Effect of endothelial differentiated adipose-derived stem cells on vascularity and osteogenesis in poly(D,L-lactide) scaffolds in vivo.

Prevascularization of engineered bony constructs can potentially improve in vivo viability. However, the effect of endothelial cells on osteogenesis is unknown when placed in poly(D,L-lactide) (PLA) scaffolds alone. Adipose-derived stem cells (ASCs) have the ability to differentiate into both osteoblasts and endothelial cells by culture in specific media. We hypothesized that ASC-derived endothelial cells would improve vascularity with minimal contribution to bone formation when placed in scaffold alone. ASCs were successfully differentiated into endothelial cells (ASC-Endo) and osteoblasts (ASC-Osteo) using media supplemented with vascular endothelial growth factor and bone morphogenic protein 2, respectively. Tissue-engineered constructs were created with PLA matrices containing no cells (control), undifferentiated ASCs (ASCs), osteogenic-differentiated ASCs (ASC-Osteo), or endothelial differentiated ASCs (ASC-Endo), and these constructs were evaluated in critical-size Lewis rat calvarial defect model (n = 34). Eight weeks after implantation, the bone volume and microvessel population of bony constructs were evaluated by micro-computed tomography analysis and histologic staining. Bone volumes for ASCs and ASC-Osteo constructs, 0.7 and 0.91 mm(3), respectively, were statistically greater than that for ASC-Endo, 0.28 mm(3) (P < 0.05). There was no statistical difference between the PLA control (0.5 mm(3)) and ASC-Endo (0.28 mm(3)) constructs in bone formation. The percent area of microvessels within constructs was highest in the ASC-Endo group, although it did not reach statistical significance (0.065). Prevascularization of PLA scaffold with ASC-Endo cells will not increase bone formation by itself but may be used as a cell source for improving vascularization and potentially improving existing osteoblast function.

Broad-spectrum CRISPR-mediated inhibition of SARS-CoV-2 variants and endemic coronaviruses in vitro.

A major challenge in coronavirus vaccination and treatment is to counteract rapid viral evolution and mutations. Here we demonstrate that CRISPR-Cas13d offers a broad-spectrum antiviral (BSA) to inhibit many SARS-CoV-2 variants and diverse human coronavirus strains with >99% reduction of the viral titer. We show that Cas13d-mediated coronavirus inhibition is dependent on the crRNA cellular spatial colocalization with Cas13d and target viral RNA. Cas13d can significantly enhance the therapeutic effects of diverse small molecule drugs against coronaviruses for prophylaxis or treatment purposes, and the best combination reduced viral titer by over four orders of magnitude. Using lipid nanoparticle-mediated RNA delivery, we demonstrate that the Cas13d system can effectively treat infection from multiple variants of coronavirus, including Omicron SARS-CoV-2, in human primary airway epithelium air-liquid interface (ALI) cultures. Our study establishes CRISPR-Cas13 as a BSA which is highly complementary to existing vaccination and antiviral treatment strategies.

In vivo performance of bilayer hydroxyapatite scaffolds for bone tissue regeneration in the rabbit radius.

The objective of this study was to investigate the in vivo biomechanical performance of bone defects implanted with novel bilayer hydroxyapatite (HAp) scaffolds that mimic the cortical and cancellous organization of bone. The scaffolds maintained architectural continuity in a rabbit radius segmental defect model and were compared to an untreated defect group (negative control) and autologous bone grafts (positive control). Micro-CT evaluations indicated total bone and scaffold volume in the experimental group was significantly greater than the defect group but lesser than the autologous bone graft treatment. The flexural toughness of the scaffold and the autograft groups was significantly greater than the flexural toughness of the defect group. Interestingly, the absolute density of the bone mineral as well as calcium to phosphorus (Ca/P) ratio in that mineral for the scaffold and autograft contralateral bones was significantly higher than those for the defect contralaterals suggesting that the scaffolds contributed to calcium homeostasis. It was concluded from this study that new bone regenerated in the bilayer HAp scaffolds was comparable to the empty defects and while the HAp scaffolds provided significant increase in modulus when compared to empty defect and their flexural toughness was comparable to autografts after 8 weeks of implantation.

Characterization of craniomaxillofacial battle injuries sustained by United States service members in the current conflicts of Iraq and Afghanistan.

PURPOSE: To characterize and describe the craniomaxillofacial (CMF) battlefield injuries sustained by US Service Members in Operation Iraqi Freedom and Operation Enduring Freedom. PATIENTS AND METHODS: The Joint Theater Trauma Registry was queried from October 19, 2001, to December 11, 2007, for CMF battlefield injuries. The CMF injuries were identified using the "International Classification of Diseases, Ninth Revision, Clinical Modification" codes and the data compiled for battlefield injury service members. Nonbattlefield injuries, killed in action, and return to duty cases were excluded. RESULTS: CMF battlefield injuries were found in 2,014 of the 7,770 battlefield-injured US service members. In the 2,014 injured service members were 4,783 CMF injuries (2.4 injuries per soldier). The incidence of CMF battlefield injuries by branch of service was Army, 72%; Marines, 24%; Navy, 2%; and Air Force, 1%. The incidence of penetrating soft-tissue injuries and fractures was 58% and 27%, respectively. Of the fractures, 76% were open. The location of the facial fractures was the mandible in 36%, maxilla/zygoma in 19%, nasal in 14%, and orbit in 11%. The remaining 20% were not otherwise specified. The primary mechanism of injury involved explosive devices (84%). CONCLUSIONS: Of the injured US service members, 26% had injuries to the CMF region in the Operation Iraqi Freedom/Operation Enduring Freedom conflicts during a 6-year period. Multiple penetrating soft-tissue injuries and fractures caused by explosive devices were frequently seen. Increased survivability because of body armor, advanced battlefield medicine, and the increased use of explosive devices is probably related to the elevated incidence of CMF battlefield injuries. The current use of "International Classification of Diseases, Ninth Revision, Clinical Modification" codes with the Joint Theater Trauma Registry failed to characterize the severity of facial wounds.

An experimental design to study adipocyte stem cells for reconstruction of calvarial defects.

Autogenous osteogenic differentiated rat adipose stromal cells (ASCs) were used to repopulate cadaveric processed bone in a critical calvarial defect model to improve bone healing as compared with the standard treatment of cadaveric bone implantation alone. Forty-two skeletally mature Sprague-Dawley rats were randomized to 5 groups including bone graft only, bone/osteogenic differentiated ASCs, fibrin glue/osteogenic differentiated ASCs, and bone/fibrin glue/osteogenic differentiated ASCs; 2 animal calvarias were left empty. Adipose stromal cells were isolated from the inguinal fat pad of rats and differentiated into osteogenic cells, verified using von Kossa and alkaline phosphatase staining, and osteocalcin immunohistochemistry. These cells were added to sterilized, 8-mm cadaveric bone graft disks and placed back into calvarial defect for 6 weeks. The rat calvarias then underwent bone density analysis and histology. Intact cells were observed in the bone graft of the bone/osteogenic differentiated ASC group only. Islands of bone were seen in the bone-graft-only group, the bone/osteogenic differentiated ASC group, and the bone/fibrin/osteogenic differentiated ASC group. The bone-graft-only group and bone/osteogenic differentiated ASC group were similar in bone mineral density (1397 +/- 184.5 vs 1365 +/- 160.4). The bone/fibrin/osteogenic differentiated ASC group density was less than the bone and bone/osteogenic differentiated ASC groups at 835.2 +/- 319.5 (P < 0.001). Allograft bone scaffolds with autogenous osteogenic differentiated ASCs showed cellularity within the bone grafts and had larger bone islands. The presence of osteogenic differentiated ASCs did not increase overall bone density compared with bone graft only.

Guided bone regeneration in long-bone defects with a structural hydroxyapatite graft and collagen membrane.

There are few synthetic graft alternatives to treat large long-bone defects resulting from trauma or disease that do not incorporate osteogenic or osteoinductive factors. The aim of this study was to test the additional benefit of including a permeable collagen membrane guide in conjunction with a preformed porous hydroxyapatite bone graft to serve as an improved osteoconductive scaffold for bone regeneration. A 10-mm-segmental long-bone defect model in the rabbit radius was used. The hydroxyapatite scaffolds alone or with a collagen wrap were compared as experimental treatment groups to an empty untreated defect as a negative control or a defect filled with autologous bone grafts as a positive control. All groups were evaluated after 4 and 8 weeks of in vivo implantation using microcomputed tomography, mechanical testing in flexure, and histomorphometry. It was observed that the use of the wrap resulted in an increased bone volume regenerated when compared to the scaffold-only group (59% greater at 4 weeks and 27% greater after 8 weeks). Additionally, the increase in density of the regenerated bone from 4 to 8 weeks in the wrap group was threefold than that in the scaffold group. The use of the collagen wrap showed significant benefits of increased interfacial bone in-growth (149% greater) and periosteal remodeling (49%) after 4 weeks compared to the scaffold-alone with the two groups being comparable after 8 weeks, by when the collagen membrane showed close-to-complete resorption. While the autograft and wrap groups showed significantly greater flexural strength than the defect group after 8 weeks, the scaffold-alone group was not significantly different from the other three groups. It is most likely that the wrap shows improvement of function by acting like a scaffold for periosteal callus ossification, maintaining the local bone-healing environment while reducing fibrous infiltration (15% less than scaffold only at 4 weeks). This study indicates that the use of a collagen membrane with a hydroxyapatite structural graft provides benefits for bone tissue regeneration in terms of early interfacial integration.