improved performance of collagen scaffolds crosslinked by Traut's reagent and Sulfo-SMCC.

Collagen scaffolds are frequently employed for applications in regenerative medicine. In previous studies, we affirmed that Traut's reagent (2-Iminothiolane hydrochloride) and Sulfo-SMCC (4-(N-Maleimidomethyl) cyclohexane-1-carboxylic acid 3-sulpho-N-hydroxysuccinimide ester sodium salt) could covalently bind growth factors on collagen scaffolds. We also observed that crosslinking formed within the collagen scaffolds with excess dosage of Sulfo-SMCC, which improved the biological performance of collagen scaffolds together with growth factors. In order to evaluate changes in capacity caused by crosslinking, Traut's reagent and adjusted different concentrations of Sulfo-SMCC (0.263, 1.315, 2.63 and 5.26 mM) were used to construct collagen scaffolds with differing extents of crosslinking in this study. The results demonstrated that resistance of collagen scaffolds to enzymatic digestion, cellularization and vascularization in vivo were enhanced by the crosslinking procedure. The cell culture studies indicated that the crosslinking procedure did not influence biocompatibility. Moreover, there were no statistical differences in the degradation rate, cellularization or vascularization among 1.315, 2.63 and 5.26 mM crosslinked groups. These results demonstrated that crosslinking collagen scaffolds with an appropriate amount of Traut's reagent and Sulfo-SMCC was an effective and safe method to modify naturally derived collagen scaffolds with notable potential uses in tissue regeneration.

Critical contribution of adenosine A2A receptors in bone marrow-derived cells to white matter lesions induced by chronic cerebral hypoperfusion.

Adenosine A2A receptors (A2ARs) in distinct cellular types may exert different and even opposite effects on many neurologic disorders; A2ARs in bone marrow-derived cells (BMDCs) have been shown to play important roles in various brain injuries. We previously showed that global A2AR inactivation aggravates chronic cerebral hypoperfusion-induced white matter lesions (WMLs); however, the specific cell populations responsible for A2AR-mediated signaling remain unknown. In the present study, we developed chimeric mice in which A2ARs were either selectively inactivated or reconstituted in BMDCs by transplanting bone marrow from global A2AR gene knockout or wild-type mice into wild-type or gene knockout mice, respectively. Chimeric mice were subsequently subjected to chronic cerebral hypoperfusion by bilateral common carotid artery stenosis, and the effects of BMDC A2ARs on WMLs were evaluated. The selective inactivation of A2AR in BMDCs aggravated chronic cerebral hypoperfusion-induced WMLs, promoted microglial activation, and increased proinflammatory cytokine expression, whereas the selective reconstitution or activation of A2AR in BMDCs using the agonist CGS21680 produced the opposite effects. These results demonstrate that A2ARs in BMDCs are important modulators of WMLs induced by chronic cerebral hypoperfusion; this modulation might be associated with the regulation of inflammatory cytokine production.

Inactivation of Vhl in osteochondral progenitor cells causes high bone mass phenotype and protects against age-related bone loss in adult mice.

Previous studies have shown that disruption of von Hippel-Lindau gene (Vhl) coincides with activation of hypoxia-inducible factor α (HIFα) signaling in bone cells and plays an important role in bone development, homeostasis, and regeneration. It is known that activation of HIF1α signaling in mature osteoblasts is central to the coupling between angiogenesis and bone formation. However, the precise mechanisms responsible for the coupling between skeletal angiogenesis and osteogenesis during bone remodeling are only partially elucidated. To evaluate the role of Vhl in bone homeostasis and the coupling between vascular physiology and bone, we generated mice lacking Vhl in osteochondral progenitor cells (referred to as Vhl cKO mice) at postnatal and adult stages in a tamoxifen-inducible manner and changes in skeletal morphology were assessed by micro-computed tomography (µCT), histology, and bone histomorphometry. We found that mice with inactivation of Vhl in osteochondral progenitor cells at the postnatal stage largely phenocopied that of mice lacking Vhl in mature osteoblasts, developing striking and progressive accumulation of cancellous bone with increased microvascular density and bone formation. These were accompanied with a significant increase in osteoblast proliferation, upregulation of differentiation marker Runx2 and osteocalcin, and elevated expression of vascular endothelial growth factor (VEGF) and phosphorylation of Smad1/5/8. In addition, we found that Vhl deletion in osteochondral progenitor cells in adult bone protects mice from aging-induced bone loss. Our data suggest that the VHL-mediated signaling in osteochondral progenitor cells plays a critical role in bone remodeling at postnatal/adult stages through coupling osteogenesis and angiogenesis. © 2014 American Society for Bone and Mineral Research.

Overexpression of H1 calponin in osteoblast lineage cells leads to a decrease in bone mass by disrupting osteoblast function and promoting osteoclast formation.

H1 calponin (CNN1) is known as a smooth muscle-specific, actin-binding protein which regulates smooth muscle contractive activity. Although previous studies have shown that CNN1 has effect on bone, the mechanism is not well defined. To investigate the role of CNN1 in maintaining bone homeostasis, we generated transgenic mice overexpressing Cnn1 under the control of the osteoblast-specific 3.6-kb Col1a1 promoter. Col1a1-Cnn1 transgenic mice showed delayed bone formation at embryonic stage and decreased bone mass at adult stage. Morphology analyses showed reduced trabecular number, thickness and defects in bone formation. The proliferation and migration of osteoblasts were decreased in Col1a1-Cnn1 mice due to alterations in cytoskeleton. The early osteoblast differentiation of Col1a1-Cnn1 mice was increased, but the late stage differentiation and mineralization of osteoblasts derived from Col1a1-Cnn1 mice were significantly decreased. In addition to impaired bone formation, the decreased bone mass was also associated with enhanced osteoclastogenesis. Tartrate-resistant acid phosphatase (TRAP) staining revealed increased osteoclast numbers in tibias of 2-month-old Col1a1-Cnn1 mice, and increased numbers of osteoclasts co-cultured with Col1a1-Cnn1 osteoblasts. The ratio of RANKL to OPG was significantly increased in Col1a1-Cnn1 osteoblasts. These findings reveal a novel function of CNN1 in maintaining bone homeostasis by coupling bone formation to bone resorption.

Improvement in the delivery system of bone morphogenetic protein-2: a new approach to promote bone formation.

Much research has been focused on developing bone morphogenetic protein-2(BMP-2) delivery systems to enhance bone formation in bone defect repair and bone tissue engineering. However, many of these current systems have several drawbacks associated with low loading efficiencies and reduced biological activities after release. Collagen scaffolds can be used as in delivery systems because of their biocompatibility. However, growth factors have naturally low affinity to collagen, which is disadvantageous for maintaining a sufficient growth factor concentration at the delivery sites. To enhance BMP-2 binding to collagen scaffolds, we chose a porous collagen scaffold that was chemically modified using Traut's reagent. The modified collagen scaffold allows cross-linking of the collagen fibers and is able to immobilize more BMP-2 after treatment with Sulfo-SMCC. We demonstrated that cross-linking led to a slower release rate of BMP-2, but did not reduce its biological activity. Moreover, more ectopic bone formation was induced by subcutaneous implants of cross-linked collagen treated with BMP-2. We concluded that collagen scaffolds chemically conjugated with BMP-2 using Traut's reagent and Sulfo-SMCC was an effective delivery system for use in bone defect repair and in bone tissue engineering.

Adenosine A2A receptor deficiency up-regulates cystatin F expression in white matter lesions induced by chronic cerebral hypoperfusion.

In previous studies, we have shown that the inactivation of the adenosine A2A receptor exacerbates chronic cerebral hypoperfusion-induced white matter lesions (WMLs) by enhancing neuroinflammatory responses. However, the molecular mechanism underlying the effect of the adenosine A2A receptor remains unknown. Recent studies have demonstrated that cystatin F, a potent endogenous cysteine protease inhibitor, is selectively expressed in immune cells in association with inflammatory demyelination in central nervous system diseases. To understand the expression of cystatin F and its potential role in the effect of A2A receptor on WMLs induced through chronic cerebral hypoperfusion, we investigated cystatin F expression in the WMLs of A2A receptor gene knockout mice, the littermate wild-type mice and wild-type mice treated daily with the A2A receptor agonist CGS21680 or both CGS21680 and A2A receptor antagonist SCH58261 after chronic cerebral hypoperfusion. The results of quantitative-PCR and western blot analysis revealed that cystatin F mRNA and protein expression were significantly up-regulated in the WMLs after chronic cerebral hypoperfusion. In addition, cystatin F expression in the corpus callosum was significantly increased in A2A receptor gene knockout mice and markedly decreased in mice treated with CGS21680 on both the mRNA and protein levels. Additionally, SCH58261 counteracted the attenuation of cystatin F expression produced by CGS21680 after chronic cerebral hypoperfusion. Moreover, double immunofluorescence staining revealed that cystatin F was co-localized with the activated microglia marker CD11b. In conclusion, the cystatin F expression in the activated microglia is closely associated with the effect of the A2A receptors, which may be related to the neuroinflammatory responses occurring during the pathological process.

Improved cellularization and angiogenesis using collagen scaffolds chemically conjugated with vascular endothelial growth factor.

Much research has focused on developing vascular endothelial growth factor (VEGF) delivery systems to enhance angiogenesis in wound repair and in tissue engineering. Collagen can be used as a delivery system because of its biocompatibility, but its fast degradation rate and limited affinity with growth factors are disadvantageous for maintaining a sufficient growth factor concentration at injury sites. To enhance VEGF binding to collagen scaffolds and reduce the collagen degradation rate we found a simple way to modify porous collagen scaffolds by chemical addition of sulfhydryl groups, which then allow both cross-linking of the collagen fibers with each other and the immobilization of more VEGF in the scaffold after treatment with sulfo-SMCC. We demonstrated that cross-linking led to a slower degradation rate of the collagen scaffolds, while cellularization was improved by both cross-linking and the presence of VEGF. On the other hand, angiogenesis was increased only moderately by cross-linking, but significantly more by the presence of immobilized VEGF. We conclude that collagen scaffolds chemically conjugated to VEGF by Traut's reagent and sulfo-SMCC is an effective delivery system in wound repair and tissue engineering.

A comparison of clinical outcomes for implants placed in fresh extraction sockets versus healed sites in periodontally compromised patients: a 1-year follow-up report.

PURPOSE: The aim of this 1-year prospective study was to evaluate, in patients with severe periodontitis, the clinical performance of implants placed immediately after extraction of remaining teeth or in healed sockets and immediately loaded for prosthetic oral rehabilitation, and to compare the clinical outcomes for implants placed in fresh extraction sockets versus healed sites. MATERIALS AND METHODS: All patients in this study had received periodontal treatment; however, the teeth were eventually deemed hopeless. The remaining teeth were extracted, the periodontally compromised sites were debrided, implants were inserted guided by a surgical template, and a provisional restoration was delivered immediately. The opposing arch was restored with a complete denture. Definitive prostheses were inserted after 6 months. Initial implant and prosthesis stability and the inflammatory response were evaluated. Clinical and radiographic analyses were performed. RESULTS: A total of 84 rough-surfaced implants were placed (50 in the maxilla and 34 in the mandible). Of these, 32 were placed in fresh extraction sockets. Four implants in three patients failed within the first 6 months, resulting in an implant survival rate of 95.2%. All of the failed implants had been placed in fresh maxillary extraction sockets. The survival rates were 92% (46/50) in the maxilla and 100% (34/34) in the mandible. Prosthetic success was 100%. The mean bone level change (± SE) between baseline and 12 months was -1.12 ± 0.18 mm. There were no statistically significant differences in insertion torque and alveolar bone loss between postextraction sites and healed sites. CONCLUSIONS: The implant failure rate was higher in maxillary postextraction sites. This indicates a heightened risk of failure for immediate implants placed in periodontally compromised maxillae. Nevertheless, a satisfactory prosthetic success was achieved after 1 year.

Generation of Fgfr3 conditional knockout mice.

Fibroblast growth factor receptor 3 (FGFR3), highly conserved in both humans and murine, is one of key tyrosine kinase receptors for FGF. FGFR3 is expressed in different tissues, including cartilage, brain, kidney, and intestine at different development stages. Conventional knockout of Fgfr3 alleles leads to short life span, and overgrowth of bone. In clinic, human FGFR3 mutations are responsible for three different types of chondrodysplasia syndromes including achondroplasia (ACH), hypochondroplasia (HCH) and thanatophoric dysplasia (TD). For better understanding of the roles of FGFR3 in different tissues at different stages of development and in pathological conditions, we generated Fgfr3 conditional knockout mice in which loxp sites flank exons 9-10 in the Fgfr3 allele. We also demonstrated that Cre-mediated recombination using Col2a1-Cre, a Cre line expressed in chondrocyte during bone development, results in specific deletion of the gene in tissues containing cartilage. This animal model will be useful to study distinct roles of FGFR3 in different tissues at different ages.

Dynamic morphological changes in the skulls of mice mimicking human Apert syndrome resulting from gain-of-function mutation of FGFR2 (P253R).

Apert syndrome is caused mainly by gain-of-function mutations of fibroblast growth factor receptor 2. We have generated a mouse model (Fgfr2(+/P253R)) mimicking human Apert syndrome resulting from fibroblast growth factor receptor 2 Pro253Arg mutation using the knock-in approach. This mouse model in general has the characteristic skull morphology similar to that in humans with Apert syndrome. To characterize the detailed changes of form in the overall skull and its major anatomic structures, euclidean distance matrix analysis was used to quantitatively compare the form and growth difference between the skulls of mutants and their wild-type controls. There were substantial morphological differences between the skulls of mutants and their controls at 4 and 8 weeks of age (P < 0.01). The mutants showed shortened skull dimensions along the rostrocaudal axis, especially in their face. The width of the frontal bone and the distance between the two orbits were broadened mediolaterally. The neurocrania were significantly increased along the dorsoventral axis and slightly increased along the mediolateral axis, and also had anteriorly displayed opisthion along the rostrocaudal axis. Compared with wild-type, the mutant mandible had an anteriorly displaced coronoid process and mandibular condyle along the rostrocaudal axis. We further found that there was catch-up growth in the nasal bone, maxilla, zygomatic bone and some regions of the mandible of the mutant skulls during the 4-8-week interval. The above-mentioned findings further validate the Fgfr2(+/P253R) mouse strain as a good model for human Apert syndrome. The changes in form characterized in this study will help to elucidate the mechanisms through which the Pro253Arg mutation in fibroblast growth factor receptor 2 affects craniofacial development and causes Apert syndrome.

Gain-of-function mutation in FGFR3 in mice leads to decreased bone mass by affecting both osteoblastogenesis and osteoclastogenesis.

Achondroplasia (ACH) is a short-limbed dwarfism resulting from gain-of-function mutations in fibroblast growth factor receptor 3 (FGFR3). Previous studies have shown that ACH patients have impaired chondrogenesis, but the effects of FGFR3 on bone formation and bone remodeling at adult stages of ACH have not been fully investigated. Using micro-computed tomography and histomorphometric analyses, we found that 2-month-old Fgfr3(G369C/+) mice (mouse model mimicking human ACH) showed decreased bone mass due to reduced trabecular bone volume and bone mineral density, defect in bone mineralization and increased osteoclast numbers and activity. Compared with primary cultures of bone marrow stromal cells (BMSCs) from wild-type mice, Fgfr3(G369C/+) cultures showed decreased cell proliferation, increased osteogenic differentiation including up-regulation of alkaline phosphatase activity and expressions of osteoblast marker genes, and reduced bone matrix mineralization. Furthermore, our studies also suggest that decreased cell proliferation and enhanced osteogenic differentiation observed in Fgfr3(G369C/+) BMSCs are caused by up-regulation of p38 phosphorylation and that enhanced Erk1/2 activity is responsible for the impaired bone matrix mineralization. In addition, in vitro osteoclast formation and bone resorption assays demonstrated that osteoclast numbers and bone resorption area were increased in cultured bone marrow cells derived from Fgfr3(G369C/+) mice. These findings demonstrate that gain-of-function mutation in FGFR3 leads to decreased bone mass by regulating both osteoblast and osteoclast activities. Our studies provide new insight into the mechanism underlying the development of ACH.

Fibroblast growth factor receptor 1 regulates the differentiation and activation of osteoclasts through Erk1/2 pathway.

To elucidate the direct role and mechanism of FGFR1 signaling in the differentiation and activation of osteoclasts, we conditionally inactivated FGFR1 in bone marrow monocytes and mature osteoclasts of mice. Mice deficient in FGFR1 (Fgfr1(-/-)) exhibited misregulated bone remodeling with reduced osteoclast number and impaired osteoclast function. In vitro assay demonstrated that the number of tartrate-resistant acid phosphatase (TRAP) positive osteoclasts derived from bone marrow monocytes of Fgfr1(-/-) mice was significantly diminished. The bone resorption activity of mature osteoclasts derived from Fgfr1(-/-) mice was also suppressed. Further analysis showed that the osteoclasts with FGFR1 deficiency exhibited downregulated expression of genes related to osteoclastic activity including TRAP and MMP-9. The phosphorylation of Erk1/2 mitogen-activated protein (MAP) kinase was also decreased. Our results suggest that FGFR1 is indispensable for complete differentiation and activation of osteoclasts in mice.

[Generation of bone morphogenetic protein 4 conditional RNA interference mice].

To explore bone morphogenetic protein 4 (BMP4) function in the developing bone, a BMP4 conditional RNA interference (CRNAi) vector was constructed based on the pBSK/U6 vector with a LoxPneo cassette. The transgene fragment targeting bmp4 was obtained by Kpn and Afl double digestion and was purified before being microinjected into fertilized eggs from FVB/NJ mice. BMP4CRNAi transgenic mice were genotyped by PCR. And the PCR positive mice were crossed with Col2a1-Cre transgenic mice, whose Cre recombinase was specifically expressed in osteo-chondro-progenitor cells. Bmp4 mRNA expression in primary chondrocytes were examined by semi-quantitive RT-PCR to determine RNA interference efficiency. Results showed that BMP4(CRNAi) mice and BMP4 (Col2a1-CRNAi) mice were produced successfully, and bmp4 knockdown efficiency in primary chondrocytes of BMP4 Col2a1-CRNAi mice was 81%. This transgenic mouse line provides excellent model for studying the role of BMP4 in chondrocyte development, and BMP4CRNAi mouse may be a good model for studying the role of BMP4 in different cells, tissues and organs through crossing with different Cre transgenic mice.