[Show abstract][Hide abstract] ABSTRACT: New strategies to fulfill craniofacial bone defects have gained attention in recent years due to the morbidity of autologous bone graft harvesting. We aimed to evaluate the in vivo efficacy of bone tissue engineering strategy using mesenchymal stem cells associated with two matrices (bovine bone mineral and α-tricalcium phosphate), compared to an autologous bone transfer. A total of 28 adult, male, non-immunosuppressed Wistar rats underwent a critical-sized osseous defect of 5 mm diameter in the alveolar region. Animals were divided into five groups. Group 1 (n = 7) defects were repaired with autogenous bone grafts; Group 2 (n = 5) defects were repaired with bovine bone mineral free of cells; Group 3 (n = 5) defects were repaired with bovine bone mineral loaded with mesenchymal stem cells; Group 4 (n = 5) defects were repaired with α-tricalcium phosphate free of cells; and Group 5 (n = 6) defects were repaired with α-tricalcium phosphate loaded with mesenchymal stem cells. Groups 2-5 were compared to Group 1, the reference group. Healing response was evaluated by histomorphometry and computerized tomography. Histomorphometrically, Group 1 showed 60.27% ± 16.13% of bone in the defect. Groups 2 and 3 showed 23.02% ± 8.6% (p = 0.01) and 38.35% ± 19.59% (p = 0.06) of bone in the defect, respectively. Groups 4 and 5 showed 51.48% ± 11.7% (p = 0.30) and 61.80% ± 2.14% (p = 0.88) of bone in the defect, respectively. Animals whose bone defects were repaired with α-tricalcium phosphate and mesenchymal stem cells presented the highest bone volume filling the defects; both were not statistically different from autogenous bone.
[Show abstract][Hide abstract] ABSTRACT: Although first reports of the clinical use of adipose-derived stromal cells suggest that this approach may be feasible and effective for soft-tissue augmentation, there is a lack of randomized, controlled clinical trials in the literature. Thus, this study aimed to investigate whether a faster protocol for isolation of adipose-derived stromal cells and their use in combination with fat tissue improve the long-term retention of the grafts in patients with craniofacial microsomia.
Patients with craniofacial microsomia (n = 14) were grafted either with supplementation of adipose-derived stromal cells (experimental group) or without supplementation of adipose-derived stromal cells (control group). The number of viable cells isolated before and after the supplementation of the grafts was calculated, and these cells were examined for mesenchymal cell surface markers using flow cytometry. Computed tomography was performed to assess both hemifaces preoperatively and at 6 months postoperatively.
The average number of viable cells isolated before and after the supplementation of the grafts was 5.6 × 10 and 9.9 × 10 cells/ml of fat tissue (p = 0.015). Flow cytometric analysis revealed that the adipose-derived stromal cells were positive for mesenchymal cell markers (>95 percent for CD73 and CD105). Surviving fat volume at 6 months was 88 percent for the experimental group and 54 percent for the control group (p = 0.003).
These results suggest that this strategy for isolation and supplementation of adipose-derived stromal cells is effective, safe, and superior to conventional lipoinjection for facial recontouring in patients with craniofacial microsomia.
Plastic and Reconstructive Surgery 07/2013; 132(1):141-152. DOI:10.1097/PRS.0b013e3182910a82 · 3.33 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: Non-syndromic cleft lip/palate (NSCL/P) is a complex, frequent congenital malformation, determined by the interplay between genetic and environmental factors during embryonic development. Previous findings have appointed an aetiological overlap between NSCL/P and cancer, and alterations in similar biological pathways may underpin both conditions. Here, using a combination of transcriptomic profiling and functional approaches, we report that NSCL/P dental pulp stem cells exhibit dysregulation of a co-expressed gene network mainly associated with DNA double-strand break repair and cell cycle control (p = 2.88×10(-2)-5.02×10(-9)). This network included important genes for these cellular processes, such as BRCA1, RAD51, and MSH2, which are predicted to be regulated by transcription factor E2F1. Functional assays support these findings, revealing that NSCL/P cells accumulate DNA double-strand breaks upon exposure to H2O2. Furthermore, we show that E2f1, Brca1 and Rad51 are co-expressed in the developing embryonic orofacial primordia, and may act as a molecular hub playing a role in lip and palate morphogenesis. In conclusion, we show for the first time that cellular defences against DNA damage may take part in determining the susceptibility to NSCL/P. These results are in accordance with the hypothesis of aetiological overlap between this malformation and cancer, and suggest a new pathogenic mechanism for the disease.
PLoS ONE 06/2013; 8(6):e65677. DOI:10.1371/journal.pone.0065677 · 3.23 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: DNA methylation is known to be a heritable regulatory mechanism in gene expression and influenced by both genetic and environmental factors. It is also known that impairment in gene methylation status may lead to gene expression dysregulation and thus disease. In this context, cleft lip and palate (CL/P) is a congenital craniofacial malformation with high incidence (1:700 live births) strongly determined by the genetic and environmental interplay in which epigenetic factors such as DNA methylation are very plausible factors in the malformation etiology. Aiming to investigate DNA methylation at specific sites to CL/P, we investigated whether BRCA1 , previously associated to CL/P (Kobayashi and Alvizi et al, 2013 PloS ONE), and 8q24.21 CL/P risk region were differentially methylated in CL/P samples in comparison to control samples. Methods: Bisulfite sequencing analysis for BRCA1 promoter was performed in a DNA sample set obtained from dental pulp stem cells (DPSC) of 18 CL/P and 12 controls and for 8q24 region in a DNA sample set from white blood cells DNA of 34 CL/P and 44 controls. A total of 300 clones for BRCA1 promoter and 780 clones for 8q24.21 were sequenced and analysis was performed using BISMA (Bisulfite Methylation Analysis - BPCD online tool). BRCA1 expression was also assessed by qRT-PCR in the DPSC sample. Results/Conclusions: Total BRCA1 promoter methylation was significantly higher (+1,4%) in the DPSC CL/P sample. Besides, BRCA1 promoter CpGs 1, 2 and 11 were the most hipermethylated in the CL/P sample (17.8%, 30.2% and 23.1%, respectively). As expected, BRCA1 expression was significantly reduced in comparison to controls (p=0.001). No evidence of differential methylation at the 8q24.21 cleft lip risk locus was found in the white blood cells DNA sample of CLP patients as compared to controls. Our results suggest that downregulation of BRCA1 in CL/P samples may be driven by increased BRCA1 promoter methylation and the causative factors in this hypermethylation should be next investigated. BRCA1 expression rescue by promoter demethylation is being conducted in DPSC CL/P samples. FAPESP/CNPq-MCT.
63rd American Society of Human Genetics Meeting, Boston-USA; 01/2013
[Show abstract][Hide abstract] ABSTRACT: Nonsyndromic cleft lip with or without cleft palate (NSCL/P) is a complex disorder with a worldwide incidence estimated at 1:700. Among the putative susceptibility loci, the IRF6 gene and a region at 8q24.21 have been corroborated in different populations. To test the role of IRF6 in NSCL/P predisposition in the Brazilian population, we conducted a structured association study with the SNPs rs642961 and rs590223, respectively, located at 5' and 3' of the IRF6 gene and not in strong linkage disequilibrium (LD), in patients from five different Brazilian locations. We also evaluated the effect of these SNPs in IRF6 expression in mesenchymal stem cells (MSC). We observed association between rs642961 and cleft lip only (CLO) (P=0.009; odds ratio (OR) for AA genotype=1.83 [95% Confidence interval (CI), 0.64-5.31]; OR for AG genotype=1.72 [95% CI, 1.03-2.84]). This association seems to be driven by the affected patients from Barbalha, a location which presents the highest heritability estimate (H2=0.85), and the A allele at rs642961 is acting through a dominant model. No association was detected for the SNP rs590223. We did not find any correlation between expression levels and genotypes of the two loci, and it is possible that these SNPs have a functional role in some specific period of embryogenesis.
American Journal of Medical Genetics Part A 09/2012; 158A(9):2170-5. DOI:10.1002/ajmg.a.35526 · 2.05 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: Non-syndromic cleft lip/palate (NSCL/P) is a multifactorial disease that arises from errors during embryonic development. Although much effort has been put into identifying genetic and environmental factors underlying disease susceptibility, the aetiology of this complex malformation remains obscure. Since a tight regulation of ontogenetic mechanisms is required to ensure appropriate orofacial morphogenesis, our objective was to identify dysregulated pathways involved in the pathogenesis of NSCL/P. Methods: We performed a global transcriptome profiling of 7 dental pulp mesenchymal stem cell cultures from NSCL/P patients by comparison to 6 controls, using Affymetrix HuGene 1.0 ST chips. Differentially expressed genes were obtained using SAM and RankProd algorithms. Functional annotation and gene network analysis were performed with Ingenuity Pathways Analysis, while gene clustering, transcription factor and functional enrichment procedures were carried out using EXPANDER. Quantification of H2O2-induced DNA damage was assessed with flow cytometry for anti-γH2AX in 6 NSCL/P, 2 van der Woude syndrome (VWS), and 7 control stem cell cultures. Finally, RNA in situ hybridisation studies were carried out to CD1 mouse embryos at E10.5, E11.5, E12.5 and E13.5, using riboprobes for Brca1, Rad51, and E2f1. Results/Conclusion: We identified, in NSCL/P cells, a dysregulated transcriptional network associated with cell cycle progression and response to DNA damage, comprising abnormal expression of important molecules for these processes (e.g. BRCA1, BRIP1, MSH2, RAD51 and BLM). These genes, also validated with qRT-PCR, exhibited a marked pattern of co-expression, and suggest that E2F1 may be a putative upstream regulator. By quantifying γ-H2AX in H2O2-exposed cells, we were able to confirm that NSCL/P cells exhibit abnormal response to DNA damage, which was also observed in IRF6-haploinsufficient (VWS) cells. In situ hybridisation studies revealed co-localised expression of Brca1, Rad51 and E2f1 in the facial primordia and developing palatal shelves of mouse embryos. Our results suggest that impairment of DNA damage response may be involved in the aetiology of NSCL/P and indicate that IRF6 may be functionally relevant in this process. Financial support: CEPID/FAPESP, CNPq, MCT.
62nd Annual Meeting of the American Society of Human Genetics, San Francisco-USA; 01/2012
[Show abstract][Hide abstract] ABSTRACT: To quantify the amount of bone formation in the calvarial region of Wistar rats after craniotomy using bone wax as a haemostatic agent.
Surgery to produce bilateral, symmetric, full-thickness cranial defects (area: 18 mm(2)) was performed in eight animals. The right side of the cranium remained open and the edges of the left side osseous defect was covered with bone wax. Calvaria were imaged immediately after surgery and 12 weeks postoperatively by computerized tomography. The areas of the bone defects were measured in three-dimensional images using Magics 13.0 (Materialise-Belgic, software CAD).
The average amount of bone formation on the left and right side respectively was 4.85 mm(2) and 8.16 mm(2). Statistically significant differences between the amount of bone formation on the left and right sides were seen.
Bone wax significantly diminishes the rate of bone formation in calvarial defects in a rat model.
Acta cirurgica brasileira / Sociedade Brasileira para Desenvolvimento Pesquisa em Cirurgia 08/2011; 26(4):274-8. DOI:10.1590/S0102-86502011000400005 · 0.48 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: We have recently reported that human fallopian tubes, which are discarded during surgical procedures of women submitted to sterilization or hysterectomies, are a rich source of human fallopian tube mesenchymal stromal cells (htMSCs). It has been previously shown that human mesenchymal stromal cells may be useful in enhancing the speed of bone regeneration. This prompted us to investigate whether htMSCs might be useful for the treatment of osteoporosis or other bone diseases, since they present a pronounced capacity for osteogenic differentiation in vitro. Based on this prior knowledge, our aim was to evaluate, in vivo, the osteogenic capacity of htMSCs to regenerate bone through an already described xenotransplantation model: nonimmunosuppressed (NIS) rats with cranial defects. htMSCs were obtained from five 30-50 years old healthy women and characterized by flow cytometry and for their multipotenciality in vitro capacity (osteogenic, chondrogenic and adipogenic differentiations). Two symmetric full-thickness cranial defects on each parietal region of seven NIS rats were performed. The left side (LS) of six animals was covered with CellCeram (Scaffdex)-a bioabsorbable ceramic composite scaffold that contains 60% hydroxyapatite and 40% β-tricalciumphosphate-only, and the right side (RS) with the CellCeram and htMSCs (10(6) cells/scaffold). The animals were euthanized at 30, 60 and 90 days postoperatively and cranial tissue samples were taken for histological analysis. After 90 days we observed neobone formation in both sides. However, in animals euthanized 30 and 60 days after the procedure, a mature bone was observed only on the side with htMSCs. PCR and immunofluorescence analysis confirmed the presence of human DNA and thus that human cells were not rejected, which further supports the imunomodulatory property of htMSCs. In conclusion, htMSCs can be used successfully to enhance bone regeneration in vivo, opening a new field for future treatments of osteoporosis and bone reconstruction.
[Show abstract][Hide abstract] ABSTRACT: Non-syndromic cleft lip with or without cleft palate (NS CL/P) is a complex disease in which heritability estimates vary widely depending on the population studied. To evaluate the importance of genetic contribution to NS CL/P in the Brazilian population, we conducted a study with 1,042 families from five different locations (Santarém, Fortaleza, Barbalha, Maceió, and Rio de Janeiro). We also evaluated the role of consanguinity and ethnic background. The proportion of familial cases varied significantly across locations, with the highest values found in Santarém (44%) and the lowest in Maceió (23%). Heritability estimates showed a higher genetic contribution to NS CL/P in Barbalha (85%), followed by Santarém (71%), Rio de Janeiro (70%), Fortaleza (64%), and Maceió (45%). Ancestry was not correlated with the occurrence of NS CL/P or with the variability in heritability. Only in Rio de Janeiro was the coefficient of inbreeding significantly larger in NS CL/P families than in the local population. Recurrence risk for the total sample was approximately 1.5-1.6%, varying according to the location studied (0.6-0.7% in Maceió to 2.2-2.8% in Barbalha). Our findings show that the degree of genetic contribution to NS CL/P varies according to the geographic region studied, and this difference cannot be attributed to consanguinity or ancestry. These findings suggest that Barbalha is a promising region for genetic studies. The data presented here will be useful in interpreting results from molecular analyses and show that care must be taken when pooling samples from different populations for association studies.
American Journal of Medical Genetics Part A 07/2011; 155A(7):1581-7. DOI:10.1002/ajmg.a.34036 · 2.05 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: Nonsyndromic cleft lip and palate (NSCL/P) is a complex disease resulting from failure of fusion of facial primordia, a complex developmental process that includes the epithelial-mesenchymal transition (EMT). Detection of differential gene transcription between NSCL/P patients and control individuals offers an interesting alternative for investigating pathways involved in disease manifestation. Here we compared the transcriptome of 6 dental pulp stem cell (DPSC) cultures from NSCL/P patients and 6 controls. Eighty-seven differentially expressed genes (DEGs) were identified. The most significant putative gene network comprised 13 out of 87 DEGs of which 8 encode extracellular proteins: ACAN, COL4A1, COL4A2, GDF15, IGF2, MMP1, MMP3 and PDGFa. Through clustering analyses we also observed that MMP3, ACAN, COL4A1 and COL4A2 exhibit co-regulated expression. Interestingly, it is known that MMP3 cleavages a wide range of extracellular proteins, including the collagens IV, V, IX, X, proteoglycans, fibronectin and laminin. It is also capable of activating other MMPs. Moreover, MMP3 had previously been associated with NSCL/P. The same general pattern was observed in a further sample, confirming involvement of synchronized gene expression patterns which differed between NSCL/P patients and controls. These results show the robustness of our methodology for the detection of differentially expressed genes using the RankProd method. In conclusion, DPSCs from NSCL/P patients exhibit gene expression signatures involving genes associated with mechanisms of extracellular matrix modeling and palate EMT processes which differ from those observed in controls. This comparative approach should lead to a more rapid identification of gene networks predisposing to this complex malformation syndrome than conventional gene mapping technologies.
Electronic supplementary material
The online version of this article (doi:10.1007/s12015-010-9197-3) contains supplementary material, which is available to authorized users.
[Show abstract][Hide abstract] ABSTRACT: To study were to reproduce an alveolar bone defect model in Wistar rats to be used for testing the efficacy of stem cell therapies. Additionally, we also aimed to determine the osteogenesis process of this osseous defect in the 1 month period post-surgery.
The animals were randomly divided into two groups of 7 animals each. A gingivobuccal incision was made, and a bone defect of 28 mm(2) of area was performed in the alveolar region. Animals were killed at 2 weeks after surgery (n=7) and 4 weeks after surgery (n=7).
The average area of the alveolar defect at time point of 2 weeks was 22.27 +/- 1.31 mm(2) and the average area of alveolar defect at time point of 4 weeks was 9.03 +/- 1.17 mm(2). The average amount of bone formation at time point of 2 weeks was 5.73 +/- 1.31 mm(2) and the average amount of bone formation at time point of 4 weeks was 19 +/- 1.17 mm(2). Statistically significant differences between the amount of bone formation at 2 weeks and 4 weeks after surgery were seen (p=0.003).
The highest rate of ossification occurred mostly from 2 to 4 weeks after surgery. This observation suggests that 4 weeks after the bone defect creation should be a satisfactory timing to assess the potential of bone inductive stem cells to accelerate bone regeneration in Wistar rats.
Acta cirurgica brasileira / Sociedade Brasileira para Desenvolvimento Pesquisa em Cirurgia 08/2010; 25(4):313-7. DOI:10.1590/S0102-86502010000400002 · 0.48 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: To develop an experimental surgical model in rats for the study of craniofacial abnormalities.
Full thickness calvarial defects with 10x10-mm and 5x8-mm dimensions were created in 40 male NIS Wistar rats, body weight ranging from 320 to 420 g. The animals were equally divided into two groups. The periosteum was removed and dura mater was left intact. Animals were killed at 8 and 16 weeks postoperatively and cranial tissue samples were taken from the defects for histological analysis.
Cranial defects remained open even after 16 weeks postoperatively.
The experimental model with 5x8-mm defects in the parietal region with the removal of the periosteum and maintenance of the integrity of the dura mater are critical and might be used for the study of cranial bone defects in craniofacial abnormalities.
Acta cirurgica brasileira / Sociedade Brasileira para Desenvolvimento Pesquisa em Cirurgia 06/2010; 25(3):264-8. DOI:10.1590/S0102-86502010000300008 · 0.48 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: The possibility of using stem cells for regenerative medicine has opened a new field of investigation. The search for sources to obtain multipotent stem cells from discarded tissues or through non-invasive procedures is of great interest. It has been shown that mesenchymal stem cells (MSCs) obtained from umbilical cords, dental pulp and adipose tissue, which are all biological discards, are able to differentiate into muscle, fat, bone and cartilage cell lineages. The aim of this study was to isolate, expand, characterize and assess the differentiation potential of MSCs from human fallopian tubes (hFTs).
Lineages of hFTs were expanded, had their karyotype analyzed, were characterized by flow cytometry and underwent in vitro adipogenic, chondrogenic, osteogenic, and myogenic differentiation.
Here we show for the first time that hFTs, which are discarded after some gynecological procedures, are a rich additional source of MSCs, which we designated as human tube MSCs (htMSCs).
Human tube MSCs can be easily isolated, expanded in vitro, present a mesenchymal profile and are able to differentiate into muscle, fat, cartilage and bone in vitro.
Journal of Translational Medicine 07/2009; 7:46. DOI:10.1186/1479-5876-7-46 · 3.99 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: The canine model provides a large animal system to evaluate many treatment modalities using stem cells (SCs). However, only bone marrow (BM) protocols have been widely used in dogs for preclinical approaches. BM donation consists of an invasive procedure and the number and differentiation potential of its mesenchymal stem cells (MSCs) decline with age. More recently, umbilical cord was introduced as an alternative source to BM since it is obtained from a sample that is routinely discarded. Here, we describe the isolation of MSCs from canine umbilical cord vein (cUCV). These cells can be obtained from every cord received and grow successfully in culture. Their multipotent plasticity was demonstrated by their capacity to differentiate in adipocytic, chondrocytic, and osteocytic lineages. Furthermore, our results open possibilities to use cUCV cells in preclinical trials for many well-characterized canine model conditions homologs to human diseases.
Stem cells and development 04/2009; 19(3):395-402. DOI:10.1089/scd.2008.0314 · 4.20 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: Cleft lip and palate (CLP), one of the most frequent congenital malformations, affects the alveolar bone in the great majority of the cases, and the reconstruction of this defect still represents a challenge in the rehabilitation of these patients. One of the current most promising strategy to achieve this goal is the use of bone marrow stem cells (BMSC); however, isolation of BMSC or iliac bone, which is still the mostly used graft in the surgical repair of these patients, confers site morbidity to the donor. Therefore, in order to identify a new alternative source of stem cells with osteogenic potential without conferring morbidity to the donor, we have used orbicular oris muscle (OOM) fragments, which are regularly discarded during surgery repair (cheiloplasty) of CLP patients. We obtained cells from OOM fragments of four unrelated CLP patients (CLPMDSC) using previously described preplating technique. These cells, through flow cytometry analysis, were mainly positively marked for five mesenchymal stem cell antigens (CD29, CD90, CD105, SH3, and SH4), while negative for hematopoietic cell markers, CD14, CD34, CD45, and CD117, and for endothelial cell marker, CD31. After induction under appropriate cell culture conditions, these cells were capable to undergo chondrogenic, adipogenic, osteogenic, and skeletal muscle cell differentiation, as evidenced by immunohistochemistry. We also demonstrated that these cells together with a collagen membrane lead to bone tissue reconstruction in a critical-size cranial defects previously induced in nonimmunocompromised rats. The presence of human DNA in the new bone was confirmed by PCR with human-specific primers and immunohistochemistry with human nuclei antibodies. In conclusion, we showed that cells from OOM have phenotypic and behavior characteristics similar to other adult stem cells, both in vitro and in vivo. Our findings suggest that these cells represent a promising source of stem cells for alveolar bone grafting treatment, particularly in young CLP patients.
Tissue Engineering Part A 10/2008; 15(2):427-35. DOI:10.1089/ten.tea.2007.0417 · 4.70 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: Phototherapy with low intensity laser irradiation has shown to be effective in promoting the proliferation of different cells. The aim of this in vitro study was to evaluate the potential effect of laser phototherapy (660 nm) on human dental pulp stem cell (hDPSC) proliferation.
The hDPSC cell strain was used. Cells cultured under nutritional deficit (10% FBS) were either irradiated or not (control) using two different power settings (20 mW/6 seconds to 40 mW/3 seconds), with an InGaAIP diode laser. The cell growth was indirectly assessed by measuring the cell mitochondrial activity through the MTT reduction-based cytotoxicity assay.
The group irradiated with the 20 mW setting presented significantly higher MTT activity at 72 hours than the other two groups (negative control--10% FBS--and lased 40 mW with 3 seconds exposure time). After 24 hours of the first irradiation, cultures grown under nutritional deficit (10% FBS) and irradiated presented significantly higher viable cells than the non-irradiated cultures grown under the same nutritional conditions.
Under the conditions of this study it was possible to conclude that the cell strain hDPSC responds positively to laser phototherapy by improving the cell growth when cultured under nutritional deficit conditions. Thus, the association of laser phototherapy and hDPSC cells could be of importance for future tissue engineering and regenerative medicine. Moreover, it opens the possibility of using laser phototherapy for improving the cell growth of other types of stem cells.
Lasers in Surgery and Medicine 08/2008; 40(6):433-8. DOI:10.1002/lsm.20646 · 2.61 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: The main aim of this study is to evaluate the capacity of human dental pulp stem cells (hDPSC), isolated from deciduous teeth, to reconstruct large-sized cranial bone defects in nonimmunosuppressed (NIS) rats. To our knowledge, these cells were not used before in similar experiments. We performed two symmetric full-thickness cranial defects (5 x 8 mm) on each parietal region of eight NIS rats. In six of them, the left side was supplied with collagen membrane only and the right side (RS) with collagen membrane and hDPSC. In two rats, the RS had collagen membrane only and nothing was added at the left side (controls). Cells were used after in vitro characterization as mesenchymal cells. Animals were euthanized at 7, 20, 30, 60, and 120 days postoperatively and cranial tissue samples were taken from the defects for histologic analysis. Analysis of the presence of human cells in the new bone was confirmed by molecular analysis. The hDPSC lineage was positive for the four mesenchymal cell markers tested and showed osteogenic, adipogenic, and myogenic in vitro differentiation. We observed bone formation 1 month after surgery in both sides, but a more mature bone was present in the RS. Human DNA was polymerase chain reaction-amplified only at the RS, indicating that this new bone had human cells. The use of hDPSC in NIS rats did not cause any graft rejection. Our findings suggest that hDPSC is an additional cell resource for correcting large cranial defects in rats and constitutes a promising model for reconstruction of human large cranial defects in craniofacial surgery.
Journal of Craniofacial Surgery 02/2008; 19(1):204-10. DOI:10.1097/scs.0b013e31815c8a54 · 0.68 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: Carpenter syndrome is a pleiotropic disorder with autosomal recessive inheritance, the cardinal features of which include craniosynostosis, polysyndactyly, obesity, and cardiac defects. Using homozygosity mapping, we found linkage to chromosome 6p12.1-q12 and, in 15 independent families, identified five different mutations (four truncating and one missense) in RAB23, which encodes a member of the RAB guanosine triphosphatase (GTPase) family of vesicle transport proteins and acts as a negative regulator of hedgehog (HH) signaling. In 10 patients, the disease was caused by homozygosity for the same nonsense mutation, L145X, that resides on a common haplotype, indicative of a founder effect in patients of northern European descent. Surprisingly, nonsense mutations of Rab23 in open brain mice cause recessive embryonic lethality with neural-tube defects, suggesting a species difference in the requirement for RAB23 during early development. The discovery of RAB23 mutations in patients with Carpenter syndrome implicates HH signaling in cranial-suture biogenesis--an unexpected finding, given that craniosynostosis is not usually associated with mutations of other HH-pathway components--and provides a new molecular target for studies of obesity.
The American Journal of Human Genetics 07/2007; 80(6):1162-70. DOI:10.1086/518047 · 10.99 Impact Factor