Post-Adipose-Derived Stem Cells (ADSC) Stimulated by Collagen Type V (Col V) Mitigate the Progression of Osteoarthritic Rabbit Articular Cartilage.

Collagen is essential for cartilage adhesion and formation. In the present study, histology, immunofluorescence, morphometry, and qRT-PCR suggested that adipose-derived stem cells (ADSCs) stimulated by type V collagen (Col V) induce a significant increase of type II collagen (Col II) in the degenerative area of surgical-induced osteoarthritic rabbit articular cartilage (OA). In vitro, the effects of Col V on the proliferation and differentiation of ADSC were investigated. The expression of the cartilage-related genes Col2a1 and Acan was significantly upregulated and Pou5fl was downregulated post-ADSC/Col V treatment. Post-ADSC/Col V treatment, in vivo analyses revealed that rabbits showed typical signs of osteoarthritic articular cartilage regeneration by hematoxylin and eosin (H&E) and Safranin O/Fast Green staining. Immunohistochemical staining demonstrated that the volume of Col II fibers and the expression of Col II protein were significantly increased, and apoptosis Fas ligand positive significantly decreased post-ADSC/Col V treatment. In conclusion, the expression of Col II was higher in rabbits with surgical-induced osteoarthritic articular cartilage; hence, ADSC/Col V may be a promising therapeutic target for OA treatment.

Human levator veli palatini muscle: a novel source of mesenchymal stromal cells for use in the rehabilitation of patients with congenital craniofacial malformations.

BACKGROUND: Bone reconstruction in congenital craniofacial differences, which affect about 2-3% of newborns, has long been the focus of intensive research in the field of bone tissue engineering. The possibility of using mesenchymal stromal cells in regenerative medicine protocols has opened a new field of investigation aimed at finding optimal sources of multipotent cells that can be isolated via non-invasive procedures. In this study, we analyzed whether levator veli palatini muscle fragments, which can be readily obtained in non-invasive manner during palatoplasty in cleft palate patients, represent a novel source of MSCs with osteogenic potential. METHODS: We obtained levator veli palatini muscle fragments (3-5 mm3), during surgical repair of cleft palate in 5 unrelated patients. Mesenchymal stromal cells were isolated from the muscle using a pre-plating technique and other standard practices. The multipotent nature of the isolated stromal cells was demonstrated via flow cytometry analysis and by induction along osteogenic, adipogenic, and chondrogenic differentiation pathways. To demonstrate the osteogenic potential of these cells in vivo, they were used to reconstruct a critical-sized full-thickness calvarial defect model in immunocompetent rats. RESULTS: Flow cytometry analysis showed that the isolated stromal cells were positive for mesenchymal stem cell antigens (CD29, CD44, CD73, CD90, and CD105) and negative for hematopoietic (CD34 and CD45) or endothelial cell markers (CD31). The cells successfully underwent osteogenic, chondrogenic, and adipogenic cell differentiation under appropriate cell culture conditions. Calvarial defects treated with CellCeram™ scaffolds seeded with the isolated levator veli palatini muscle cells showed greater bone healing compared to defects treated with acellular scaffolds. CONCLUSION: Cells derived from levator veli palatini muscle have phenotypic characteristics similar to other mesenchymal stromal cells, both in vitro and in vivo. Our findings suggest that these cells may have clinical relevance in the surgical rehabilitation of patients with cleft palate and other craniofacial anomalies characterized by significant bone deficit.

Deciduous Dental Pulp Stem Cells for Maxillary Alveolar Reconstruction in Cleft Lip and Palate Patients.

BACKGROUND: To reduce morbidity to cleft patients, new approaches have been developed and here, we report for the first time the use of deciduous dental pulp stem cells (DDPSC) associated with a hydroxyapatite-collagen sponge (Bio-Oss Collagen® 250 mg, Geistlich) for closing alveolar defects during secondary dental eruption, further comparing these results to historical controls. METHODS: Six patients, aged 8 to 12, were selected. Autologous DDPSC were isolated from each patient, then associated with the biomaterial and this bone tissue engineered set was used to fill the alveolar defect. Computed tomography was performed to assess both preoperative and 6- and 12-month postoperative outcomes. Overall morbidity was recorded. Historical controls consisted of sixteen patients previously selected and randomly assigned to group one (rhBMP-2) or group two (iliac crest bone graft). RESULTS: DDPSC could be isolated and characterized as mesenchymal stem cells. Progressive alveolar bone union has occurred in all patients. Similarly to group two 75.4%, SD ± 4.0, p > 0.999, but statistically different from group one (59.6%, SD ± 9.9, p > 0.999, but statistically different from group one (59.6%, SD ± 9.9. CONCLUSION: For this selected group of patients, DDPSC therapy resulted in satisfactory bone healing with excellent feasibility and safety, which adds significantly to the prospect of stem cell use in clinical settings. Clinical Question/Level of Evidence. Therapeutic, II. This trial is registered with https://clinicaltrials.gov/ct2/show/NCT01932164?term=NCT01932164&rank=1.

Mesenchymal Stem Cells from Human Exfoliated Deciduous Teeth and the Orbicularis Oris Muscle: How Do They Behave When Exposed to a Proinflammatory Stimulus?

Mesenchymal stem cells (MSCs) have been studied as a promising type of stem cell for use in cell therapies because of their ability to regulate the immune response. Although they are classically isolated from the bone marrow, many studies have sought to isolate MSCs from noninvasive sources. The objective of this study was to evaluate how MSCs isolated from the dental pulp of human exfoliated deciduous teeth (SHED) and fragments of the orbicularis oris muscle (OOMDSCs) behave when treated with an inflammatory IFN-γ stimulus, specifically regarding their proliferative, osteogenic, and immunomodulatory potentials. The results demonstrated that the proliferation of SHED and OOMDSCs was inhibited by the addition of IFN-γ to their culture medium and that treatment with IFN-γ at higher concentrations resulted in a greater inhibition of the proliferation of these cells than treatment with IFN-γ at lower concentrations. SHED and OOMDSCs maintained their osteogenic differentiation potential after stimulation with IFN-γ. Additionally, SHED and OOMDSCs have been shown to have low immunogenicity because they lack expression of HLA-DR and costimulatory molecules such as CD40, CD80, and CD86 before and after IFN-γ treatment. Last, SHED and OOMDSCs expressed the immunoregulatory molecule HLA-G, and the expression of this antigen increased after IFN-γ treatment. In particular, an increase in intracellular HLA-G expression was observed. The results obtained suggest that SHED and OOMDSCs lack immunogenicity and have immunomodulatory properties that are enhanced when they undergo inflammatory stimulation with IFN-γ, which opens new perspectives for the therapeutic use of these cells.

Macrophage: A Potential Target on Cartilage Regeneration.

Cartilage lesions and osteoarthritis (OA) presents an ever-increasing clinical and socioeconomic burden. Synovial inflammation and articular inflammatory environment are the key factor for chondrocytes apoptosis and hypertrophy, ectopic bone formation and OA progression. To effectively treat OA, it is critical to develop a drug that skews inflammation toward a pro-chondrogenic microenvironment. In this narrative and critical review, we aim to see the potential use of immune cells modulation or cell therapy as therapeutic alternatives to OA patients. Macrophages are immune cells that are present in synovial lining, with different roles depending on their subtypes. These cells can polarize to pro-inflammatory (M1) and anti-inflammatory (M2) phenotypes, being the latter associated with wound-healing by the production of ARG-1 and pro-chondrogenic cytokines, such as IL-10, IL-1RA, and TGF-b. Emerging evidence reveals that macrophage shift can be determined by several stimuli, apart from the conventional in vitro IL-4, IL-13, and IL-10. Evidences show the potential of physical exercise to induce type 2 response, favoring M2 polarization. Moreover, macrophages in contact with oxLDL have effect on the production of anabolic mediators as TGF-b. In the same direction, type II collagen, that plays a critical role in development and maturation process of chondrocytes, can also induce M2 macrophages, increasing TGF-b. The mTOR pathway activation in macrophages was shown to be able to polarize macrophages in vitro, though further studies are required. The possibility to use mesenchymal stem cells (MSCs) in cartilage restoration have a more concrete literature, besides, MSCs also have the capability to induce M2 macrophages. In the other direction, M1 polarized macrophages inhibit the proliferation and viability of MSCs and impair their ability to immunosuppress the environment, preventing cartilage repair. Therefore, even though MSCs therapeutic researches advances, other sources of M2 polarization are attractive issues, and further studies will contribute to the possibility to manipulate this polarization and to use it as a therapeutic approach in OA patients.

Human Synovial Mesenchymal Stem Cells Good Manufacturing Practices for Articular Cartilage Regeneration.

Background: Cartilage restoration is a desperately needed bridge for patients with symptomatic cartilage lesions. Chondral lesion is a pathology with high prevalence, reaching as much as 63% of general population and 36% among athletes. Despite autologous chondrocyte implantation versatility, it still fails to fully reproduce hyaline articular cartilage characteristics. Mesenchymal stem cells (MSCs) may be isolated from various known tissues, including discarded fragments at arthroscopy such as synovial membrane. Choice of harvesting site is motivated by MSCs' abilities to modulate immunologic and inflammatory response through paracrine communication. Synovial MSCs have a greater proliferation and strong chondrogenic potential than bone and adipose MSCs and a less hypertrophic differentiation than bone MSCs. Good manufacturing practice (GMP) laboratory techniques for human clinical trials are still novel. To our knowledge, there are only two clinical trials in humans published since today. Purpose: Therefore, this work aimed to isolate and characterize synovial MSCs and evaluated their differentiation properties according to GMP standards. Materials and Methods: One-gram tissue sample from three patients of synovia was harvested at the beginning of arthroscopy surgery. MSCs were isolated, expanded, and characterized by flow cytometry. Results: It was possible to isolate and expand MSCs cultures from synovia, characterize MSCs by flow cytometry using proper monoclonal antibodies, and differentiate MSCs by coloring technique after chondrogenic, adipogenic, and osteogenic differentiations. Cartilage treatment may benefit from these tissue engineering protocols since arthroscopic procedures are routinely performed for different purposes in a previous stage and a favorable chondronegic differentiation cell lineage may be collected and stored in a less invasive way. Conclusion: Laboratory protocols established according to presented GMP were able to isolate and characterize MSCs obtained from synovia. Impact Statement Articular cartilage restoration is a desperately needed bridge for patients with symptomatic cartilage lesions and it rises as a socioeconomic issue with a considerable economic burden. Synovial mesenchymal stem cells (MSCs) have a greater proliferation rate and strong chondrogenic potential than bone and adipose MSCs and a less hypertrophic differentiation than bone MSCs. To our knowledge, there are only two human clinical trials with good manufacturing practice laboratory techniques for synovial MSCs harvesting and differentiation. Cartilage treatment may benefit from these tissue engineering protocols since arthroscopic procedures are routinely performed for different purposes in a previous stage.

Nonsyndromic cleft lip and/or palate: A multicenter study of the dental anomalies involved.

BACKGROUND: Nonsyndromic cleft lip and/or palate (NSCL/P) is the most common craniofacial malformation. Due to the anatomical defect present in the alveolar process, these patients tend to exhibit more dental anomalies. The aim of this study was to identify the prevalence of dental anomalies in patients with NSCL/P by obtaining orthodontic documentation from Brazilian Centers for cleft lip and palate treatment. MATERIAL AND METHODS: A retrospective analysis (2000-2014) was conducted on orthodontic archives, radiographs and medical records of NSCL/P of 524 patients under orthodontic treatment. Panoramic radiographs and intra-oral photographs were examined to identify these anomalies. Categorical variables were expressed in terms of frequencies and percentages and analyzed using the Chi-Square test. The level of significance was set at p≤0.05 in all analyses. RESULTS: Approximately 83.3% of the individuals had at least one dental anomaly. Tooth agenesis was the most common abnormality found in those patients (87.8%) (p<0.001). Also, the largest number of dental anomalies was detected in the group of unilateral left clefts. The prevalence of dental anomalies in the present sample of NSCL/P patients was high and reached the highest levels in patients with alveolar bone clefts. CONCLUSIONS: This study describes the most common dental anomalies observed in patients with NSCL/P. These abnormalities can cause significant problems that may be solved or minimized by early diagnosis and treatment. Key words:Cleft lip and/or palate, dental care for children, epidemiology, craniofacial abnormalities.

The Use of Human Mesenchymal Stem Cells as Therapeutic Agents for the in vivo Treatment of Immune-Related Diseases: A Systematic Review.

Background: One of the greatest challenges for medicine is to find a safe and effective treatment for immune-related diseases. However, due to the low efficacy of the treatment available and the occurrence of serious adverse effects, many groups are currently searching for alternatives to the traditional therapy. In this regard, the use of human mesenchymal stem cells (hMSCs) represents a great promise for the treatment of a variety of immune-related diseases due to their potent immunomodulatory properties. The main objective of this study is, therefore, to present and summarize, through a systematic review of the literature, in vivo studies in which the efficacy of the administration of hMSCs for the treatment of immune-related diseases was evaluated. Methods: The article search was conducted in PubMed/MEDLINE, Scopus and Web of Science databases. Original research articles assessing the therapeutic potential of hMSCs administration for the in vivo treatment immune-related diseases, published from 1984 to December 2017, were selected and evaluated. Results: A total of 132 manuscripts formed the basis of this systematic review. Most of the studies analyzed reported positive results after hMSCs administration. Clinical effects commonly observed include an increase in the survival rates and a reduction in the severity and incidence of the immune-related diseases studied. In addition, hMSCs administration resulted in an inhibition in the proliferation and activation of CD19+ B cells, CD4+ Th1 and Th17 cells, CD8+ T cells, NK cells, macrophages, monocytes, and neutrophils. The clonal expansion of both Bregs and Tregs cells, however, was stimulated. Administration of hMSCs also resulted in a reduction in the levels of pro-inflammatory cytokines such as IFN-γ, TNF-α, IL-1, IL-2, IL-12, and IL-17 and in an increase in the levels of immunoregulatory cytokines such as IL-4, IL-10, and IL-13. Conclusions: The results obtained in this study open new avenues for the treatment of immune-related diseases through the administration of hMSCs and emphasize the importance of the conduction of further studies in this area.

Development of a Novel Large Animal Model to Evaluate Human Dental Pulp Stem Cells for Articular Cartilage Treatment.

PURPOSE: Chondral lesion is a pathology with high prevalence, reaching as much as 63% of general population and 36% among athletes. The ability of human Dental Pulp Stem Cells (DPSCs) to differentiate into chondroblasts in vitro suggests that this stem cell type may be useful for tissue bioengineering. However, we have yet to identify a study of large animal models in which DPSCs were used to repair articular cartilage. Therefore, this study aimed to describe a novel treatment for cartilage lesion with DPSCs on a large animal model. METHODS: Mesenchymal stem cells (MSC) were obtained from deciduous teeth and characterized by flow cytometry. DPSCs were cultured and added to a collagen type I/III biomaterial composite scaffold. Brazilian miniature pig (BR-1) was used. A 6-mm diameter, full-thickness chondral defect was created in each posterior medial condyle. The defects were covered with scaffold alone or scaffold + DPSCs on the contralateral side. Animals were euthanized 6 weeks post-surgery. Cartilage defects were analyzed macroscopically and histology according to modified O'Driscoll scoring system. RESULTS: Flow cytometry confirmed characterization of DPSCs as MSCs. Macroscopic and histological findings suggested that this time period was reasonable for evaluating cartilage repair. To our knowledge, this study provides the first description of an animal model using DPSCs to study the differentiation of hyaline articular cartilage in vivo. CONCLUSION: The animals tolerated the procedure well and did not show clinical or histological rejection of the DPSCs, reinforcing the feasibility of this descriptive miniature pig model for pre-clinical studies.

Is bone transplantation the gold standard for repair of alveolar bone defects?

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.

Susceptibility to DNA damage as a molecular mechanism for non-syndromic cleft lip and palate.

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.

Histological and radiological changes in cranial bone in the presence of bone wax.

PURPOSE: To quantify the amount of bone formation in the calvarial region of Wistar rats after craniotomy using bone wax as a haemostatic agent. METHODS: 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). RESULTS: 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. CONCLUSIONS: Bone wax significantly diminishes the rate of bone formation in calvarial defects in a rat model.

Histological and radiological changes in cranial bone in the presence of bone wax / Alterações histológicas e radiológicas no osso craniano na presença de cera de osso

PURPOSE: To quantify the amount of bone formation in the calvarial region of Wistar rats after craniotomy using bone wax as a haemostatic agent. METHODS: Surgery to produce bilateral, symmetric, full-thickness cranial defects (area: 18 mm²) 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). RESULTS: The average amount of bone formation on the left and right side respectively was 4.85 mm² and 8.16 mm². Statistically significant differences between the amount of bone formation on the left and right sides were seen. CONCLUSIONS: Bone wax significantly diminishes the rate of bone formation in calvarial defects in a rat model.

OBJETIVO: Quantificar a formação óssea da região da calvaria de ratos Wistar submetidos à craniotomia com a utilização de cera de osso como agente hemostático. MÉTODOS: Cirurgia para realizar um defeito ósseo craniano bilateral, simétrico (área: 18 mm²) e com espessura total foi realizado em oito animais. O lado direito do crânio permaneceu aberto e as extremidades do defeito ósseo do lado esquerdo foram recobertas com cera de osso. O crânio foi submetido à avaliação radiológica imediatamente após a cirurgia e 12 semanas após a cirurgia com a utilização de tomografia computadorizada. As áreas dos defeitos ósseos foram medidas através de imagens tridimensionais e utilizando o programa de computador Magics 13.0 (Materialise-Belgic, software CAD). RESULTADOS: A quantidade média de formação óssea no lado esquerdo e direito foi respectivamente de 4.85 mm² e 8.16 mm². Diferença estatisticamente significante foi observada entre o lado direito e esquerdo. CONCLUSÕES: A cera de osso diminuiu significativamente a formação óssea nos defeitos ósseos em modelo animal.

Human stem cell cultures from cleft lip/palate patients show enrichment of transcripts involved in extracellular matrix modeling by comparison to controls.

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.

An experimental model for the study of craniofacial deformities / Modelo experimental para o estudo de deformidades craniofaciais

PURPOSE: To develop an experimental surgical model in rats for the study of craniofacial abnormalities. METHODS: 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. RESULTS: Cranial defects remained open even after 16 weeks postoperatively. CONCLUSION: 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.

OBJETIVO: Desenvolver um modelo experimental em ratos para o estudo de deformidades craniofaciais. MÉTODOS: Foram realizados defeitos ósseos de espessura total com diâmetro de 5 x 8 mm e 10 x 10mm na calota craniana em 40 ratos, machos, NIS Wistar, com peso de 320 a 420 g divididos igualmente em dois grupos. O periósteo foi retirado e a dura-máter mantida intacta. Os animais foram sacrificados na 8ª e 16ª semana de pós-operatório e amostras de tecido ósseo foram extraídas para realização da análise histológica. RESULTADOS: Os defeitos cranianos permaneceram abertos mesmo após 16 semanas após a cirurgia. CONCLUSÃO: O modelo experimental com defeitos de 5x8mm na região parietal, com remoção do periósteo e manutenção da integridade da dura-máter são considerados críticos, e poderá ser utilizado para o estudo dos defeitos ósseos cranianos nas anomalias craniofaciais.

An experimental model for the study of craniofacial deformities.

PURPOSE: To develop an experimental surgical model in rats for the study of craniofacial abnormalities. METHODS: 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. RESULTS: Cranial defects remained open even after 16 weeks postoperatively. CONCLUSION: 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.

New source of muscle-derived stem cells with potential for alveolar bone reconstruction in cleft lip and/or palate patients.

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.

Allogenous bone grafts improved by bone marrow stem cells and platelet growth factors: clinical case reports.

In order to increase the amount of available bone where dental implants must be placed, the present study has associated platelet-rich plasma (PRP) and mononuclear cells (MNCs) from bone marrow aspirate and bone scaffold (BS) in 32 patients aged between 45 and 75 years old. The MNC attainment and the adherence to the BS were confirmed through histology, cell culture, and scanning electron microscopy. The clinical results, analyzed by computed tomography, have showed that the scaffolds were well integrated and adapted to the cortical bone. We can conclude that the process of healing observed in the patients was due to the presence of mesenchymal stem cell in MNC fraction in the bone grafts.