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In our previous study, serum soluble T-cell immunoglobulin and mucin structure-3 (sTim-3) and galactosin-9 (sGal-9) were found to be associated with renal function after kidney transplantation. However, it is unclear whether these two indicators can predict adverse outcomes after transplantation.Ninety-one recipients of kidney transplantation were enrolled and divided into a stable group and an adverse outcome group (consisting of biopsy-proven rejection, graft loss, death and clinically diagnosed rejection). The expression levels of sTim-3 and sGal-9 before (pre-Tim-3 and pre-Gal-9) and one month after transplantation (post-Tim-3 and post-Gal-9) were measured by ELISA.The level of pre-Tim-3 was significantly higher in the stable group than in the adverse outcome group [median (range), 2275 (840-4236) pg/mL vs. 1589 (353-3094) pg/mL, P = 0.002]. The level of post-Gal-9 was significantly lower in the stable group than in the adverse outcome group [median (range), 4869 (1418-13080) pg/mL vs. 6852: (4128-10760) pg/mL, P = 0.003]. The areas under the curve (AUCs) for pre-Tim-3 and post-Gal-9 were 0.737 (P = 0.002) and 0.751 (P = 0.003), respectively, better than AUC of post-eGFR (0.633) (P = 0.071), according to the receiver operating characteristic (ROC) curve. Through Cox regression analysis, including pre-Tim-3, post-Gal-9, post-eGFR, sex, age, BMI of recipients and donors, pre-Tim-3 and post-Gal-9 were independent risk factors for adverse outcomes after kidney transplantation (P = 0.016, P = 0.033, respectively).Serum sTim-3 and sGal-9 can predict adverse outcomes within two years after kidney transplantation.

Di-Fei Duan,1,2,&ast; Min Liu,1,2,&ast; Yi Chen,1,2 Yue-Yang Huang,1,2 Yun-Ying Shi1 1Department of Nephrology, West China Hospital of Sichuan University, Chengdu, People’s Republic of China; 2West China School of Nursing, Sichuan University, Chengdu, People’s Republic of China&ast;These authors contributed equally to this workCorrespondence: Yun-Ying Shi, Department of Nephrology, West China Hospital of Sichuan University, No. 37 Guoxuexiang Wuhou District, Chengdu, Sichuan, People’s Republic of China, Email yyshi0130@163.comPurpose: Nephrology professionals’ understanding of food literacy (FL) and influencing factors is significant for nutrition management, which is key to controlling disease progress among non-dialysis patients with chronic kidney disease (CKD). However, few studies have explored FL in CKD patients. Hence, this study aimed to investigate the level of FL of non-dialysis patients with CKD and to analyze influencing factors in China.Patients and Methods: A total of 203 CKD patients without dialysis were recruited from August to December 2020 to participate in a cross-sectional study. Food literacy was assessed by a modified short food literacy questionnaire for adults. Related influencing factors were measured by the motivation for dietary self-control scale and the satisfaction with dietary behavior scale. Data were also collected regarding patients’ health information-seeking behavior (four items), satisfaction and compliance with dietary advice from healthcare professionals (two items), and demographics and clinical diagnoses.Results: The mean age of patients was 44.5 years (range 18 to 75), and 42.5% were male. Most (50.2%) were in stages 1– 2 of CKD. The mean FL score of these patients was 38.75± 0.38. The multivariable linear regression analysis shows that secondary educational level (β=0.221, p=0.004), motivation for controlling diet (β=0.198, p=0.003), satisfaction with dietary behaviors (β=0.319, p< 0.001), and health information-seeking behavior (β=0.146, p=0.019) were significant influencing factors.Conclusion: Food literacy of Chinese CKD patients without dialysis should be improved. Patients with higher education levels exhibit more active information-seeking behaviors, have greater satisfaction with dietary behaviors, and their motivation for dietary self-control is more likely to be associated with better FL. Healthcare workers should be aware of factors influencing FL and attempt to integrate assessment of FL into routine food-related education for CKD patients.Keywords: motivation, satisfaction, information-seeking behavior, health literacy, nutrition

The combination of multivariate calibration and spectroscopic analysis technology can achieve the rapid analysis of substance. However, when the external environmental conditions such as instruments or accessories are replaced or temperature changed, the current master calibration may be “invalid”. Recalibration usually takes a lot of time and financial resources. The development of calibration transfer techniques can solve this problem well, and the adaptability and robustness of the calibration are enhanced by correcting the spectra or selecting variables, therefore it is very important for popularizing the use of calibrations. This review describes a number of new calibration transfer methods and their applications in recent years, and introduces them according to traditional transfer methods, new methods and new strategies. The applications of each method as well as both its advantages and disadvantages are evaluated.

Background T cell immunoglobulin mucin-3 (Tim-3) has been reported to participate in the regulation of immune response and the induction of allograft tolerance. However, the association between Tim-3 and renal allograft dysfunction is unclear. We studied the expression of cellular and soluble Tim-3 (sTim-3), soluble galectin-9 (sGal-9) and carcinoembryonic antigen-related cell adhesion molecule-1 (sCEACAM-1) in kidney transplantation recipients (KTRs) to explore their roles in allograft dysfunction. Methods 96 KTRs (53 with stable graft and 43 with graft dysfunction) and 30 healthy controls (HC) were enrolled. Among the KTRs, 55 used Tacrolimus (TAC) and 41 used Sirolimus (SRL). In the dysfunction group, 29 recipients have undergone graft biopsy and 14 were classified as biopsy-proven rejection (BPR). Cellular Tim-3 was determined by flow cytometry. sTim-3 was determined by ELISA. sGal-9 and sCEACAM-1 were determined by Bio-Plex® suspension array system. Results KTRs with renal dysfunction showed significantly higher levels of sTim-3 and sGal-9 but similar levels of cellular Tim-3 and sCEACAM-1 compared with stable recipients. Correlation analysis revealed that estimated glomerular filtration rate (eGFR) was negatively associated with sTim-3 and sGal-9. Both BPR and non-BPR groups showed comparable levels of Tim-3, Gal-9 and CEACAM-1. Moreover, SRL group showed significantly higher levels of sCEACAM-1 than TAC and HC groups. Conclusions sTim-3 and sGal-9 were promising biomarkers for allograft dysfunction, but unable to differentiate allograft rejection from other causes of renal dysfunction in KTRs. Moreover, long-term administration of sirolimus would up-regulate sCEACAM-1 level, while exert similar regulatory effects on Tim-3 and Gal-9 compared to tacrolimus.

Background Neutrophil gelatinase-assoicated lipocalin (NGAL) appears to be a promising proximal tubular injury biomarker for early prediction of delayed graft function (DGF) in kidney transplant recipients. However, its predictive values in urine and blood were varied among different studies. Here, we performed the meta-analysis to compare the predictive values of urine NGAL (uNGAL) and blood NGAL (bNGAL) for DGF in adult kidney transplant recipients. Methods We systematically searched Medline, Cochrane library and Embase for relevant studies from inception to May 2018. The summary receiver operating characteristic (SROC) curves, the pooled sensitivity, specificity and diagnostic odds ratio (DOR) were used to evaluate the prognostic performance of uNGAL and bNGAL for the identification of DGF. Results A total of 1036 patients from 14 eligible studies were included in the analysis. 8 studies focused on NGAL in urine and 6 reported NGAL in serum or plasma. The composite area under the ROC (AUC) for 24 h uNGAL was 0.91 (95% CI, 0.89–0.94) and the overall DOR for 24 h uNGAL was 24.17(95% CI, 9.94–58.75) with a sensitivity of 0.88 (95% CI, 0.75–0.94) and a specificity of 0.81 (95% CI, 0.68–0.89). The composite AUC for 24 h bNGAL was 0.95 (95% CI, 0.93–0.97) and the overall DOR for 24 h bNGAL was 43.11 (95% CI, 16.43–113.12) with a sensitivity of 0.91 (95% CI, 0.81–0.96) and a specificity of 0.86 (95% CI, 0.78–0.92). Conclusions Urine and serum/plasma NGAL were valuable biomarkers for early identification of DGF in kidney transplantation. In addition, the bNGAL was superior to uNGAL in early prediction of DGF. Electronic supplementary material The online version of this article (10.1186/s12882-019-1491-y) contains supplementary material, which is available to authorized users.

Infection remains a major cause of morbidity and mortality after kidney transplantation (KT). Reliable biomarkers to predict post-transplant infection are lacking. We investigated the predictive performance of pre- and post-transplant levels of T-cell immunoglobulin and mucin domain-3 (Tim-3) and Galectin-9 (Gal-9), two pleiotropic immunomodulatory molecules, in early identification of infection. Serum Tim-3 and Gal-9 were paired measured before and 30 days after transplantation (PTD 30) in 95 KT recipients (KTRs). The decline rates of Tim-3 and Gal-9 were calculated relative to pre-transplant levels. KTRs with infection history had significantly higher levels of PTD 30 Tim-3 and Gal-9, and slower decrease rates of Gal-9 compared to non-infected recipients, while no difference was observed between two groups regarding pre-transplant levels. The AUCs for predicting 1-year post-transplant infection were 0.653 and 0.711 for post-transplant Tim-3 and Gal-9, 0.664 and 0.670 for relative Tim-3 and Gal-9, respectively. After adjusting for potential confounders, PTD 30 Tim-3, Gal-9 and relative Gal-9 remained as independent risk factors for post-transplant infection. Our results suggested that PTD 30 Tim-3 and Gal-9 and relative decrease of Gal-9 were promising predictors for identifying KTRs with high risk of infection, while pre-transplant Tim-3 and Gal-9 showed no predictive power to infection.

OBJECTIVE A genome-wide association study has identified several gene polymorphisms associated with loss of renal function. The effect of these variants on renal function in kidney transplant recipients receiving immunosuppressive treatment is unknown. MATERIALS AND METHODS A cohort of 189 kidney transplant recipients and their living donors were recruited from West China Hospital of Sichuan University, on whom we assessed the association of five single nucleotide polymorphisms with renal function after kidney transplantation. RESULTS Glomerular filtration rate estimated by serum creatinine was significantly higher in recipients carrying allograft with the A allele at rs17319721 in SHROOM3 (shroom family member 3) than those in the group with the GG genotype from month 1 to month 6 after transplantation (P=0.020). Covariate adjustment analysis showed that the variant at rs17319721 in SHROOM3 was an independent risk factor for renal dysfunction after the first month after transplantation (P=0.022). The estimated glomerular filtration rate was the lowest in recipients with allograft carrying both the A allele at rs17319721 in SHROOM3 and the CC genotype at rs1045642 in ABCB1 (P

Background About one in four patients suffers from postoperative nausea and vomiting. Fortunately, risk scores have been developed to better manage this outcome in hospitalized patients, but there is currently no risk score for postdischarge nausea and vomiting (PDNV) in ambulatory surgical patients. Methods We conducted a prospective multicenter study of 2,170 adults undergoing general anesthesia at ambulatory surgery centers in the United States from 2007 to 2008. PDNV was assessed from discharge until the end of the second postoperative day. Logistic regression analysis was applied to a development dataset and the area under the receiver operating characteristic curve was calculated in a validation dataset. Results The overall incidence of PDNV was 37%. Logistic regression analysis of the development dataset (n=1,913) identified five independent predictors (odds ratio; 95% CI): female gender (1.54; 1.22 to 1.94), age less than 50 yr (2.17; 1.75 to 2.69), history of nausea and/or vomiting after previous anesthesia (1.50; 1.19 to 1.88), opioid administration in the postanesthesia care unit (1.93; 1.53 to 2.43), and nausea in the postanesthesia care unit (3.14; 2.44-4.04). In the validation dataset (n=257), zero, one, two, three, four, and five of these factors were associated with a PDNV incidence of 7%, 20%, 28%, 53%, 60%, and 89%, respectively, and an area under the receiver operating characteristic curve of 0.72 (0.69 to 0.73). Conclusions PDNV affects a substantial number of patients after ambulatory surgery. We developed and validated a simplified risk score to identify patients who would benefit from long-acting prophylactic antiemetics at discharge from the ambulatory care center.

BACKGROUND: Guided tissue regeneration is a technique that uses barrier materials to enhance tissue regeneration. Although previously demonstrated to be an effective way of enhancing craniofacial osteogenesis in several animal models, the ability of guided tissue regeneration to augment bone formation in the context of distraction osteogenesis is unknown. In the current study, the authors applied the principle of guided tissue regeneration to their rat mandibular distraction osteogenesis model in an attempt to enhance bone regeneration. METHODS: Twelve (n = 6 per group) adult Sprague-Dawley rats underwent routine gradual distraction (5 days' latency, 4-mm distraction over 8 days, 4 to 6 weeks of consolidation) and acute distraction (immediate lengthening to 4 mm, 6 to 8 weeks of consolidation). An additional 10 animals underwent acute distraction followed by application of bioabsorbable Gore Resolut XT membranes (acute distraction plus guided tissue regeneration). Membranes were completely wrapped around the distraction gap. Animals were killed 6 and 8 weeks postoperatively and mandibles analyzed radiographically and histologically. RESULTS: Quantitative histomorphometric analyses were performed to compare relative bone formation between all three groups. Gradual distraction mandibles achieved bony union by 6 weeks with 86 percent bone formation, which increased to 98 percent by 8 weeks. Acute distraction mandibles healed with a fibrous nonunion and only 37 percent bone formation by 8 weeks. In contrast, acute distraction plus guided tissue regeneration-treated mandibles formed significantly more bone than acute distraction mandibles by 6 weeks (57 percent) and achieved bony bridging by 8 weeks, with 88 percent new bone formation. CONCLUSION: The authors' data demonstrate that guided tissue regeneration can significantly enhance bone formation in a fibrous nonunion model of mandibular distraction osteogenesis.

Previous studies have documented the differences in expression of various genes associated with the process of osteogenesis in fusing and nonfusing cranial sutures, including growth factors, growth factor receptors, and extracellular matrix molecules. Most of these studies were performed in rats, and although the biology regulating cranial suture fusion in mice and rats is presumed to be similar, studies are needed to verify these expression patterns as mice become increasingly utilized for scientific inquiry into the molecular biology of suture fusion and patency. The purpose of this study was to determine the differences in expression of several genes known to be critical to osteoblast biology. Posterior frontal and sagittal suture complexes (including the associated dura mater, suture mesenchyme, and osteogenic fronts) were isolated from 5-, 15-, 25-, 35-, and 45-day-old male CD-1 mice (n = 8 per age; n = 40 total). Total cellular RNA was extracted and converted to cDNA. Quantitative real-time reverse transcriptase polymerase chain reaction was then performed for the following genes: transforming growth factor beta1 and beta3, fibroblast growth factor receptor 1, Runx2,Osteopontin, and Osteocalcin. Expression of all genes examined was increased significantly in the posterior frontal suture as compared with the sagittal suture. Peak expression for all genes was observed on day 25. These data demonstrate that the expression of osteogenic growth factors, growth factor receptors, transcription factors, and extracellular matrix molecules is increased in the fusing posterior frontal suture in mice.

Young children are capable of healing large calvarial defects, whereas adults lack this endogenous osseous tissue-engineering capacity. Despite the important clinical implications, little is known about the molecular and cell biology underlying this differential ability. Traditionally, guinea pig, rabbit, and rat models have been used to study the orchestration of calvarial healing. To harness the research potential of knockout and transgenic mice, the authors developed a mouse model for calvarial healing. Nonsuture-associated parietal defects 3, 4, and 5 mm in diameter were made in both juvenile (6-day-old, n = 15) and adult (60-day-old, n = 15) mice. Calvariae were harvested after 8 weeks and analyzed radiographically and histologically. Percentage of healing was quantified using Scion Image software analysis of calvarial radiographs. A significant difference in the ability to heal calvarial defects was seen between 6-day-old and 60-day-old mice when 3-, 4-, or 5-mm defects were created. The authors' analysis revealed that juvenile mice healed a significantly greater percentage of their calvarial defects than adult mice (juvenile mean percentage of healing: 3-mm defects, 59 percent; 4-mm defects, 65 percent; 5-mm defects, 44 percent; adult mean percentage of healing

In adults and children over two years of age, large cranial defects do not reossify successfully, posing a substantial biomedical burden. The osteogenic potential of bone marrow stromal (BMS) cells has been documented. This study investigates the in vivo osteogenic capability of adipose-derived adult stromal (ADAS) cells, BMS cells, calvarial-derived osteoblasts and dura mater cells to heal critical-size mouse calvarial defects. Implanted, apatite-coated, PLGA scaffolds seeded with ADAS or BMS cells produced significant intramembranous bone formation by 2 weeks and areas of complete bony bridging by 12 weeks as shown by X-ray analysis, histology and live micromolecular imaging. The contribution of implanted cells to new bone formation was 84-99% by chromosomal detection. These data show that ADAS cells heal critical-size skeletal defects without genetic manipulation or the addition of exogenous growth factors.

Retinoic acid has been shown to adversely affect craniofacial development. Cleft palate and craniosynostosis are two examples of craniofacial defects associated with prenatal exposure to this agent. Although the effects of retinoic acid on cephalic neural crest-derived tissues have previously been studied, the specific effects of retinoic acid on the cellular biology of osteoblasts remain unclear. The purpose of this study was to analyze in detail the effects of pharmacologic doses of retinoic acid on the differentiation and proliferation of osteoblasts derived from an intramembranous source. Primary rat calvarial osteoblasts were established in culture and treated with 1 or 10 μM all-trans-retinoic acid. Retinoic acid treatment markedly increased expression of osteopontin up to 48 h after stimulation. Consistent with this early stage of differentiation, both mRNA and protein analysis of FGF receptor isoforms demonstrated a switch in predominance from fibroblast growth factor receptor 2 (fgfr2) to fgfr1. Analysis of PCNA protein confirmed inhibition of proliferation by retinoic acid. To determine whether these alterations in osteoblast biology would lead to increased differentiation, we examined short term [alkaline phosphatase (AP) activity] and long term (von Kossa staining) surrogates of bone formation in vitro. These assays confirmed that retinoic acid increased osteogenesis, with a 4-fold increase in bone nodule formation in cells treated with 10 μM retinoic acid after 28 days. Overall, our results demonstrated that pharmacologic doses of all-trans-retinoic acid decreased osteoblast proliferation and increased differentiation, suggesting that retinoic acid may effect craniofacial development by pathologically enhancing osteogenesis. © 2005 Wiley-Liss, Inc.

Radical resection (wedge resection of the gallbladder bed and dissection of the hepatoduodenal ligament, portal, and celiac lymph nodes) has been reported to improve survival from pathologic T2 gallbladder carcinoma (pT2 GBCa; invasion through the muscularis without perforation of the serosa). We report our experience and the outcome of patients with pT2 GBCa. Between 1989 and 2000 at Vanderbilt University Medical Center ten patients were found to have pT2 disease after cholecystectomy. The patients had an average age of 64 ± 13 years and underwent either radical resection (n = 5) or no further surgical therapy (n = 5). Of the patients who underwent cholecystectomy only, one (20%) is still alive at 27 months and four (80%) died of recurrent GBCa between 6.5 and 21 months. For the patients who underwent radical resection all five are alive at 15 to 83 months with no recurrence. The proportion of patients surviving pT2 GBCa after radical resection was significantly greater than with cholecystectomy alone ( P < 0.05). The difference in length of survival between the two groups was also significant ( P < 0.05). Morbidity after radical resection was low (pancreatic leak in one patient), and there were no operative mortalities. Radical resection significantly improved survival over cholecystectomy alone for patients with pT2 GBCa. The procedure has low morbidity and mortality rates. Therefore a radical resection operation is indicated for patients with pT2 GBCa.

The aim of this study was to perform an association study between two single nucleotide polymorphisms (SNPs) rs2910164 G>C and rs3746444 T>C in pre-miRNA (hsa-mir-146a and hsa-mir-499) and rheumatoid arthritis (RA) in the Han Chinese population. 208 Han Chinese patients with RA and 240 healthy controls were recruited in this study. The SNPs was genotyped by polymerase chain reaction-restriction fragment length polymorphism. Anti-cyclic citrullinated peptide (anti-CCP) antibody was measured by enzyme linked immunosorbent assay and rheumatoid factor (RF) was measured by rate nephelometry. The genotype frequencies between cases and controls were compared by χ(2) analysis. No significant association between the SNPs (rs2910164 and rs3746444) and RA was observed (P = 0.631 and 0.775, respectively), and the SNPs did not show any association with the RF-positive (P = 0.631 and 0.775, respectively). However, there was a significant difference on the level of anti-CCP antibody between different genotypes in rs3746444 (P = 0.007). The heterozygote CT had significantly higher level of anti-CCP antibody compared with homozygote CC and TT (P = 0.054 and 0.003, respectively). We first investigated the association between the SNPs (rs2910164 G>C and rs3746444 T>C) in the pre-miRNA (hsa-mir-146a and hsa-mir-499) and RA in a Han Chinese population. We did not find a significant association between the SNPs and the susceptibility to RA, while the SNP rs3746444 may affect anti-CCP antibody production.

To investigate the proteomic characteristics of systemic lupus erythematosus (SLE) in a SLE family from Sichuan, China which consisting of 7 members with 3 SLE cases, and to find the proteins correlated with the heredity of SLE.A total of 153 serum samples were collected from 7 members including 3 SLE sisters in this SLE family, 63 individual SLE patients, as well as 83 healthy controls. The diagnosis of SLE is based on the American College of Rheumatology criteria (1997). All serum samples were analyzed using matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF-MS) combined with magnetic beads technology. Serum protein profiles were obtained by MALDI-TOF-MS combined with magnetic beads in order to identify predictive biomarkers of risk of suffering SLE. The resulting spectra were analyzed with Biomarker Wizard software 3.1.0.Four discriminative mass/charge (m/z) proteins serving as pathogenic biomarkers were identified on arrays for family SLE cases versus individual SLE and healthy controls. The protein level of peak intensities at m/z of 9342.23 was significantly greater in SLE family group compared with that in individual SLE patients and healthy controls (P0.05), those of individual SLE patients were significantly greater compared with healthy controls (P0.05); the proteins level of peak intensities at m/z of 4094.03, 5905.35 and 7973.53 in SLE family group were significantly lower compared with that in individual SLE patients and healthy controls (P0.05), those of individual SLE patients were significantly lower compared with healthy controls (P0.05).The proteins of m/z of 9342.23, 4094.03, 5905.35 and 7973.53 maybe play a great role in assemble pathogenesis of SLE and predict the risk of suffering SLE. The higher protein level of m/z of 9342.23 and the lower protein level of m/z of 4094.03, 5905.35 and 7973.53, the higher risk of sufferring with SLE.

To establish a diagnostic model for systemic lupus erythematosus (SLE) using proteiomic fingerprint techology.Blood samples were collected from 64 cases of SLE, 30 cases of rheumatoid arthritis (RA), 30 cases of Sjogren's syndrome (SS), 25 cases of systemic sclerosis (SSc), as well as 83 healthy controls. Proteomic spectra of these 232 serum samples were generated by proteomic fingerprint technology. Diagnostic model was established by a machine learning algorithm called decision boosting. The sensitivity and specificity of the diagnostic model was validated with a blinded testing set.Sixty differential protein peaks (P0.05) between SLE and control subjects were indicated, 28 of them were up regulated and 32 were down regulated in SLE patients. The algorithm identified a cluster pattern segregating SLE from non-SLE with sensitivity of 91% and specificity of 92%. The discriminatory diagnostic pattern correctly identified SLE. A sensitivity of 78% and specificity of 96% for the blinded test were obtained when comparing SLE vs non-SLE.This diagnostic model using proteiomic fingerprint techology appears to be a promising tools with high sensitivity and specificity in diagnosis of SLE.

Although the multilineage potential of human adipose-derived adult stromal cells (ADAS) has been well described, few published studies have investigated the biological and molecular mechanisms underlying osteogenic differentiation of mouse ADAS. We report here that significant osteogenesis, as determined by gene expression and histological analysis, is induced only when mouse ADAS are cultured in the presence of retinoic acid with or without recombinant human bone morphogenetic protein (BMP)-2 supplementation. Furthermore, a dynamic expression profile for the BMP receptor (BMPR) isoform IB was observed, with dramatic up-regulation during osteogenesis. Western blot analysis revealed that retinoic acid enhanced levels of BMPR-IB protein during the first 7 days of osteogenic differentiation and that RNAi-mediated suppression of BMPR-IB dramatically impaired the ability of ADAS to form bone in vitro . In contrast, absence of BMPR-IA did not significantly diminish ADAS osteogenesis. Our data therefore demonstrate that the osteogenic commitment of multipotent mouse ADAS requires retinoic acid, which enhances expression of the critical BMPR-IB isoform.

BACKGROUND: Adipose-derived mesenchymal cells are multipotent progenitor cells derived from the vascular-stromal compartment of adipose tissue. Although we have recently shown that these cells, from both juvenile and adult animals, are capable of forming bone in vivo, a detailed examination of the differences in the biology of these two populations (and in particular their ability to form bone) has not been performed. METHODS: Adipose-derived mesenchymal cells were harvested from juvenile (6-day-old) and adult (60-day-old) mice. Differences in cellular attachment, proliferation, and proliferating cell nuclear antigen production were assessed. The ability of cells to undergo adipogenic differentiation was determined by Oil Red O staining. Early osteogenic differentiation was determined with alkaline phosphatase staining, and terminal differentiation with von Kossa staining as well as determination of extracellular matrix calcium content. All experiments were performed in triplicate. RESULTS: Greater attachment, proliferation, and proliferating cell nuclear antigen production were seen in juvenile as compared with adult adipose-derived mesenchymal cells. The juvenile cells underwent significantly greater adipogenic differentiation than did adult cells (p < 0.001). Interestingly, the adult cells were capable of robust early and terminal osteogenic differentiation, with levels of all three osteo-genic assays being similar to those seen in juvenile cells. Differences were not statistically significant. CONCLUSIONS: Although biologic differences exist between adipose-derived mesenchymal cells from juveniles and adults, the osteogenic capacity of these cells appears to be minimally affected by donor age. This suggests that these cells may be a particularly useful cellular resource in the design of cell-based therapies for skeletal regeneration in an aging population.

Reconstruction of craniofacial defects presents a substantial biomedical burden, and requires complex surgery. Interestingly, children after age 2 years and adults are unable to heal large skull defects. This nonhealing paradigm provides an excellent model system for craniofacial skeletal tissueengineering strategies. Previous studies have documented the in vivo osteogenic potential of adipose-derived stromal (ADS) cells and bone marrow-derived stromal (BMS) cells. This study investigates the ability to accelerate in vivo osteogenesis on ex vivo recombinant human bone morphogenetic protein 2 (BMP-2) and retinoic acid stimulation. Mouse osteoblasts, ADS cells, and BMS cells were seeded onto apatite-coated PLGA scaffolds, stimulated with rhBMP-2 and retinoic acid ex vivo for 4 weeks, and subsequently implanted into critically sized (4 mm) calvarial defects. Samples were harvested after 2, 4, 8, and 12 weeks. Areas of complete bony bridging were noted as early as 2 weeks in vivo; however, osteoclasts were attracted to the scaffold as identified by calcitonin receptor staining and tartrate-resistant acid phosphatase activity staining. Although the optimal method of in vitro osteogenic priming for mesenchymal cells remains unknown, these results provide evidence that BMP-2 and retinoic acid stimulation of multipotent cells ex vivo can subsequently induce significant quantities of bone formation within a short time period in vivo.

Introduction: Similarities between skeletal formation and repair have led to the theory that adult fracture repair may parallel development. We hypothesize that since the mandible is derived from cranial neural crest (CNC) and forms via intramembranous ossification, non-stabilized mandibular fractures will heal in a similar fashion. Methods: Non-stabilized osteotomies were created in the right hemi-mandible of adult male mice. Mandibles were harvested at 7, 14, 21, and 28 days and processed for histology. Ossification was assessed and the presence of cartilage determined using histology and in situ hybridization (ISH). To determine the embryologic origins of the regenerate, transgenic mice were generated where CNC cells express GFP constitutively under the Wnt-1 promoter. Fractures were created as described above and processed for frozen section. CNC cells were localized using immunohistochemistry (IHC) for GFP. Closed, non-stabilized tibial fractures were also performed to determine the presence, if any, of CNC-derived cells in appendicular skeletal fractures. Results: In contrast to non-stabilized fractures of the appendicular skeleton, mandibular fractures healed predominantly via intramembranous ossification, with minimal and delayed cartilage production seen via histology and collagen2 ISH. IHC for GFP revealed that the mandibular fracture callous and regenerate was comprised primarily of GFP+, CNC-derived cells. In contrast, the tibial fracture callous was devoid of any CNC-derived cells. Conclusions: These data suggest that mandibular fractures heal by recapitulating their developmental program. These findings have important implications for designing appropriate cell-based approaches to craniofacial regeneration.

Introduction: Data indicates that TGF-betas may play an important role in craniosynostosis. In a murine organ culture model, we previously demonstrated that blocking TGF-beta signaling leads to pathologic suture patency. In this study, we investigated both the osteogenic properties of TGF-beta1 and determined whether adenoviral overexpression of TGF-beta1 in a normally patent sagittal (SAG) suture would lead to pathologic suture fusion. Methods: Primary calvarial rat osteoblasts were treated with TGF-beta1 adenovirus or vehicle and cultured for four weeks. Expression of TGF-beta1 was confirmed using quantitative real-time PCR (QRT-PCR) and bone nodule formation assessed with von Kossa staining. The dura mater of 22 day CD-1 mouse calvarial explants were then transfected with adenovirus overexpressing TGF-beta1 or lacZ. SAG sutures were harvested weekly for four weeks and analyzed with H&E as well as beta-galactosidase staining. Results: QRT-PCR data confirmed the marked up regulation of TGF-beta1 mRNA in osteoblasts transfected with the TGF-beta1 overexpression adenovirus. TGF-beta1 adenoviral overexpression led to a 5-fold increase in bone nodule formation compared to untreated osteoblasts. Interestingly TGF-beta1 adenoviral transfection in the underlying dura mater did not lead to pathologic SAG suture fusion in any specimens. Conclusions: TGF-beta1 has potent osteogenic capabilities as demonstrated by a significant increase in bone nodule formation. Although blocking TGF-beta signaling prevents physiologic suture fusion, overexpression of TGF-beta in a normally patent cranial suture does not lead to pathologic craniosynostosis. TGF-beta is potentially necessary but not sufficient in the mechanism of programmed cranial suture fusion.

Introduction: Although the multilineage potential of human processed lipoaspirate has previously been demonstrated, few studies exist utilizing mouse adipose-derived mesenchymal cells (ADMSCs) in vitro or in vivo. Here, we develop a method for osteogenic differentiation of ADMSCs in vitro and utilize them to repair calvarial defects in vivo. Methods: ADMSCs were harvested from 3 week old female FVB mice. ADMSCs were cultured in standard osteogenic media with various concentrations of BMP-2 or retinoic acid (RA) for 3 weeks. Growth and differentiation were assessed via cell counting, Oil Red O (ORO), alkaline phosphatase (AP), and von Kossa (VK) staining. Apatite-coated poly-lactic co-glycolic acid (PLGA) scaffolds were then seeded with ADMSCs and implanted into critical sized (4mm) calvarial defects in adult male mice. Calvarial regeneration was assessed using histology and radiography. Results: Only slight differences in proliferation were seen in response to BMP2, RA, or BMP2 + RA. ORO staining was dramatically decreased in treated cells. AP and von Kossa staining were induced in a synergistic fashion by BMP2 + RA. In vivo experiments revealed successful healing of calvarial defects by 12 weeks after surgery. ADMSC-seeded scaffolds produced significant intramembranous bone formation by 2 weeks, and areas of complete bony bridging by 12 weeks as demonstrated by X-ray and histology. Conclusions: Our data demonstrate successful osteogenic differentiation of ADMSCs in vitro and in vivo. This novel mouse model potentiates further investigation into the mechanisms governing osteogenesis and the epigenetic manipulations that can be used to enhance this process.

Introduction: Although a wealth of data demonstrates that regionally patterned dura mater regulates suture fusion or patency, the onset and extent of this patterning remains unknown. In this study we used microarray analyses as a starting point to investigate the differences in suture-specific dura mater from the mouse posterior frontal (PF) and sagittal (SAG) suture. Methods: Regional dura mater was harvested from the PF and SAsutures of 15 and 25 day male CD-1 mice (n = 40 each) and RNA isolated. Microarray analyses were performed using 42,000 element cDNA arrays. Slug was also evaluated in non-suture dura from young (5-day-old) and adult (60-day-old) animals. Investigation into regulation of novel genes, such as the transcription factor Slug, were carried out using rat calvarial osteoblasts stimulated with FGF-2, TGF-beta 1, and BMP2 and 4. Results: Microarray analyses demonstrated differential regulation of over 220 genes in the PF suture at d15, and over 280 genes at d25. Although the number of genes down-regulated was similar, there was a dramatic increase in the number of genes up-regulated in the PF suture at d25 vs. d15 (137 vs. 43). Distinct patterns of expression were seen for several groups of genes, including cytokines (FGF and FGF receptors) and extra-cellular matrix molecules (Col-1, osteopontin, osteocalcin). Several novel genes were identified, including the transcription factor Slug. QRT-PCR verified a > 4-fold increase in Slug mRNA in PF vs. SAG dura, and a > 3-fold increase in young vs. adult dura mater. Functional studies demonstrated a selective inhibition of Slug mRNA in osteoblasts by FGF-2 and TGF-beta 1, but not the BMPs, up to 24 hrs post stimulation (see figure). Conclusions: Our data demonstrate genomic patterning of regional dura mater as early as ten days prior to PF suture fusion. In addition we show that the transcription factor Slug, an evolutionarily conserved gene involved in epithelial-to-mesenchymal cell transitions, is regulated by two growth factors known to be expressed in dura mater.