Measurements and morphometric landmarks of the human spinal cord: A cadaveric study.

The topographical neuroanatomy of the human spinal cord (SC) is currently based on the adjacent vertebrae. This morphometric study sought to develop a dataset allowing for statistical analysis of human SC segment characteristics. Overall, 32 human SCs were dissected (18 female and 14 male donors), and individual SC segments were identified. Anterior and posterior lengths, thicknesses and widths were measured by two examiners. Statistical analyses included t-tests, as well as intraclass and Pearson's correlation coefficients. The SC length was significantly shorter in females than males. The cranial (C4) and caudal (T1/T2) limits of the cervical enlargement, along with its maximal width (C6-C7), were identified by combining widths and thicknesses of the segments. The thoracic region, T2 to T12, could be identified using segments widths and thicknesses values. The length of the lumbosacral region, from segments L2 to S5, was particularly stable, independently of SC length and sex. The lumbar enlargement was characterized by a thickness increase between the segments L2 and S1 which reached its maximum at the level of L3, L4, and L5, whereas the width was not significantly increased. From the S2 to S5 segments, width and thickness were equal, with both decreasing of 1 mm per segment. The morphometrical analysis of 32 human SCs provided a dataset allowing for statistical analysis of segmental measures with significant results. A combined approach mostly using widths and thicknesses provided landmarks of potential interest for the localization of SC segments in a clinical MRI setting.

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.

Biomechanical, Microstructural and Material Properties of Tendon and Bone in the Young Oim Mice Model of Osteogenesis Imperfecta.

Osteogenesis imperfecta (OI) is a genetic disorder of connective tissue characterized by low bone mass and spontaneous fractures, as well as extra-skeletal manifestations, such as dental abnormalities, blue sclera, hearing loss and joint hypermobility. Tendon ruptures have been reported in OI patients. Here, we characterized the biomechanical, structural and tissue material properties of bone and tendon in 5-week-old female osteogenesis imperfecta mice (oim), a validated model of severe type III OI, and compared these data with age- and sex-matched WT littermates. Oim tendons were less rigid and less resistant than those of WT mice. They also presented a significantly higher rate of pentosidine, without significant modification of enzymatic crosslinking. The oim bones were less resistant and avulsion fractures were evident at high tendinous stress areas. Alterations of trabecular and cortical bone microarchitectures were noticed in young female oim. Bone tissue material properties were also modified, with a less mature and more mineralized matrix in association with lower collagen maturity. Our data suggest that the tendon-to-bone unit is affected in young oim mice, which could explain tendon ruptures and bone fragility observed in OI patients.

A Comparison of Genicular Nerve Blockade With Corticosteroids Using Either Classical Anatomical Targets vs Revised Targets for Pain and Function in Knee Osteoarthritis: A Double-Blind, Randomized Controlled Trial.

OBJECTIVE: Compare the effectiveness of genicular nerve blockade (GNB) using classical anatomical targets (CT) versus revised targets (RT) in patients suffering from chronic knee osteoarthritis pain. DESIGN: Double-blinded randomized controlled trial. SETTING: Pain medicine center of a teaching hospital. METHODS: We randomly assigned 55 patients with chronic knee osteoarthritis pain to receive a GNB (using a fluid mixture of 2 mL: lidocaine 1% + 20 mg triamcinolone) with either classical targets (CT-group, n = 28) or revised targets (RT-group, n = 27). Numeric rating pain scale (NRS), Oxford knee score (OKS), Western Ontario and McMaster Universities osteoarthritis index score (WOMAC), Quantitative analgesic questionnaire (QAQ) and global perceived effects were assessed at baseline, and at 1-hour, 24-hours, 1, 4, and 12 weeks post-intervention. RESULTS: The RT-group showed greater reduction in NRS mean score at 1-hour post-intervention (2.4 ± 2.1 vs 0.4 ± 0.9, 95% confidence interval (CI) [.0-.8] vs [1.6-3.2], P < .001). The proportion of patients achieving more than 50% knee pain reduction was higher in the RT-group at each follow up interval, yet these differences were statistically significant only at 1-hour post intervention (82.1% [95% CI = 63.1-93.9] vs 100% [95% CI = 97.2-100] P = .02). Both protocols resulted in significant pain reduction and joint function improvement up to 12 weeks post-intervention. CONCLUSIONS: The revised technique allowed more pain relief as well as greater proportion of successful responders at 1-hour post intervention. The large volume injected during therapeutic GNB could have compensated the lack of precision of the classical anatomical targets, mitigating differences in outcomes between both techniques.

Anatomical study of the descending genicular artery and implications for image-guided interventions for knee pain.

INTRODUCTION: The descending genicular artery (DGA) has recently been mentioned as accompanying some nerves in the medial aspect of the knee joint. This could be clinically relevant as the arteries could serve as landmarks for accurate nerve capture during ultrasound-guided nerve blockade or ablation. The aim of this cadaveric study was to investigate the anatomical distribution of the DGA, assess the nerves running alongside its branches, and discuss the implications for regional anesthesia and knee pain interventions. METHODS: We dissected the femoral artery (FA) all along its course to identify the origin of the DGA, from which we carefully dissected all branches, in 27 fresh-frozen human specimens. Simultaneously, we systematically dissected the nerves supplying the medial aspect of the knee from proximally to distally and identified those running alongside the branches of the DGA. The surrounding anatomical landmarks were identified and measurements were recorded. RESULTS: The DGA was found in all specimens, arising from the FA 130.5 ± 17.5 mm (mean ± SD) proximally to the knee joint line. Seven distribution patterns of the DGA were observed. We found three consistent branches from the DGA running alongside their corresponding nerves at the level of the medial aspect of the knee: the artery of the superior-medial genicular nerve, the artery of the infrapatellar branch of the saphenous nerve, and the saphenous branch of the DGA. CONCLUSION: The consistent arteries and surrounding landmarks found in this study could help to improve the capture of the targeted nerves during ultrasound-guided interventions.

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.

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.

Sclerostin-Antibody Treatment Decreases Fracture Rates in Axial Skeleton and Improves the Skeletal Phenotype in Growing oim/oim Mice.

In osteogenesis imperfecta (OI), vertebrae brittleness causes thorax deformations and leads to cardiopulmonary failure. As sclerostin-neutralizing antibodies increase bone mass and strength in animal models of osteoporosis, their administration in two murine models of severe OI enhanced the strength of vertebrae in growing female Crtap-/- mice but not in growing male Col1a1Jrt/+ mice. However, these two studies ignored the impact of antibodies on spine growth, fracture rates, and compressive mechanical properties. Here, we conducted a randomized controlled trial in oim/oim mice, an established model of human severe OI type III due to a mutation in Col1a2. Five-week-old female WT and oim/oim mice received either PBS or sclerostin antibody (Scl-Ab) for 9 weeks. Analyses included radiography, histomorphometry, pQCT, microcomputed tomography, and biomechanical testing. Though it did not modify vertebral axial growth, Scl-Ab treatment markedly reduced the fracture prevalence in the pelvis and caudal vertebrae, enhanced osteoblast activity (L4), increased cervico-sacral spine BMD, and improved the lumbosacral spine bone cross-sectional area. Scl-Ab did not impact vertebral height and body size but enhanced the cortical thickness and trabecular bone volume significantly in the two Scl-Ab groups. At lumbar vertebrae and tibial metaphysis, the absolute increase in cortical and trabecular bone mass was higher in Scl-Ab WT than in Scl-Ab oim/oim. The effects on trabecular bone mass were mainly due to changes in trabecular number at vertebrae and in trabecular thickness at metaphyses. Additionally, Scl-Ab did not restore a standard trabecular network, but improved bone compressive ultimate load with more robust effects at vertebrae than at metaphysis. Overall, Scl-Ab treatment may be beneficial for reducing vertebral fractures and spine deformities in patients with severe OI.

Distribution of sensory nerves supplying the knee joint capsule and implications for genicular blockade and radiofrequency ablation: an anatomical study.

BACKGROUND: Despite their emerging therapeutic relevance, there are many discrepancies in anatomical description and terminology of the articular nerves supplying the human knee capsule. This cadaveric study aimed to determine their origin, trajectory, relationship and landmarks for therapeutic purpose. METHODS: We dissected 21 lower limbs from 21 cadavers, to investigate the anatomical distribution of all the articular nerves supplying the knee joint capsule. We identified constant genicular nerves according to their anatomical landmarks at their entering point to knee capsule and inserted Kirschner wires through the nerves in underlying bone at those target points. Measurements were taken, and both antero-posterior and lateral radiographs were obtained. RESULTS: The nerve to vastus medialis, saphenous nerve, anterior branch of obturator nerve and a branch from sciatic nerve provide substantial innervation to the medial knee capsule and retinaculum. The sciatic nerve and the nerve to the vastus lateralis supply sensory innervation to the supero-lateral aspect of the knee joint while the fibular nerve supplies its infero-lateral quadrant. Tibial nerve and posterior branch of obturator nerve supply posterior aspect of knee capsule. According to our findings, five constant genicular nerves with accurate landmarks could be targeted for therapeutic purpose. CONCLUSION: The pattern of distribution of sensitive nerves supplying the knee joint capsule allows accurate and safe targeting of five constant genicular nerves for therapeutic purpose. This study provides robust anatomical foundations for genicular nerve blockade and radiofrequency ablation.

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.

Specific branches of hypoglossal nerve to genioglossus muscle as a potential target of selective neurostimulation in obstructive sleep apnea: anatomical and morphometric study.

PURPOSE: To determine the ideal implantation site for selective tongue neurostimulation in obstructive sleep apnea, anatomy of the distal branching of the hypoglossal nerve (HGN) was revisited. METHODS: The HGN distal course and intramuscular distribution to the tongue muscles were studied in 17 embalmed and 5 fresh heads (age 60-98, BMI 20-35). Medial branches supplying selectively the genioglossus (GG) muscle were identified. Then, the distinct bundles entering the oblique (GGo) and horizontal (GGh) parts of the GG were located. Morphometric data were compared to similar measurements made on MRI sections from 12 patients (age 43-71, BMI 18-47). RESULTS: The key facts relevant to optimize stimulation and electrode design are the following: the mean width of both GG muscles in embalmed and fresh cadavers was 20.7 ± 2.9 and 21.4 ± 5 mm, respectively; it is significantly (p < 0.05) superior to the MRI value of 18.26 ± 2.0 mm. Selective nervous branches for GGh and GGo were located at 52 ± 8% of hyoid bone-mandibular symphysis distance and at 5.8 ± 1.1 mm from the inferior border of the GG muscle. The surface of stimulation is a 4.4 ± 1.1 × 6.9 ± 3.8 mm ellipse. CONCLUSIONS: According to our observations, the optimal selective or supra-selective stimulation of the tongue protractor muscles can be performed on the lateral surface of the GG at roughly equal distance between the mandibular symphysis and the hyoid bone, at a depth of about 0.6 cm above the GG lower border.

Isolated bilateral transverse agenesis of the distal segments of the lower limbs at the level of the knee joint in a human fetus.

Congenital limb anomalies occur in Europe with a prevalence of 3.81/1,000 births and can have a major impact on patients and their families. The present study concerned a female fetus aborted at 23 weeks of gestation because she was affected by non-syndromic bilateral absence of the zeugopod (leg) and autopod (foot). Autopsy of the aborted fetus, X-ray imaging, MRI, and histochemical analysis showed that the distal extremity of both femurs was continued by a cartilage-like mass, without joint cavitation. Karyotype was normal. Moreover, no damaging variant was detected by exome sequencing. The limb characteristics of the fetus, which to our knowledge have not yet been reported in humans, suggest a developmental arrest similar to anomalies described in chicks following surgical experiments on the apical ectodermal ridge of the lower limbs.

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.

Microradiographic and histological evaluation of the bone-screw and bone-plate interface of orthodontic miniplates in patients.

OBJECTIVES: To describe the tissue reactions at the bone-titanium interface of orthodontic miniplates in humans. MATERIALS AND METHODS: Forty-two samples, consisting of tissue fragments attached or not to miniplates or their fixation screws, were collected from 24 orthodontic patients treated with miniplate anchorage, at the time of removal of their miniplates. The samples were embedded in methylmethacrylate and cut into undecalcified sections which were submitted to microradiographic analysis. The sections were also stained and examined under ordinary light. RESULTS: Three types of reactions were observed both on the histological sections and on the microradiographs. 1. The majority of the stable miniplates were easy to remove (34/42). The tissue samples collected consisted mainly in mature lamellar bone with some medullary spaces containing blood vessels, 2. two screws were highly osseointegrated and required the surgeon to remove them by trephining (2/42). They were surrounded by bone tissue which extended to the miniplate. The histological features were similar to the previous group, though the bone-screw contact was higher, and 3. in six samples obtained after unstable miniplate removal during the treatment, we observed either some woven bone trabeculae or loose connective tissue, without any histological sign of inflammation. LIMITATIONS AND CONCLUSION: For evident ethical reasons, our data were limited by the size of the tissue fragments and the limited number of patients and variety of clinical presentations. The healing reactions consisted mainly in mature lamellar bone tissue sparsely in contact with the screw or the miniplate, with signs of a moderate remodelling activity.

Collagen molecular organization preservation in human fascia lata and periosteum after tissue engineering.

Large bone defect regeneration remains a major challenge for orthopedic surgeons. Tissue engineering approaches are therefore emerging in order to overcome this limitation. However, these processes can alter some of essential native tissue properties such as intermolecular crosslinks of collagen triple helices, which are known for their essential role in tissue structure and function. We assessed the persistence of extracellular matrix (ECM) properties in human fascia lata (HFL) and periosteum (HP) after tissue engineering processes such as decellularization and sterilization. Harvested from cadaveric donors (N = 3), samples from each HFL and HP were decellularized following five different chemical protocols with and without detergents (D1-D4 and D5, respectively). D1 to D4 consisted of different combinations of Triton, Sodium dodecyl sulfate and Deoxyribonuclease, while D5 is routinely used in the institutional tissue bank. Decellularized HFL tissues were further gamma-irradiated (minimum 25 kGy) in order to study the impact of sterilization on the ECM. Polarized light microscopy (PLM) was used to estimate the thickness and density of collagen fibers. Tissue hydration and content of hydroxyproline, enzymatic crosslinks, and non-enzymatic crosslinks (pentosidine) were semi-quantified with Raman spectroscopy. ELISA was also used to analyze the maintenance of the decorin (DCN), an important small leucine rich proteoglycan for fibrillogenesis. Among the decellularization protocols, detergent-free treatments tended to further disorganize HFL samples, as more thin fibers (+53.7%) and less thick ones (-32.6%) were recorded, as well as less collagen enzymatic crosslinks (-25.2%, p = 0.19) and a significant decrease of DCN (p = 0.036). GAG content was significantly reduced in both tissue types after all decellularization protocols. On the other hand, HP samples were more sensitive to the D1 detergent-based treatments, with more disrupted collagen organization and greater, though not significant loss of enzymatic crosslinks (-37.4%, p = 0.137). Irradiation of D5 HFL samples, led to a further and significant loss in the content of enzymatic crosslinks (-29.4%, p = 0.037) than what was observed with the decellularization process. Overall, the results suggest that the decellularization processes did not significantly alter the matrix. However, the addition of a gamma-irradiation is deleterious to the collagen structural integrity of the tissue.

Postnatal growth defect in mice upon persistent Hoxa2 expression in the chondrogenic cell lineage.

Hoxa2 is a homeotic transcription factor, which is downregulated once chondrogenic differentiation is initiated. We previously generated a transgenic mouse model, which turns Hoxa2 on in cells expressing Collagen II A1, i.e. in cells entering chondrogenesis. As a consequence, mice display a general embryonic delay of ossification and then a postnatal growth defect. Col2a1-Cre mice were crossed with an inducible ß-actin driven Hoxa2 transgene. Spines, vertebrae and limbs were measured and skeletal elements were studied by X-ray, microCT, pQCT, TEM, western-blotting, histomorphometry and immunohistochemistry. Mice expressing Hoxa2 in chondrogenic cells feature a proportionate short stature phenotype with a severe lordosis, which appeared significant from postnatal day 4. Analysis of both cartilage and bone development in affected embryos and mice from birth till P35 did not reveal any major defect in histogenesis, except a reduced number of chondrocytes in the vertebral anlage at E13.5. In conclusion, the sustained expression of Hoxa2 in the chondrocyte lineage is characterized by a proportionate short stature resulting from skeletal growth defect. The indepth analysis of cartilage and bone histogenesis points towards an initial deficit in cell mobilization to enter chondrogenesis.

Molecular study of a Hoxa2 gain-of-function in chondrogenesis: a model of idiopathic proportionate short stature.

In a previous study using transgenic mice ectopically expressing Hoxa2 during chondrogenesis, we associated the animal phenotype to human idiopathic proportionate short stature. Our analysis showed that this overall size reduction was correlated with a negative influence of Hoxa2 at the first step of endochondral ossification. However, the molecular pathways leading to such phenotype are still unknown. Using protein immunodetection and histological techniques comparing transgenic mice to controls, we show here that the persistent expression of Hoxa2 in chondrogenic territories provokes a general down-regulation of the main factors controlling the differentiation cascade, such as Bapx1, Bmp7, Bmpr1a, Ihh, Msx1, Pax9, Sox6, Sox9 and Wnt5a. These data confirm the impairment of chondrogenic differentiation by Hoxa2 overexpression. They also show a selective effect of Hoxa2 on endochondral ossification processes since Gdf5 and Gdf10, and Bmp4 or PthrP were up-regulated and unmodified, respectively. Since Hoxa2 deregulation in mice induces a proportionate short stature phenotype mimicking human idiopathic conditions, our results give an insight into understanding proportionate short stature pathogenesis by highlighting molecular factors whose combined deregulation may be involved in such a disease.

Beneficial Effects of Zoledronic Acid on Tendons of the Osteogenesis Imperfecta Mouse (Oim).

Osteogenesis imperfecta (OI) is a genetic disorder of connective tissue characterized by spontaneous fractures, bone deformities, impaired growth and posture, as well as extra-skeletal manifestations. Recent studies have underlined an impairment of the osteotendinous complex in mice models of OI. The first objective of the present work was to further investigate the properties of tendons in the osteogenesis imperfecta mouse (oim), a model characterized by a mutation in the COL1A2 gene. The second objective was to identify the possible beneficial effects of zoledronic acid on tendons. Oim received a single intravenous injection of zoledronic acid (ZA group) at 5 weeks and were euthanized at 14 weeks. Their tendons were compared with those of untreated oim (oim group) and control mice (WT group) by histology, mechanical tests, western blotting and Raman spectroscopy. The ulnar epiphysis had a significantly lower relative bone surface (BV/TV) in oim than WT mice. The tendon of the triceps brachii was also significantly less birefringent and displayed numerous chondrocytes aligned along the fibers. ZA mice showed an increase in BV/TV of the ulnar epiphysis and in tendon birefringence. The tendon of the flexor digitorum longus was significantly less viscous in oim than WT mice; in ZA-treated mice, there was an improvement of viscoelastic properties, especially in the toe region of stress-strain curve, which corresponds to collagen crimp. The tendons of both oim and ZA groups did not show any significant change in the expression of decorin or tenomodulin. Finally, Raman spectroscopy highlighted differences in material properties between ZA and WT tendons. There was also a significant increase in the rate of hydroxyproline in the tendons of ZA mice compared with oim ones. This study highlighted changes in matrix organization and an alteration of mechanical properties in oim tendons; zoledronic acid treatment had beneficial effects on these parameters. In the future, it will be interesting to better understand the underlying mechanisms which are possibly linked to a greater solicitation of the musculoskeletal system.

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.