Micro-CT Evaluation Research Articles

Australian Dental Research Foundation Special Research Supplement 2017

BRONJ was considered to be due to direct suppression of bone remodelling and cytotoxic effects of zoledronic acid (ZA) on bone and oral mucosal cells. However, recent literature suggests that suppressed angiogenesis contributes significantly to the pathogenesis of this condition. This in vitro and in vivo study examined the effect of locally delivered VEGF-hydrogel and local gene expression in rat BRONJ model. In vitro release study with 200 ng of rat VEGF-165 (Biovision) loaded into 500 lL hydrogel (Hystem-HP) prior to cross-linking and incubated in release media that was sampled and quantified using ELISA, over 28-days. In vivo, a BRONJ model using Sprague-Dawley rats was established by weekly intraperitoneal injection of ZA (three weeks), followed by extraction of maxillary first two molars and creation of a 5 mm defect which received either VEGF-containing hydrogel, HA gel alone or no gel. ZA was continued for four weeks post-surgery, prior to sacrifice. Gross examination, micro-CT evaluation and histological assessment was conducted to evaluate the effect of VEGF local delivery. Histological assessment involved quantification of osteonecrotic areas, total vascularity (von Willebrand Factor, vWF immunostaining) and specifically, the microvessel density (CD105 immunostaining). Furthermore, to assess molecular level changes during bone and soft tissue healing, gene expression analysis was conducted on the bone and gingival tissue samples from defect area.

Micro-CT and nano-CT analysis of filling quality of three different endodontic sealers.

To investigate voids in different root canal sealers using micro-CT and nano-CT, and to explore the feasibility of using nano-CT for quantitative analysis of sealer filling quality. 30 extracted mandibular central incisors were randomly assigned into three groups according to the applied root canal sealers (Total BC Sealer, Sure Seal Root, AH Plus) by the single cone technique. Subsequently, micro-CT and nano-CT were performed to analyse the incidence rate of voids, void fraction, void volume and their distribution in each sample. Micro-CT evaluation showed no significant difference among sealers for the incidence rate of voids or void fraction in the whole filling materials (p > 0.05), whereas a significant difference was found between AH Plus and the other two sealers using nano-CT (p < 0.05). All three sealers presented less void volume in the apical third; however, higher void volumes were observed in the apical and coronal thirds in AH Plus using micro-CT (p < 0.05), while nano-CT results displayed higher void volume in AH Plus among all the sealers and regions (p < 0.05). Bioactive sealers showed higher root filling rate, lower incidence rate of voids, void fraction and void volume than AH Plus under nano-CT analysis, when round root canals were treated by the single cone technique. The disparate results suggest that the higher resolution of nano-CT have a greater ability of distinguishing internal porosity, and therefore suggesting the potential use of nano-CT in quantitative analysis of filling quality of sealers.

Injectable and thermosensitive TGF-\u03b21-loaded PCEC hydrogel system for in vivo cartilage repair

Chondral defects pose a great challenge for clinicians to manage owing to the limited capacity for self-healing. Various traditional approaches have been adopted for the repair of these defects with unsatisfactory results. Cartilage tissue engineering techniques have emerged as promising strategies to enhance regeneration and overcome these traditional shortcomings. The cell-homing based technique is considered the most promising owing to its unique advantages. Thermosensitive hydrogels have been applied as scaffolds for biomedical applications with smart sol–gel response for altering environmental temperature. Transforming growth factor (TGF)-β1 is considered to be capable of promoting chondrogenesis. In this study, a novel TGF-β1-loaded poly(ε-caprolactone)–poly(ethylene glycol)–poly(ε-caprolactone) (PCEC) hydrogel was fabricated using simple procedures. Hydrogel characterization, rheological testing, component analysis, and assessment of sol–gel transition, in vitro degradation, and TGF-β1 release confirmed that this material possesses a porous microstructure with favorable injectability and sustained drug release. Full-thickness cartilage defects were induced on rat knees for in vivo cartilage repair for eight weeks. Micro-CT and histological evaluation provided further evidence of the optimal capacity of this novel hydrogel for cartilage regeneration with respect to that of other methods. Moreover, our results demonstrated that the cell-free hydrogel is thermosensitive, injectable, biodegradable, and capable of in vivo cartilage repair and possesses high potential and benefits for acellular cartilage tissue engineering and clinical application in the future.

In Vitro and in Vivo Study of Poly(Lactic⁻co⁻Glycolic) (PLGA) Membranes Treated with Oxygen Plasma and Coated with Nanostructured Hydroxyapatite Ultrathin Films for Guided Bone Regeneration Processes.

The novelty of this study is the addition of an ultrathin layer of nanostructured hydroxyapatite (HA) on oxygen plasma modified poly(lactic–co–glycolic) (PLGA) membranes (PO2) in order to evaluate the efficiency of this novel material in bone regeneration. Methods: Two groups of regenerative membranes were prepared: PLGA (control) and PLGA/PO2/HA (experimental). These membranes were subjected to cell cultures and then used to cover bone defects prepared on the skulls of eight experimental rabbits. Results: Cell morphology and adhesion of the osteoblasts to the membranes showed that the osteoblasts bound to PLGA were smaller and with a lower number of adhered cells than the osteoblasts bound to the PLGA/PO2/HA membrane (p < 0.05). The PLGA/PO2/HA membrane had a higher percentage of viable cells bound than the control membrane (p < 0.05). Both micro-CT and histological evaluation confirmed that PLGA/PO2/HA membranes enhance bone regeneration. A statistically significant difference in the percentage of osteoid area in relation to the total area between both groups was found. Conclusions: The incorporation of nanometric layers of nanostructured HA into PLGA membranes modified with PO2 might be considered for the regeneration of bone defects. PLGA/PO2/HA membranes promote higher osteosynthetic activity, new bone formation, and mineralisation than the PLGA control group.

Micro-CT Evaluation of Root and Canal Morphology of Mandibular First Premolars with Radicular Grooves.

The aim of this study was to evaluate morphological features of 70 single-rooted mandibular first premolars with radicular grooves (RG) using micro-CT technology. Teeth were scanned and evaluated regarding the morphology of the roots and root canals as well as length, depth and percentage frequency location of the RG. Volume, surface area and Structure Model Index (SMI) of the canals were measured for the full root length. Two-dimensional parameters and frequency of canal orifices were evaluated at 1, 2, and 3 mm levels from the apical foramen. The number of accessory canals, the dentinal thickness, and cross-sectional appearance of the canal at different root levels were also recorded. Expression of deep grooves was observed in 21.42% of the sample. Mean lengths of root and RG were 13.43 mm and 8.5 mm, respectively, while depth of the RG ranged from 0.75 to 1.13 mm. Mean canal volume, surface area and SMI were 10.78 mm3, 58.51 mm2, and 2.84, respectively. Apical delta was present in 4.35% of the sample and accessory canals were observed mostly at the middle and apical thirds. Two-dimensional parameters indicated an oval-shaped cross-sectional appearance of the root canal with a high percentage frequency of canal divisions (87.15%). Canal configuration type V (58.57%) was the most prevalent. C-shaped configuration was observed in 13 premolars (18.57%), whereas dentinal thickness ranged from 1.0 to 1.31 mm. Radicular grooves in mandibular first premolars was associated with the occurrence of several anatomical complexities, including C-shaped canals and divisions of the main root canal.

Magnetic hyperthermia enhance the treatment efficacy of peri-implant osteomyelitis

BackgroundWhen bacteria colony persist within a biofilm, suitable drugs are not yet available for the eradication of biofilm-producing bacteria. The aim of this study is to study the effect of magnetic nano-particles-induced hyperthermia on destroying biofilm and promoting bactericidal effects of antibiotics in the treatment of osteomyelitis.MethodsSixty 12-weeks-old male Wistar rats were used. A metallic 18G needle was implanted into the bone marrow cavity of distal femur after the injection of Methicillin-sensitive Staphylococcus aureus (MSSA). All animals were divided into 5 different treatment modalities. The microbiological evaluation, scanning electron microscope examination, radiographic examination and then micro-CT evaluation of peri-implant bone resorption were analyzed.ResultsThe pathomorphological characteristics of biofilm formation were completed after 40-days induction of osteomyelitis. The inserted implants can be heated upto 75 °C by magnetic heating without any significant thermal damage on the surrounding tissue. We also demonstrated that systemic administration of vancomycin [VC (i.m.)] could not eradicate the bacteria; but, local administration of vancomycin into the femoral canal and the presence of magnetic nanoparticles hyperthermia did enhance the eradication of bacteria in a biofilm-based colony. In these two groups, the percent bone volume (BV/TV: %) was significantly higher than that of the positive control.ConclusionsFor the treatment of chronic osteomyelitis, we developed a new modality to improve antibiotic efficacy; the protection effect of biofilms on bacteria could be destroyed by magnetic nanoparticles-induced hyperthermia and therapeutic effect of systemic antibiotics could be enhanced.

Sustained release of recombinant human bone morphogenetic protein-2 combined with stromal vascular fraction cells in promoting posterolateral spinal fusion in rat model

To observe the effect of stromal vascular fraction cells (SVFs) from rat fat tissue combined with sustained release of recombinant human bone morphogenetic protein-2 (rhBMP-2) in promoting the lumbar fusion in rat model. SVFs were harvested from subcutaneous fat of bilateral inguinal region of 4-month-old rat through the collagenase I digestion. The sustained release carrier was prepared via covalent bond of the rhBMP-2 and β-tricalcium phosphate (β-TCP) by the biominetic apatite coating process. The sustained release effect was measured by BCA method. Thirty-two rats were selected to establish the posterolateral lumbar fusion model and were divided into 4 groups, 8 rats each group. The decalcified bone matrix (DBX) scaffold+PBS, DBX scaffold+rhBMP-2/β-TCP sustained release carrier, DBX scaffold+SVFs, and DBX scaffold+rhBMP-2/β-TCP sustained release carrier+SVFs were implanted in groups A, B, C, and D respectively. X-ray films, manual spine palpation, and high-resolution micro-CT were used to evaluate spinal fusion at 8 weeks after operation; bone mineral density (BMD) and bone volume fraction were analyzed; the new bone formation was evaluated by HE staining and Masson's trichrome staining, osteocalcin (OCN) was detected by immunohistochemical staining. The cumulative release amount of rhBMP-2 was about 40% at 2 weeks, indicating sustained release effect of rhBMP-2; while the control group was almost released within 2 weeks. At 8 weeks, the combination of manual spine palpation, X-ray, and micro-CT evaluation showed that group D had the strongest bone formation (100%, 8/8), followed by group B (75%, 6/8), group C (37.5%, 3/8), and group A (12.5%, 1/8). Micro-CT analysis showed BMD and bone volume fraction were significantly higher in group D than groups A, B, and C ( P<0.05), and in group B than groups A and C ( P<0.05). HE staining, Masson's trichrome staining, and immunohistochemistry staining for OCN staining exhibited a large number of cartilage cells with bone matrix deposition, and an active osteogenic process similar to the mineralization of long bones in group D. The bone formation of group B was weaker than that of group D, and there was no effective new bone formation in groups A and C. The combination of sustained release of rhBMP-2 and freshly SVFs can significantly promote spinal fusion in rat model, providing a theoretical basis for further clinical applications.

Rhein and polydimethylsiloxane functionalized carbon/carbon composites as prosthetic implants for bone repair applications

A major issue in bone tissue engineering is the selection of biocompatible materials for implants, to reduce unwanted inflammatory reactions and promote cell adhesion. Bone tissue growth on suitable biomedical implants can shorten recovery and hospitalization after surgery. Therefore, a method to improve tissue–implant integration and healing would be of scientific and clinical interest. In this work, we permeated polydimethylsiloxane (PDMS) into carbon/carbon (C/C) composites (PDMS–C/C) and then coated it with 4,5-dihydroxyanthraquinone-2-carboxylic acid (rhein) to create rhein–PDMS–C/C to increase its biocompatibility and reduce the occurrence of inflammatory reactions. We measured in vitro adhesion and proliferation of MC3T3-E1 cells and bacteria to evaluate the biocompatibility and antimicrobial properties of C/C, PDMS–C/C, and rhein–PDMS–C/C. In vivo, x-ray and micro-CT evaluation three, six and nine weeks after surgery revealed that rhein–PDMS–C/C was more effective than PDMS–C/C and C/C composite in terms of antibacterial activity, cell adhesion and tissue growth. Compared with C/C and PDMS–C/C, rhein–PDMS–C/C could be suitable for clinical applications for bone tissue engineering.

New nano-hydroxyapatite in bone defect regeneration: A histological study in rats

Many types of bone substitute materials are available on the market. Researchers are refining new bone substitutes to make them comparable to autologous grafting materials in treatment of bone defects. The purpose of the study was to evaluate the osseoconductive potential and bone defect regeneration in rat calvaria bone defects treated with new synthetic nano-hydroxyapatite. The study was performed on 30 rats divided into 5 equal groups. New preproduction of experimental nano-hydroxyapatite material by NanoSynHap (Poznań, Poland) was tested and compared with commercially available materials. Five mm critical size defects were created and filled with the following bone grafting materials: 1) Geistlich Bio-Oss®; 2) nano-hydroxyapatite+β-TCP; 3) nano-hydroxyapatite; 4) nano-hydroxyapatite+collagen membrane. The last group served as controls without any augmentation. Bone samples from calvaria were harvested for histological and micro-ct evaluation after 8 weeks. New bone formation was observed in all groups. Histomorphometric analysis revealed an amount of regenerated bone between 34.2 and 44.4% in treated bone defects, whereas only 13.0% regenerated bone was found in controls. Interestingly, in group 3, no significant particles of the nano-HA material were found. In contrast, residual bone substitute material could be detected in all other test groups. Micro-CT study confirmed the results of the histological examinations. The new nano-hydroxyapatite provides comparable results to other grafts in the field of bone regeneration.

Cell Sheets of Co-cultured Endothelial Progenitor Cells and Mesenchymal Stromal Cells Promote Osseointegration in Irradiated Rat Bone

Irradiated bone has a greater risk of implant failure than nonirradiated bone. The purpose of this study was to investigate the influence of cell sheets composed of co-cultured bone marrow mesenchymal stromal cells (BMSCs) and endothelial progenitor cells (EPCs) on implant osseointegration in irradiated bone. Cell sheets (EPCs, BMSCs or co-cultured EPCs and BMSCs) were wrapped around titanium implants to make cell sheet-implant complexes. The co-cultured group showed the highest osteogenic differentiation potential in vitro, as indicated by the extracellular matrix mineralization and the expression of osteogenesis related genes at both mRNA and protein levels. The co-cultured cells promoted ectopic bone formation as indicated by micro-computed tomography (Micro-CT) and histological analysis. In the irradiated tibias of rats, implants of the co-cultured group showed enhanced osseointegration by Micro-CT evaluation and histological observation. Co-cultured EPCs and BMSCs also up-regulated the expression of osteogenesis related genes in bone fragments in close contact with implants. In conclusion, cell sheets of co-cultured EPCs and BMSCs could promote osseous healing around implants and are potentially useful to improve osseointegration process for patients after radiotherapy.

Synchrotron radiation μCT and histology evaluation of bone-to-implant contact

The purpose of this study was to evaluate bone-to-implant contact (BIC) in two-dimensional (2D) histology compared to high-resolution three-dimensional (3D) synchrotron radiation micro computed tomography (SR micro-CT). High spatial resolution, excellent signal-to-noise ratio, and contrast establish SR micro-CT as the leading imaging modality for hard X-ray microtomography. Using SR micro-CT at voxel size 5 μm in an experimental goat mandible model, no statistically significant difference was found between the different treatment modalities nor between recipient and reconstructed bone. The histological evaluation showed a statistically significant difference between BIC in reconstructed and recipient bone (p < 0.0001). Further, no statistically significant difference was found between the different treatment modalities which we found was due to large variation and subsequently due to low power. Comparing histology and SR micro-CT evaluation a bias of 5.2% was found in reconstructed area, and 15.3% in recipient bone. We conclude that for evaluation of BIC with histology and SR micro-CT, SR micro-CT cannot be proven more precise than histology for evaluation of BIC, however, with this SR micro-CT method, one histologic bone section is comparable to the 3D evaluation. Further, the two methods complement each other with knowledge on BIC in 2D and 3D.

ST1926 Attenuates Steroid-Induced Osteoporosis in Rats by Inhibiting Inflammation Response.

Steroid, also known as glucocorticoid, induced osteonecrosis of the femoral head in young adults, which has been a challenging disorder for the frequent incidence of collapse of femoral head, leading to dysfunction of hip joint and impairing life quality of human. Bioavailable and less toxic synthetic retinoids, such as the atypical adamantyl retinoid ST1926, have been developed and investigated in clinical trials for many diseases. Serum lipid-related indicators were assessed to elucidate the role of ST1926 in regulating lipid metabolism. Microfocal computed tomography (Micro-CT) was included to explore the effects of ST1926 treatment on microstructure and bone mass. Then, the role of ST1926 treatment in regulating osteoclast differentiation was also evaluated in vivo and in vitro. In addition, alkaline phosphatase (ALP), osteoprotegerin (OPG), bone alkaline phosphatase (BAP) and bone morphogenic protein (BMP) expression in serum and cells were detected at protein or mRNA levels. The ratio of empty lacuna in the bone tissue samples was significantly low in ST1926-treated groups than in the control group. Micro-CT evaluation suggested that ST1926 treatment could ameliorate the microstructure of the bone and up-regulate bone mineral density in steroid-induced rats. Moreover, ST1926 treatment suppressed osteoclast differentiation and promoted bone formation markers. Also, OPG, ALP, and Wnt3a/β-catenin down-regulation as well as inflammation up-regulation could be reversed by ST1926 administration through NFκB inhibition. Hence, ST1926 may inhibit steroid-induced osteoporosis and promote steroid-induced bone remodeling by regulating the Wnt3a/β-catenin/NFκB signaling pathway. J. Cell. Biochem. 118: 2072-2086, 2017. © 2017 Wiley Periodicals, Inc.

Micro-CT evaluation of asymmetrical ovine intervertebral disc height loss from surgical approach.

The primary goal of this study is to clearly define and evaluate new intervertebral disc height parameters in analysing the morphological pathology of disc degeneration for application in damage model and regeneration therapy development, as well as applying traditional variables to 3-D characterization methods. A posterolateral surgical approach was used to induce disc degeneration in an ovine model. At 12-months post-operation, sheep vertebral segments were removed and characterized using micro-CT to evaluate disc height parameters in regard to injury localization. Statistically significant differences between the disc height loss of the left and right side of the disc, consistent with the lateral surgical approach used were seen using the modified average disc height method by Dabbs et al. However, convexity index and the newly proposed Cross Tilt Index did not conclusively demonstrate a difference. Two-dimensional morphological evaluations can be applied in 3-D to provide a more complete picture of disc height loss for injury models. New 3-D parameters that are tailored to the type of surgical approach used should be investigated, with the 9-point system described herein providing a useful basis for derived values. Additionally, the surgical approach chosen when artificially injuring the disc can result in asymmetrical degeneration, as indicated by uneven disc height loss.

Comparison of nanoparticular hydroxyapatite pastes of different particle content and size in a novel scapula defect model

Nanocrystalline hydroxyapatite (HA) has good biocompatibility and the potential to support bone formation. It represents a promising alternative to autologous bone grafting, which is considered the current gold standard for the treatment of low weight bearing bone defects. The purpose of this study was to compare three bone substitute pastes of different HA content and particle size with autologous bone and empty defects, at two time points (6 and 12 months) in an ovine scapula drillhole model using micro-CT, histology and histomorphometry evaluation. The nHA-LC (38% HA content) paste supported bone formation with a high defect bridging-rate. Compared to nHA-LC, Ostim® (35% HA content) showed less and smaller particle agglomerates but also a reduced defect bridging-rate due to its fast degradation The highly concentrated nHA-HC paste (48% HA content) formed oversized particle agglomerates which supported the defect bridging but left little space for bone formation in the defect site. Interestingly, the gold standard treatment of the defect site with autologous bone tissue did not improve bone formation or defect bridging compared to the empty control. We concluded that the material resorption and bone formation was highly impacted by the particle-specific agglomeration behaviour in this study.

Efficacy and safety of human mesenchymal stromal cells in healing of critical-size bone defects in immunodeficient rats.

To evaluate the preclinical efficacy and safety of human mesenchymal stem cells (hMSC) rapidly expanded in growth medium for clinical use with human serum and recombinant growth factors, we conducted a controlled, randomized trial of plasma clots with hMSC vs. plasma clots only in critical segmental femoral defects in rnu/rnu immunodeficient rats. X-ray, microCT and histomorphometrical evaluation were performed at 8 and 16 weeks. MSC were obtained from healthy volunteers and patients with lymphoid malignancy. Human MSC survived in the defect for the entire duration of the trial. MSC from healthy volunteers, in contrast to hMSC from cancer patients, significantly improved bone healing at 8, but not 16 weeks. However, at 16 weeks, hMSC significantly improved vasculogenesis in residual defect. We conclude that hMSC from healthy donors significantly contributed to the healing of bone defects at 8 weeks and to the vascularisation of residual connective tissue for up to 16 weeks. We found the administration of hMSC to be safe, as no adverse reaction to human cells at the site of implantation and no evidence of migration of hMSC to distant organs was detected.

Improvement of Bone and Dental Phenotype of Murine Hypophosphatasia Mediated by a Single Injection of Lentiviral Gene Therapy

Background: Alkaline phosphatase has 4 isozymes, tissue non-specific alkaline phosphatase (TNAP), placental alkaline phosphatase (PLAP), intestinal alkaline phosphatase and germ-cell alkaline phosphatase. Hypophosphatasia (HPP) is an inherited skeletal disease caused by mutations of the gene encoding TNAP. Although TNAP is expressed in various tissues, the primary HPP symptoms appear in bones and teeth. The clinical severity of HPP varies widely from the most severe (perinatal, infantile and childhood) to the mildest forms (adult, and odonto-hypophosphatasia). We reported that gene therapy using a single injection of lentiviral vector expressing bone-targeted TNAP (TNAP-D10) is effective in preventing all the skeletal of HPP in TNAP knockout (Alpl–/–) mice as the model of infantile HPP. Objective: In this study we focus on evaluating the efficacy of treatment with gene therapy on the bone and teeth using TNAP-D10 and also we investigate the feasibility of gene therapy using bone-targeted PLAP (PLAP-D10). Methods and Findings: We used Alpl–/–mice that develop skeletal disease at postnatal days 6-8 mimicking the infantile form of human HPP. We injected 100 μl of lentiviral vectors harboring TNALP-D10 (5.0 × 107 TU) or PLAP-D10 (5.0 × 107 TU) to 1-day-old Alpl–/– mice via the jugular vein. We performed histological analysis and micro-CT evaluation on bone and mandible of Alpl–/– mice. The alveolar bone, enamel and dentin defects were corrected on treated Alpl–/– mice by this treatment. Additionally the long bone growth rates (LGR) of long bones were encouraged on treated Alpl–/– mice compared with untreated mice. Conclusions: These results indicate that the bone-targeted TNAP treatment mediated by lentivirus can correct not only the bone disorder but also the dental symptoms in Alpl–/–. This study also shows that PLAP-D10 can potentially be used to correct HPP disease.

Multifunctions of dual Zn/Mg ion co-implanted titanium on osteogenesis, angiogenesis and bacteria inhibition for dental implants.

In order to enhance the rapid osseointegration and long-term survival of dental implants, various works on titanium surface modification have been carried out. However, only improving osteogenic activity of implants is not enough, because angiogenesis and bacteria inhibition are also very important for dental implants. In the present study, a novel dental implant material-dual Zn/Mg ion co-implanted titanium (Zn/Mg-PIII) was developed, which was found to have superior osteoinductivity, pro-angiogenic effects and inhibitory effects against oral anaerobes. Furthermore, synergistic effects of Zn/Mg ions on osteogenic differentiation of rBMSCs and the possible mechanism were discovered. In addition, rapid osseointegration and sustained biomechanical stability are greatly enhanced by Zn/Mg-PIII implants, which may have the potential application in dental implantation in the future. We believe this paper may be of particular interest to the readers.

Lethal lung hypoplasia and vascular defects in mice with conditional Foxf1 overexpression.

ABSTRACTFOXF1 heterozygous point mutations and genomic deletions have been reported in newborns with the neonatally lethal lung developmental disorder, alveolar capillary dysplasia with misalignment of pulmonary veins (ACDMPV). However, no gain-of-function mutations in FOXF1 have been identified yet in any human disease conditions. To study the effects of FOXF1 overexpression in lung development, we generated a Foxf1 overexpression mouse model by knocking-in a Cre-inducible Foxf1 allele into the ROSA26 (R26) locus. The mice were phenotyped using micro-computed tomography (micro-CT), head-out plethysmography, ChIP-seq and transcriptome analyses, immunohistochemistry, and lung histopathology. Thirty-five percent of heterozygous R26-Lox-Stop-Lox (LSL)-Foxf1 embryonic day (E)15.5 embryos exhibit subcutaneous edema, hemorrhages and die perinatally when bred to Tie2-cre mice, which targets Foxf1 overexpression to endothelial and hematopoietic cells. Histopathological and micro-CT evaluations revealed that R26Foxf1; Tie2-cre embryos have immature lungs with a diminished vascular network. Neonates exhibited respiratory deficits verified by detailed plethysmography studies. ChIP-seq and transcriptome analyses in E18.5 lungs identified Sox11, Ghr, Ednrb, and Slit2 as potential downstream targets of FOXF1. Our study shows that overexpression of the highly dosage-sensitive Foxf1 impairs lung development and causes vascular abnormalities. This has important clinical implications when considering potential gene therapy approaches to treat disorders of FOXF1 abnormal dosage, such as ACDMPV.

Abstract: Radiological and Histological Assessment in Perforator Flap Microvasculature Following Pretreatment with Topical Negative Pressure Therapy: An Experimental Rat Model

BACKGROUND: Surgical delay and ischemic preconditioning have been traditionally used to precondition flaps to render flaps more vascular and resistant to changes in their microcirculation. Negative pressure wound therapy (NPWT) has been extensively in clinical practice and associated with angiogenesis and accelerated wound healing. This experimental model evaluated topical negative pressure as a mechanism for non-invasive preconditioning for perforator flap microvascularity and perfusion. METHODS: Two gluteal perforator flaps (matched control and intervention) were designed on sixteen 400g Sprague-Dawley male rats. NPWT was applied to the flap area directly continuously at -125mmHg for 7 days, after which the rats were divided into two principal groups. Group A (n=8) underwent 4D computed tomographic and angiography (CTA) with a body volume perfusion protocol after NPWT and euthanized. Group B (n=8), control and intervention flaps were raised, isolated on a single pedicle and laid back down and monitored for a further 7 days. Group B flaps were assessed using laser-assisted indocyanine fluorescence angiography before surgery, following flap harvest and at 7 days prior to euthanasia. Half of all rats in each group were analyzed with Micro-CT to assess the microvasculature. All paired specimens were assessed histologically with H&E and immunohistochemistry (IHC). RESULTS: There was a 17% increase in CT tissue perfusion in skin treated with NPWT versus matched controls (P=0.001). LA-ICGFA demonstrated higher perfusion following NPWT treatment (P=0.006), however no significant difference immediate post flap harvest (P=0.19) but a difference was seen 7 days postoperatively (P=0.03). Micro-CT evaluation showed an increase in average vessel volume (%) from 0.005 in control to 0.009 in the NPWT flaps (P=0.04).H&E analysis showed significant difference in the epidermal thickness (P<0.001), but comparable dermal thickness (P=0.34).Quantitative analysis of CD31 IHC demonstrated a mean area fraction percentage of 4.30 and 2.68 in the NPWT and control flaps respectively (P=0.002).There was partial necrosis in the control (n=3) and NPWT flaps (N=1), however this was <5% in the NPWT flap. CONCLUSION: We present novel multimodal approaches using static and dynamic imaging and histological assessment to provide a proof of concept on the use of NPWT for non-invasive conditioning of flaps. The study provides the basis for further investigation and clinical studies with potential for direct translation into clinical practice. DISCLOSURE/FINANCIAL SUPPORT:None of the authors has a financial interest in any of the products, devices, or drugs mentioned in this manuscript.

The use of a cartilage decellularized matrix scaffold for the repair of osteochondral defects: the importance of long-term studies in a large animal model

To investigate the effect of decellularized cartilage-derived matrix (CDM) scaffolds, by itself and as a composite scaffold with a calcium phosphate (CaP) base, for the repair of osteochondral defects. It was hypothesized that the chondral defects would heal with fibrocartilaginous tissue and that the composite scaffold would result in better bone formation. After an 8-week pilot experiment in a single horse, scaffolds were implanted in eight healthy horses in osteochondral defects on the medial trochlear ridge of the femur. In one joint a composite CDM-CaPscaffold was implanted (+P), in the contralateral joint a CDM only (-P) scaffold. After euthanasia at 6 months, tissues were analysed by histology, immunohistochemistry, micro-CT, biochemistry and biomechanical evaluation. The 8-week pilot showed encouraging formation of bone and cartilage, but incomplete defect filling. At 6 months, micro-CT and histology showed much more limited filling of the defect, but the CaP component of the+P scaffolds was well integrated with the surrounding bone. The repair tissue was fibrotic with high collagen type I and low type II content and with no differences between the groups. There were also no biochemical differences between the groups and repair tissue was much less stiff than normal tissue (P<0.0001). The implants failed to produce reasonable repair tissue in this osteochondral defect model, although the CaP base in the-P group integrated well with the recipient bone. The study stresses the importance of long-term invivo studies to assess the efficacy of cartilage repair techniques.