Col1A-2 Mutation in Osteogenesis Imperfecta Mice Contributes to Long Bone Fragility by Modifying Cell-Matrix Organization.

Osteogenesis imperfecta (OI) is a rare congenital bone dysplasia generally caused by a mutation of one of the type I collagen genes and characterized by low bone mass, numerous fractures, and bone deformities. The collagen organization and osteocyte lacuna arrangement were investigated in the long bones of 17-week-old wildtype (WT, n = 17) and osteogenesis imperfecta mice (OIM, n = 16) that is a validated model of severe human OI in order to assess their possible role in bone fragility. Fractures were counted after in vivo scanning at weeks 5, 11, and 17. Humerus, femur, and tibia diaphyses from both groups were analyzed ex vivo with pQCT, polarized and ordinary light histology, and Nano-CT. The fractures observed in the OIM were more numerous in the humerus and femur than in the tibia, whereas the quantitative bone parameters were altered in different ways among these bones. Collagen fiber organization appeared disrupted, with a lower birefringence in OIM than WT bones, whereas the osteocyte lacunae were more numerous, more spherical, and not aligned in a lamellar pattern. These modifications, which are typical of immature and less mechanically competent bone, attest to the reciprocal alteration of collagen matrix and osteocyte lacuna organization in the OIM, thereby contributing to bone fragility.

A New Osteogenic Membrane to Enhance Bone Healing: At the Crossroads between the Periosteum, the Induced Membrane, and the Diamond Concept.

The lack of viability of massive bone allografts for critical-size bone defect treatment remains a challenge in orthopedic surgery. The literature has reviewed the advantages of a multi-combined treatment with the synergy of an osteoconductive extracellular matrix (ECM), osteogenic stem cells, and growth factors (GFs). Questions are still open about the need for ECM components, the influence of the decellularization process on the latter, the related potential loss of function, and the necessity of using pre-differentiated cells. In order to fill in this gap, a bone allograft surrounded by an osteogenic membrane made of a decellularized collagen matrix from human fascia lata and seeded with periosteal mesenchymal stem cells (PMSCs) was analyzed in terms of de-/recellularization, osteogenic properties, PMSC self-differentiation, and angiogenic potential. While the decellularization processes altered the ECM content differently, the main GF content was decreased in soft tissues but relatively increased in hard bone tissues. The spontaneous osteogenic differentiation was necessarily obtained through contact with a mineralized bone matrix. Trying to deepen the knowledge on the complex matrix-cell interplay could further propel these tissue engineering concepts and lead us to provide the biological elements that allow bone integration in vivo.

Reconstruction of the human nipple-areolar complex: a tissue engineering approach.

Introduction: Nipple-areolar complex (NAC) reconstruction after breast cancer surgery is challenging and does not always provide optimal long-term esthetic results. Therefore, generating a NAC using tissue engineering techniques, such as a decellularization-recellularization process, is an alternative option to recreate a specific 3D NAC morphological unit, which is then covered with an in vitro regenerated epidermis and, thereafter, skin-grafted on the reconstructed breast. Materials and methods: Human NACs were harvested from cadaveric donors and decellularized using sequential detergent baths. Cellular clearance and extracellular matrix (ECM) preservation were analyzed by histology, as well as by DNA, ECM proteins, growth factors, and residual sodium dodecyl sulfate (SDS) quantification. In vivo biocompatibility was evaluated 30 days after the subcutaneous implantation of native and decellularized human NACs in rats. In vitro scaffold cytocompatibility was assessed by static seeding of human fibroblasts on their hypodermal side for 7 days, while human keratinocytes were seeded on the scaffold epidermal side for 10 days by using the reconstructed human epidermis (RHE) technique to investigate the regeneration of a new epidermis. Results: The decellularized NAC showed a preserved 3D morphology and appeared white. After decellularization, a DNA reduction of 98.3% and the absence of nuclear and HLA staining in histological sections confirmed complete cellular clearance. The ECM architecture and main ECM proteins were preserved, associated with the detection and decrease in growth factors, while a very low amount of residual SDS was detected after decellularization. The decellularized scaffolds were in vivo biocompatible, fully revascularized, and did not induce the production of rat anti-human antibodies after 30 days of subcutaneous implantation. Scaffold in vitro cytocompatibility was confirmed by the increasing proliferation of seeded human fibroblasts during 7 days of culture, associated with a high number of living cells and a similar viability compared to the control cells after 7 days of static culture. Moreover, the RHE technique allowed us to recreate a keratinized pluristratified epithelium after 10 days of culture. Conclusion: Tissue engineering allowed us to create an acellular and biocompatible NAC with a preserved morphology, microarchitecture, and matrix proteins while maintaining their cell growth potential and ability to regenerate the skin epidermis. Thus, tissue engineering could provide a novel alternative to personalized and natural NAC reconstruction.

Periosteum and fascia lata: Are they so different?

Introduction: The human fascia lata (HFL) is used widely in reconstructive surgery in indications other than fracture repair. The goal of this study was to compare microscopic, molecular, and mechanical properties of HFL and periosteum (HP) from a bone tissue engineering perspective. Material and Methods: Cadaveric HP and HFL (N = 4 each) microscopic morphology was characterized using histology and immunohistochemistry (IHC), and the extracellular matrix (ECM) ultrastructure assessed by means of scanning electron microscopy (SEM). DNA, collagen, elastin, glycosaminoglycans, major histocompatibility complex Type 1, and bone morphogenetic protein (BMP) contents were quantified. HP (N = 6) and HFL (N = 11) were submitted to stretch tests. Results: Histology and IHC highlighted similarities (Type I collagen fibers and two-layer organization) but also differences (fiber thickness and compaction and cell type) between both tissues, as confirmed using SEM. The collagen content was statistically higher in HFL than HP (735 vs. 160.2 µg/mg dry weight, respectively, p < 0.0001). On the contrary, DNA content was lower in HFL than HP (404.75 vs. 1,102.2 µg/mg dry weight, respectively, p = 0.0032), as was the immunogenic potential (p = 0.0033). BMP-2 and BMP-7 contents did not differ between both tissues (p = 0.132 and p = 0.699, respectively). HFL supported a significantly higher tension stress than HP. Conclusion: HP and HFL display morphological differences, despite their similar molecular ECM components. The stronger stretching resistance of HFL can specifically be explained by its higher collagen content. However, HFL contains many fewer cells and is less immunogenic than HP, as latter is rich in periosteal stem cells. In conclusion, HFL is likely suitable to replace HP architecture to confer a guide for bone consolidation, with an absence of osteogenicity. This study could pave the way to a bio-engineered periosteum built from HFL.

Oxidative Stress-Induced Sirtuin1 Downregulation Correlates to HIF-1α, GLUT-1, and VEGF-A Upregulation in Th1 Autoimmune Hashimoto's Thyroiditis.

In Hashimoto's thyroiditis (HT), oxidative stress (OS) is driven by Th1 cytokines' response interfering with the normal function of thyrocytes. OS results from an imbalance between an excessive production of reactive oxygen species (ROS) and a lowering of antioxidant production. Moreover, OS has been shown to inhibit Sirtuin 1 (SIRT1), which is able to prevent hypoxia-inducible factor (HIF)-1α stabilization. The aims of this study were to determine the involvement of NADPH-oxidases (NOX), SIRT1, and HIF-1α in HT pathophysiology as well as the status of antioxidant proteins such as peroxiredoxin 1 (PRDX1), catalase, and superoxide dismutase 1 (SOD1). The protein expressions of NOX2, NOX4, antioxidant enzymes, SIRT1, and HIF-1α, as well as glucose transporter-1 (GLUT-1) and vascular endothelial growth factor A (VEGF-A), were analyzed by Western blot in primary cultures of human thyrocytes that were or were not incubated with Th1 cytokines. The same proteins were also analyzed by immunohistochemistry in thyroid samples from control and HT patients. In human thyrocytes incubated with Th1 cytokines, NOX4 expression was increased whereas antioxidants, such as PRDX1, catalase, and SOD1, were reduced. Th1 cytokines also induced a significant decrease of SIRT1 protein expression associated with an upregulation of HIF-1α, GLUT-1, and VEGF-A proteins. With the exception of PRDX1 and SOD1, similar results were obtained in HT thyroids. OS due to an increase of ROS produced by NOX4 and a loss of antioxidant defenses (PRDX1, catalase, SOD1) correlates to a reduction of SIRT1 and an upregulation of HIF 1α, GLUT-1, and VEGF-A. Our study placed SIRT1 as a key regulator of OS and we, therefore, believe it could be considered as a potential therapeutic target in HT.

miR-199a Downregulation as a Driver of the NOX4/HIF-1α/VEGF-A Pathway in Thyroid and Orbital Adipose Tissues from Graves' Patients.

Graves' disease (GD) is an autoimmune thyroiditis often associated with Graves' orbitopathy (GO). GD thyroid and GO orbital fat share high oxidative stress (OS) and hypervascularization. We investigated the metabolic pathways leading to OS and angiogenesis, aiming to further decipher the link between local and systemic GD manifestations. Plasma and thyroid samples were obtained from patients operated on for multinodular goiters (controls) or GD. Orbital fats were from GO or control patients. The NADPH-oxidase-4 (NOX4)/HIF-1α/VEGF-A signaling pathway was investigated by Western blotting and immunostaining. miR-199a family expression was evaluated following quantitative real-time PCR and/or in situ hybridization. In GD thyroids and GO orbital fats, NOX4 was upregulated and correlated with HIF-1α stabilization and VEGF-A overexpression. The biotin assay identified NOX4, HIF-1α and VEGF-A as direct targets of miR-199a-5p in cultured thyrocytes. Interestingly, GD thyroids, GD plasmas and GO orbital fats showed a downregulation of miR-199a-3p/-5p. Our results also highlighted an activation of STAT-3 signaling in GD thyroids and GO orbital fats, a transcription factor known to negatively regulate miR-199a expression. We identified NOX4/HIF-1α/VEGF-A as critical actors in GD and GO. STAT-3-dependent regulation of miR-199a is proposed as a common driver leading to these events in GD thyroids and GO orbital fats.

Downregulation of Caveolin-1 and Upregulation of Deiodinase 3, Associated with Hypoxia-Inducible Factor-1α Increase, Are Involved in the Oxidative Stress of Graves' Orbital Adipocytes.

Background: Even though the clinical features of Graves' orbitopathy (GO) are well known, its exact pathogenesis remains controversial. The imbalance of redox homeostasis in the connective tissue could play a crucial role leading to an inflammatory state and edema of soft orbital tissues, thus contributing to orbital hypoxia and increase in hypoxia-inducible factor (HIF)-1α. This oxidative stress appears to target the orbital cells such as fibroblasts and also adipocytes. This study aims to explore which pathways can lead to the aforementioned oxidative stress in GO adipose cells and therefore offers new plausible therapeutic targets. Methods: Orbital fat samples were obtained from patients with GO (Western blot [WB]: n = 8, immunohistochemistry [IHC]: n = 8) and from control patients (WB: n = 5, IHC: n = 3-5). They were processed for WB analysis and IHC of the antioxidants (catalase, superoxide dismutase 1) and for HIF-1α. The expression of caveolin-1 (Cav-1) and deiodinase 3 (DIO3), known to be regulated by HIF-1α, was also analyzed by WB and IHC, as well as the targets of Cav-1: glucose transporter type 4 (Glut-4), NADPH oxidase (NOX)-2, and endothelial nitric oxide synthase (eNOS). Triiodothyronine (T3) expression was also analyzed by IHC. Results: In GO adipocytes, the expression of catalase was reduced, whereas that of HIF-1α was strongly increased. A decreased local T3 supply was associated with DIO3 upregulation. The low expression of Cav-1 in GO adipocytes was associated not only with low expression of Glut-4 but also with an increased expression of NOX-2 and active eNOS phosphorylated on serine 1177. Conclusions: Cav-1 and DIO3, both sensitive to hypoxia and to the increase of HIF-1α, play a pivotal role in the oxidative stress in GO adipocytes. DIO3 regulates the cellular supply of T3, which is essential for the cell homeostasis. Cav-1 determines the cellular glucose supply through Glut-4 and regulates the activity of NOX-2 generating superoxide anions and that of eNOS generating nitric oxide (NO).

A cadaveric model of anterior compartment leg syndrome: Subcutaneous minimally invasive fasciotomy versus open fasciotomy.

OBJECTIVE: Because of disabling sequelae of open fasciotomy in anterior compartment syndrome (ACS) of the leg, we wanted to describe and validate a cadaveric model of ACS. We hypothesized that, first, anterior compartment syndrome (ACS) could be reproduced in cadaveric leg and, second, fasciotomy without complete skin incision could lower the intramuscular pressure (IMP) in an equivalent range to complete dermatofasciotomy. MATERIALS AND METHODS: Lower limb ACS was reproduced by progressive injection of physiologic serum in the anterior compartment of 23 fresh frozen cadaveric legs with monitoring of IMP, in order to reach a maximal stabilised IMP higher than 30mmHg. Subcutaneous minimally invasive fasciotomy was performed on 14 legs through 5 transversal mini-incisions of the skin (2cm) along the axis from the tibial tuberosity to the posterior aspect of the lateral malleolus. Standard open fasciotomy of the anterior compartment was performed on the remaining 9 legs as control. IMP was measured after the skin incisions and after every fasciotomy through skin incisions in the first group and after skin and fascia incisions in the control group. RESULTS: A maximal IMP of 43±2mmHg was obtained by injection of 177±9ml physiologic serum into the anterior compartment of the leg. In the control open fasciotomy group, the skin incision alone did not lower IMP significantly, whereas fasciotomy lowered IMP to 10±1mmHg, which is statistically different from maximal IMP (p<0.001). In the subcutaneous fasciotomy group, complete fasciotomy lowered significantly the IMP to 11±4mmHg (p<0.001), without statistical difference with the control group. DISCUSSION: This cadaveric model is effective to reproduce the hyperpressure encountered in ACS. In this model, IMP release after fasciotomy is as efficient through minimally invasive subcutaneous incision as with control open fasciotomy. This in vitro technique appears as an attractive alternative treatment in anterior compartment syndrome of the leg. It should be tested in the other compartments of the leg and its in vivo feasibility in acute conditions has to be clarified. LEVEL OF EVIDENCE: III, control laboratory study.

Perfusion-decellularization of human ear grafts enables ECM-based scaffolds for auricular vascularized composite tissue engineering.

INTRODUCTION: Human ear reconstruction is recognized as the emblematic enterprise in tissue engineering. Up to now, it has failed to reach human applications requiring appropriate tissue complexity along with an accessible vascular tree. We hereby propose a new method to process human auricles in order to provide a poorly immunogenic, complex and vascularized ear graft scaffold. METHODS: 12 human ears with their vascular pedicles were procured. Perfusion-decellularization was applied using a SDS/polar solvent protocol. Cell and antigen removal was examined by histology and DNA was quantified. Preservation of the extracellular matrix (ECM) was assessed by conventional and 3D-histology, proteins and cytokines quantifications. Biocompatibility was assessed by implantation in rats for up to 60 days. Adipose-derived stem cells seeding was conducted on scaffold samples and with human aortic endothelial cells whole graft seeding in a perfusion-bioreactor. RESULTS: Histology confirmed cell and antigen clearance. DNA reduction was 97.3%. ECM structure and composition were preserved. Implanted scaffolds were tolerated in vivo, with acceptable inflammation, remodeling, and anti-donor antibody formation. Seeding experiments demonstrated cell engraftment and viability. CONCLUSIONS: Vascularized and complex auricular scaffolds can be obtained from human source to provide a platform for further functional auricular tissue engineered constructs, hence providing an ideal road to the vascularized composite tissue engineering approach. STATEMENT OF SIGNIFICANCE: The ear is emblematic in the biofabrication of tissues and organs. Current regenerative medicine strategies, with matrix from donor tissues or 3D-printed, didn't reach any application for reconstruction, because critically missing a vascular tree for perfusion and transplantation. We previously described the production of vascularized and cell-compatible scaffolds, from porcine ear grafts. In this study, we ---- applied findings directly to human auricles harvested from postmortem donors, providing a perfusable matrix that retains the ear's original complexity and hosts new viable cells after seeding. This approach unlocks the ability to achieve an auricular tissue engineering approach, associated with possible clinical translation.

Face Graft Scaffold Production in a Rat Model.

BACKGROUND: As a route toward face bioengineering, the authors previously reported the production of a complete scaffold by perfusion-decellularization of a porcine ear subunit graft and partial recellularization. To extend the scaffold to the whole face and to down-scale it, they applied their findings to a rodent hemifacial graft model. METHODS: After the animals were killed, seven full-thickness rat hemiface grafts were harvested with the common carotid artery and the external jugular vein as a pedicle, and cannulated. Grafts were decellularized by a detergent-based protocol: either by perfusion through the common carotid artery, or by mechanical agitation. After decellularization, samples were analyzed for DNA quantification and histology by hematoxylin and eosin, Masson trichrome, Sirius red, or Safranin O staining. Vascular tree patency was assessed by microangiographic computed tomography after contrast injection. Cell-friendly extracellular matrix was assessed by seeding of human adipose-derived stem cells and vital staining after 7 days of culture. RESULTS: Decellularization was effective in both groups, with a cell clearance at all levels, with the exception of cartilage areas in the agitation-treated groups. Microscopic assessment found a well-preserved extracellular matrix in both groups. Vascular contrast was found in all regions of the scaffolds. After the animals were killed, seeded cells were found viable and well distributed on all scaffolds. CONCLUSIONS: The authors successfully decellularized face grafts in a rodent model, with a preserved vascular tree. Perfusion-decellularization led to better and faster results compared with mechanical agitation but is not mandatory in this model. The rat face is an interesting scaffold model for further recellularization studies, in the final goal of human face bioengineering.

Nose and Lip Graft Variants: A Subunit Anatomical Study.

BACKGROUND: In the field of vascularized composite tissue allotransplantation, the surgical design of facial subunit grafts is an evolving concept. The purpose of the present article is to study the possibility of dividing the historical nose and lip face transplant into several morphologic and functional subunit grafts, depending on their respective supply. METHODS: This study was conducted in 20 adult cadavers. The facial artery and its branches were dissected bilaterally in 16 fresh and four embalmed heads. Nasolabial perfusion was assessed by selective injection of methylene blue and eosin (n = 2) or India ink (n = 2) in the superior labial and distal facial arteries. Dynamic perfusion through the distal facial artery was illustrated by fluoroscopy (n = 3). Three nose-upper lip grafts were harvested and injected with barium sulfate for microangiography computed tomographic analysis. Finally, three isolated nasal and bilabial grafts were procured and their vascular patency assessed by fluoroscopy. RESULTS: The distal facial artery can perfuse the entire nose, septum, and upper lip, without any contribution of the superior labial artery. A dense anastomotic network indeed exists between the respective distal rami of both vessels. Furthermore, the exclusion of the superior labial artery from the harvested nasal subunit allowed safe bilabial subunit procurement, from the same specimen. CONCLUSIONS: The authors' results demonstrate the feasibility of harvesting nasal and labial subunits, in an isolated or a combined manner. These results can find applications in subunit autologous replantation, allotransplantation, allogenic face partial retransplantation, and the emerging field of vascularized composite tissue engineering.

Decellularization of the Porcine Ear Generates a Biocompatible, Nonimmunogenic Extracellular Matrix Platform for Face Subunit Bioengineering.

OBJECTIVE: The purpose of this study was to assess whether perfusion-decellularization technology could be applied to facial grafts. BACKGROUND: Facial allotransplantation remains an experimental procedure. Regenerative medicine techniques allow fabrication of transplantable organs from an individual's own cells, which are seeded into extracellular matrix (ECM) scaffolds from animal or human organs. Therefore, we hypothesized that ECM scaffolds also can be created from facial subunits. We explored the use of the porcine ear as a clinically relevant face subunit model to develop regenerative medicine-related platforms for facial bioengineering. METHODS: Porcine ear grafts were decellularized and histologic, immunologic, and cell culture studies done to determine whether scaffolds retained their 3D framework and molecular content; were biocompatible in vitro and in vivo, and triggered an anti-MHC immune response from the host. RESULTS: The cellular compartment of the porcine ear was completely removed except for a few cartilaginous cells, leaving behind an acellular ECM scaffold; this scaffold retained its complex 3D architecture and biochemical components. The framework of the vascular tree was intact at all hierarchical levels and sustained a physiologically relevant blood pressure when implanted in vivo. Scaffolds were biocompatible in vitro and in vivo, and elicited no MHC immune response from the host. Cells from different types remained viable and could even differentiate at the scale of a whole-ear scaffold. CONCLUSIONS: Acellular scaffolds were produced from the porcine ear, and may be a valuable platform to treat facial deformities using regenerative medicine approaches.

Bioengineering a Human Face Graft: The Matrix of Identity.

OBJECTIVE: During the last decade, face allotransplantation has been shown to be a revolutionary reconstructive procedure for severe disfigurements. However, offer to patients remains limited due to lifelong immunosuppression. To move forward in the field, a new pathway in tissue engineering is proposed. BACKGROUND: Our previously reported technique of matrix production of a porcine auricular subunit graft has been translated to a human face model. METHODS: 5 partial and 1 total face grafts were procured from human fresh cadavers. After arterial cannulation, the specimens were perfused using a combined detergent/polar solvent decellularization protocol. Preservation of vascular patency was assessed by imaging, cell and antigen removal by DNA quantification and histology. The main extracellular matrix proteins and associated cytokines were evaluated. Lip scaffolds were cultivated with dermal, muscle progenitor and endothelial cells, either on discs or in a bioreactor. RESULTS: Decellularization was successful in all facial grafts within 12 days revealing acellular scaffolds with full preservation of innate morphology. Imaging demonstrated a preservation of the entire vascular tree patency. Removal of cells and antigens was confirmed by reduction of DNA and antigen markers negativation. Microscopic evaluation revealed preservation of tissue structures as well as of major proteins. Seeded cells were viable and well distributed within all scaffolds. CONCLUSIONS: Complex acellular facial scaffolds were obtained, preserving simultaneously a cell-friendly extracellular matrix and a perfusable vascular tree. This step will enable further engineering of postmortem facial grafts, thereby offering new perspectives in composite tissue allotransplantation.

Single-Artery Human Ear Graft Procurement: A Simplified Approach.

In the field of experimental facial vascularized composite tissue allotransplantation, a human auricular subunit model, pedicled on both superficial temporal and posterior auricular arteries, was described. Clinical cases of extensive auricular replantation, however, suggested that a single artery could perfuse the entire flap. In this study, variants of this single-pedicle approach have been studied, aiming to develop a more versatile replantation technique, in which the question of venous drainage has also been addressed. For arterial perfusion study, the authors harvested 11 auricular grafts, either on a single superficial temporal artery pedicle (n = 3) or a double superficial temporal and posterior auricular artery pedicle (n = 8). The authors then proceeded to selective barium injections, in the superficial temporal, posterior auricular, or both superficial temporal and posterior auricular arteries. Arteriograms were acquired with a micro-computed tomographic scan and analyzed on three-dimensionally reconstructed images. Venous drainage was investigated in eight hemifaces, carefully dissected after latex injection. Observations showed a homogenous perfusion of the whole auricle in all arterial graft variants. Venous drainage was highly variable, with either a dominant superficial temporal vein (37.5 percent), dominant posterior auricular vein (12.5 percent), or co-dominant trunks (50 percent). The authors demonstrated that auricular subunit vascularized composite tissue allotransplantation can be performed on a single artery, relying on the dynamic intraauricular anastomoses between superficial temporal artery and posterior auricular branches. Potentially, this vascular versatility is prone to simplify the subunit harvest and allows various strategies for pedicle selection. Venous drainage, however, remains inconstant and thus the major issue when considering auricular transplantation. CLINICAL QUESTION/LEVEL OF EVIDENCE: Therapeutic, V.

Repair of calvarial bone defects in mice using electrospun polystyrene scaffolds combined with ß-TCP or gold nanoparticles.

Non-biodegradable porous polystyrene (PS) scaffolds, composed of microfibers, have been prepared by electrospinning for the reconstruction of large bone defects. PS microfibers were prepared by incorporating ß-TCP grains inside the polymer or grafting gold nanoparticles surface functionalized with mercaptosuccinic acid. Cytocompatibility of the three types of scaffolds (PS, ß-TCP-PS and Au-PS) was studied by seeding human mesenchymal stem cells. Biocompatibility was evaluated by implanting ß-TCP-PS and Au-PS scaffolds into a critical size (4mm) calvarial defect in mice. Calvaria were taken 6, 9, and 12 weeks after implantation; newly formed bone and cellular response was analyzed by microcomputed tomography (microCT) and histology. ß-TCP-PS scaffolds showed a significantly higher cell proliferation in vitro than on PS or Au-PS alone; clearly, the presence of ß-TCP grains improved cytocompatibility. Biocompatibility study in the mouse calvaria model showed that ß-TCP-PS scaffolds were significantly associated with more newly-formed bone than Au-PS. Bone developed by osteoconduction from the defect margins to the center. A dense fibrous connective tissue containing blood vessels was identified histologically in both types of scaffolds. There was no inflammatory foci nor giant cell in these areas. AuNPs aggregates were identified histologically in the fibrosis and also incorporated in the newly-formed bone matrix. Although the different types of PS microfibers appeared cytocompatible during the in vitro experiment, they appeared biotolerated in vivo since they induced a fibrotic reaction associated with newly formed bone.

In vitro evaluation of peri-implantitis treatment modalities on Saos-2osteoblasts.

OBJECTIVES: Mechanical treatment of the implant surface through surgical approach is recommended to control peri-implantitis. Few conclusive data exist about the physical and chemical properties of treated titanium surfaces and their biocompatibility towards osteoblasts. This in vitro study aimed to evaluate four clinical procedures: plastic curette, air-abrasive device (Perio-Flow(®) ), titanium brush (Ti-Brush(®) ) and implantoplasty in terms of biocompatibility and osteogenic effect when cultured with Saos2. MATERIALS AND METHODS: Titanium disks were treated with plastic curette, air-abrasive device (Perio-Flow(®) ), titanium brush (Ti-Brush(®) ) and implantoplasty. Their surface microtopography (SEM), chemical composition (EDX) and wettability were evaluated. After seeding with Saos-2, cell morphology (1 h, 24 h), viability (three and 6 days) and alkaline phosphatase (ALP), osteoprotegerin (OPG) and osteocalcin (OCN) production (7 days) were analyzed. RESULTS: Control, plastic curette, Perio-Flow(®) and Ti-Brush(®) groups presented complex microstructures including craters and micropits, whereas the implantoplasty group appeared much smoother (SEM). Titanium, oxygen, aluminium and carbon were identified as the main components in all disks with a decrease in the percentage of oxygen, carbon and an increase in the percentage of titanium in the implantoplasty group (EDX). Implantoplasty disks were also significantly more hydrophilic than the other ones, whose surfaces appeared hydrophobic. Saos-2 showed no morphological difference at 1 h. At 24 h, they appeared round shaped in all groups, except the implantoplasty group where the cells appeared stretched and elongated. Viability was similar in all groups, but significantly higher in the Perio-Flow(®) than the control group at day six. ALP, OPG and OCN protein expression at 7 days was similar in all groups. CONCLUSIONS: Although implantoplasty was the only modality to modify the titanium surface morphology, composition and wettability, all treatment modalities promoted ALP, OPG and OCN production and appeared as valid approaches in terms of biocompatibility.

Oxidative Stress and Upregulation of Antioxidant Proteins, Including Adiponectin, in Extraocular Muscular Cells, Orbital Adipocytes, and Thyrocytes in Graves' Disease Associated with Orbitopathy.

BACKGROUND: Graves' orbitopathy (GO) is the main extrathyroidal manifestation associated with Graves' disease (GD). It is characterized by reduced eye motility due to an increased volume of orbital fat and/or of extraocular muscles (EOMs) infiltrated by fibrosis and adipose tissue. The pathogenetic mechanisms leading to fibrosis and adipogenesis are mainly based on the interaction between orbital fibroblasts and immune cells (lymphocytes and mast cells) infiltrating the GO EOMs. METHODS: Analysis of the morphological status, oxidative stress (OS), and antioxidant defenses in the orbital muscular cells and adipocytes in GO patients compared with controls was conducted. RESULTS: Both cell types are affected by OS, as shown by the increased expression of 4-hydroxynonenal, which leads to apoptosis in muscular cells. However, the EOMs and the adipocytes possess antioxidant defenses (peroxiredoxin 5 and catalase) against the OS, which are also upregulated in thyrocytes in GD. The expression of adiponectin (ApN) and proliferator-activated receptor gamma (PPARγ) is also increased in GO muscular cells and adipocytes. OS and antioxidant proteins expression are correlated to the level of blood antithyrotropin receptor antibodies (TSHR-Ab). CONCLUSION: Even when TSHR-Ab level is normalized, OS and antioxidant protein expression is high in EOM muscular cells and adipocytes in GO compared with controls. This justifies a supplementation with antioxidants in active as well as chronic GO patients. Orbital muscular cells are also the sources of PPARγ and ApN, which have direct or indirect local protective effects against OS. Modulation of these proteins could be considered as a future therapeutic approach for GO.

Gingiva laser welding: preliminary study on an ex vivo porcine model.

OBJECTIVE: The use of lasers to fuse different tissues has been studied for 50 years. As none of these experiments concerned the oral soft tissues, our objective was to assess the feasibility of laser gingiva welding. MATERIALS AND METHODS: Porcine full-thickness gingival flaps served to prepare calibrated samples in the middle of which a 2 cm long incision was closed, either by conventional suture or by laser tissue welding (LTW). To determine the irradiation conditions yielding the best tensile strength, 13 output power values, from 0.5 to 5 W, delivered either at 10 Hz or in continuous wave mode, were tested on six indocyanine green (ICG) concentrations, from 8% to 13% (588 samples). Then, some samples served to compare the tensile strength between the laser welded and the sutured gingiva; the other samples were histologically processed in order to evaluate the thermal damage extent. The temperature rise during the LTW was measured by thermocouples. Another group of 12 samples was used to measure the temperature elevation by thermal camera. RESULTS: In the laser welding groups, the best tensile strength (p<0.05) was yielded by the 9% ICG saline solution (117 mM) at 4.5 W, 10 Hz, and a fluence of 31.3 kJ/cm(2). The apposition strength revealed no statistically significant difference (p<0.05) between the sutured and the laser welded gingiva at 4.5 W, 10 Hz, and 9% ICG solution. The mean temperature was 74±5.4°C at the upper surface and 42±8.9°C at the lower surface. The damaged zone averaged 333 µm at the upper surface. CONCLUSIONS: The 808 nm diode laser associated with ICG can achieve oral mucosa LTW, which is conceivable as a promising technique of gingival repair.

Critical size bone defect reconstruction by an autologous 3D osteogenic-like tissue derived from differentiated adipose MSCs.

For critical size bone defects and bone non-unions, bone tissue engineering using osteoblastic differentiated adipose mesenchymal stem cells (AMSCs) is limited by the need for a biomaterial to support cell transplantation. An osteoblastic three-dimensional autologous graft made of AMSCs (3D AMSC) was developed to solve this issue. This autograft was obtained by supplementing the osteoblastic differentiation medium with demineralized bone matrix. Two surgical models were developed to assess the potential of this 3D osteogenic AMSC autograft. A four-level spinal fusion using polyetheretherketone cages was designed in six pigs to assess the early phase of ossification (8-12 weeks postimplantation). In each pig, four groups were compared: cancellous bone autograft, freeze-dried irradiated cancellous pig bone, 3D AMSC, and an empty cage. A critical size femoral defect (n = 4, bone non-union confirmed 6 months postoperatively) was used to assess the 3D AMSCs' ability to achieve bone fusion. Pigs were followed by CT scan and explanted specimens were analyzed for bone tissue remodeling by micro-CT scan, micro-radiography, and histology/histomorphometry. In the spine fusion model, bone formation with the 3D AMSC was demonstrated by a significant increase in bone content. In the critical-size femoral defect model, the 3D AMSC achieved new bone formation and fusion in a poorly vascularized fibrotic environment. This custom-made 3D osteogenic AMSC autograft is a therapeutic solution for bone non-unions and for critical-size defects.

Influence of sodium hypochlorite on Er:YAG laser-irradiated dentin and its effect on the quality of adaptation of the composite restoration margins.

OBJECTIVE: The aims of this in vitro study were to evaluate: (1) the influence of 5% NaOCl application on Er:YAG-irradiated dentin; and (2) its effect on the quality of adaptation of the composite restoration margins. BACKGROUND DATA: Previous research has shown that Er:YAG dentin irradiation produces a thermally affected tissue layer that results in lower bond strength than that of nonirradiated dentin. The removal of this thermally-affected layer may enhance the quality of dentin bonding. MATERIALS AND METHODS: Forty-nine caries-free extracted human molars were transversely sectioned in order to totally expose the dentin. Four standardized cavities were created on the dentinal surface of each molar. First, two cavities were irradiated with Er:YAG laser (2.94 nm): 150 mJ, 10 Hz, variable square pulse (VSP) mode (100 µsec), beam diameter=0.9 mm, speed of irradiation=1 mm/sec, 20% air and 20% water. Then, one of irradiated cavities and one of nonirradiated cavities were treated for 30 sec with 5% NaOCl solution. Finally, they went through a standard bonding treatment for composite restoration, etching, bonding, and composite filling. We obtained four groups of cavities: (1) one control group of nonirradiated cavities not pretreated with NaOCl; (2) one group of nonirradiated cavities, pretreated with NaOCl; (3) one group of irradiated cavities, not pretreated with NaOCl; and (4) one group of irradiated cavities, pretreated with NaOCl. All samples were subjected to thermocycling. Every cavity was immersed into a 0.5% solution of methylene blue. The percentage of dye penetration (microleakage) in the composite-dentin interface was evaluated. Six molars were analyzed by scanning electron microscope. RESULTS: Dye infiltration depth was significantly reduced in irradiated cavities treated with 5% NaOCl solution. CONCLUSIONS: The application of a 5% NaOCl solution on Er:YAG irradiated cavities can significantly improve the marginal quality of composite bonding.