Role of anatomical region and hypoxia on angiogenic markers in adipose-derived stromal cells.

BACKGROUND: Recent research into adipose-derived stem cells (ASCs) suggests that anatomical location has a major impact on the metabolic profile and differentiation capacity of ASCs. By having a better understanding of how various ASCs respond to cellular stressors such as hypoxia, which are induced during routine surgical procedures, we can facilitate future development of cell-based therapies to improve wound healing. PATIENTS AND METHODS: Human ASCs were isolated from the superficial and deep adipose layers of four patients undergoing elective abdominoplasty. ASCs were cultured in hypoxic (1% O2, 5% CO2, and 94% N2) conditions. After 12 and 48 hours, ASCs were assessed for markers of angiogenesis by mRNA levels of vascular endothelial growth factor A (VEGF-A), vascular endothelial growth factor B (VEGF-B), and hypoxia inducible factor 1 α (HIF-1α). Western blot analysis was performed to assess levels of VEGF-A, p-NF-κB, and NF-κB. In addition, in vitro analysis of angiogenesis was performed using Matrigel assay (BD Biosciences, Franklin Lakes, NJ). RESULTS: We observed significant increases in deep ASC's VEGF-A, VEGF-B, and HIF-1α mRNA expression compared with the superficial layer after 24-hour hypoxia (p < 0.05). Similar results were found when examining protein expression levels, with the deep ASCs expressing significantly larger amounts of VEGF-A and p-NF-κB (p < 0.05) compared with the superficial layer. CONCLUSION: Our results suggest that significant variations exist in the angiogenic profile of superficial and deep ASCs. We demonstrate that superficial ASCs are less prone to transcribe potent chemokines for angiogenesis, such as VEGF-A, VEGF-B, and HIF-1α and are less likely to translate VEGF-A and NF-κB. This may help with the selection of specific stem cell donor sites in future models for stem cell therapy.

Burn injury enhances bone formation in heterotopic ossification model.

OBJECTIVE: To demonstrate the pro-osteogenic effect of burn injury on heterotopic bone formation using a novel burn ossicle in vivo model. BACKGROUND: Heterotopic ossification (HO), or the abnormal formation of bone in soft tissue, is a troubling sequela of burn and trauma injuries. The exact mechanism by which burn injury influences bone formation is unknown. The aim of this study was to develop a mouse model to study the effect of burn injury on heterotopic bone formation. We hypothesized that burn injury would enhance early vascularization and subsequent bone formation of subcutaneously implanted mesenchymal stem cells. METHODS: Mouse adipose-derived stem cells were harvested from C57/BL6 mice, transfected with a BMP-2 adenovirus, seeded on collagen scaffolds (ossicles), and implanted subcutaneously in the flank region of 8 adult mice. Burn and sham groups were created with exposure of 30% surface area on the dorsum to 60°C water or 30°C water for 18 seconds, respectively (n = 4/group). Heterotopic bone volume was analyzed in vivo by micro-computed tomography for 3 months. Histological analysis of vasculogenesis was performed with platelet endothelial cell adhesion molecule staining. Osteogenic histological analysis was performed by Safranin O, Picrosirius red, and aniline blue staining. Qualitative analysis of heterotopic bone composition was completed with ex vivo Raman spectroscopy. RESULTS: Subcutaneously implanted ossicles formed heterotopic bone. Ossicles from mice with burn injuries developed significantly more bone than sham control mice, analyzed by micro-computed tomography at 1, 2, and 3 months (P < 0.05), and had enhanced early and late endochondral ossification as demonstrated by Safranin O, Picrosirius red, and aniline blue staining. In addition, burn injury enhanced vascularization of the ossicles (P < 0.05). All ossicles demonstrated chemical composition characteristic of bone as demonstrated by Raman spectroscopy. CONCLUSIONS: Burn injury increases the predilection to osteogenic differentiation of ectopically implanted ossicles. Early differences in vascularity correlated with later bone development. Understanding the role of burn injury on heterotopic bone formation is an important first step toward the development of treatment strategies aimed to prevent unwanted and detrimental heterotopic bone formation.

Morphomic measurement of the temporalis muscle and zygomatic bone as novel predictors of hospital-based clinical outcomes in patients with mandible fracture.

INTRODUCTION: Patients with mandibular fracture often have comorbidities and concomitant injuries making the decision for when and how to operate a challenge. Physicians describe "temporalis wasting" as a finding that indicates frailty; however, this is a subjective finding without quantitative values. In this study, we demonstrate that decreased morphomic values of the temporalis muscle and zygomatic bone are an objective measure of frailty associated with increased injury-induced morbidity as well as negative impact on overall hospital-based clinical outcomes in patients with mandible fracture. METHODS: Computed tomographic (CT) scans from all patients with a diagnosis of a mandible fracture in the University of Michigan trauma registry and with a hospital admission were collected from the years 2004 to 2011. Automated, high-throughput CT analysis was used to reconstruct the anatomy and quantify morphomic values (temporalis volume, area and thickness, and zygomatic thickness) in these patients using MATLAB v13.0 (MathWorks Inc, Natick, MA, USA). Subsequently, a subset of 16 individuals with a Glasgow Coma Scale of 14 or 15 was analyzed to control for brain injury. Clinical data were obtained, and the association between morphomic measurements and clinical outcomes was evaluated using Pearson correlation for unadjusted analysis and multiple regression for adjusted analysis. RESULTS: The mean age of patients in the study was 47.1 years. Unadjusted analysis using Pearson correlation revealed that decreases in zygomatic bone thickness correlated strongly with increases in hospital, intensive care unit, and ventilator days (P < 0.0001, P = 0.0003, and P = 0.0017, respectively). Furthermore, we found that decreases in temporalis mean thickness correlated with increases in hospital and ventilator days (P = 0.0264 and P = 0.0306, respectively). Similarly, decreases in temporalis local mean thickness are significantly correlated with increases in hospital and ventilator days (P = 0.0232 and P = 0.0472, respectively). CONCLUSIONS: Decreased thicknesses of the zygomatic bone and temporalis muscle are significantly correlated with higher hospital, ventilator, and intensive care unit days in patients with mandibular fracture receiving reconstructive operations. This morphomic methodology provides an accurate, quantitative means to evaluate craniofacial trauma patient frailty, injury, and outcomes using routinely obtained CT scans. In the future, we plan to apply this approach to determine preoperative risk stratification and assist in surgical planning.

Bi-directional drones to strengthen healthcare provision: experiences and lessons from Madagascar, Malawi and Senegal.

Drones are increasingly being used globally for the support of healthcare programmes. Madagascar, Malawi and Senegal are among a group of early adopters piloting the use of bi-directional transport drones for health systems in sub-Saharan Africa. This article presents the experiences as well as the strengths, weaknesses, opportunities and threats (SWOT analysis) of these country projects. Methods for addressing regulatory, feasibility, acceptability, and monitoring and evaluation issues are presented to guide future implementations. Main recommendations for governments, implementers, drone providers and funders include (1) developing more reliable technologies, (2) thorough vetting of drone providers' capabilities during the selection process, (3) using and strengthening local capacity, (4) building in-country markets and businesses to maintain drone operations locally, (5) coordinating efforts among all stakeholders under government leadership, (6) implementing and identifying funding for long-term projects beyond pilots, and (7) evaluating impacts via standardised indicators. Sharing experiences and evidence from ongoing projects is needed to advance the use of drones for healthcare.

Effects of aging on osteogenic response and heterotopic ossification following burn injury in mice.

Heterotopic ossification (HO) is a common and debilitating complication of burns, traumatic brain injuries, and musculoskeletal trauma and surgery. Although the exact mechanism of ectopic bone formation is unknown, mesenchymal stem cells (MSCs) capable of osteogenic differentiation are known to play an essential role. Interestingly, the prevalence of HO in the elderly population is low despite the high overall occurrence of musculoskeletal injury and orthopedic procedures. We hypothesized that a lower osteogenicity of MSCs would be associated with blunted HO formation in old compared with young mice. In vitro osteogenic differentiation of adipose-derived MSCs from old (18-20 months) and young (6-8 weeks) C57/BL6 mice was assessed, with or without preceding burn injury. In vivo studies were then performed using an Achilles tenotomy with concurrent burn injury HO model. HO formation was quantified using µCT scans, Raman spectroscopy, and histology. MSCs from young mice had more in vitro bone formation, upregulation of bone formation pathways, and higher activation of Smad and nuclear factor kappa B (NF-κB) signaling following burn injury. This effect was absent or blunted in cells from old mice. In young mice, burn injury significantly increased HO formation, NF-κB activation, and osteoclast activity at the tenotomy site. This blunted, reactive osteogenic response in old mice follows trends seen clinically and may be related to differences in the ability to mount acute inflammatory responses. This unique characterization of HO and MSC osteogenic differentiation following inflammatory insult establishes differences between age populations and suggests potential pathways that could be targeted in the future with therapeutics.

Adipose-derived mesenchymal stem cells from ventral hernia repair patients demonstrate decreased vasculogenesis.

INTRODUCTION: In adipose tissue healing, angiogenesis is stimulated by adipose-derived stromal stem cells (ASCs). Ventral hernia repair (VHR) patients are at high risk for wound infections. We hypothesize that ASCs from VHR patients are less vasculogenic than ASCs from healthy controls. METHODS: ASCs were harvested from the subcutaneous fat of patients undergoing VHR by the component separation technique and from matched abdominoplasty patients. RNA and protein were harvested on culture days 0 and 3. Both groups of ASCs were subjected to hypoxic conditions for 12 and 24 hours. RNA was analyzed using qRT-PCR, and protein was used for western blotting. ASCs were also grown in Matrigel under hypoxic conditions and assayed for tubule formation after 24 hours. RESULTS: Hernia patient ASCs demonstrated decreased levels of VEGF-A protein and vasculogenic RNA at 3 days of growth in differentiation media. There were also decreases in VEGF-A protein and vasculogenic RNA after growth in hypoxic conditions compared to control ASCs. After 24 hours in hypoxia, VHR ASCs formed fewer tubules in Matrigel than in control patient ASCs. CONCLUSION: ASCs derived from VHR patients appear to express fewer vasculogenic markers and form fewer tubules in Matrigel than ASCs from abdominoplasty patients, suggesting decreased vasculogenic activity.

Treatment of heterotopic ossification through remote ATP hydrolysis.

Heterotopic ossification (HO) is the pathologic development of ectopic bone in soft tissues because of a local or systemic inflammatory insult, such as burn injury or trauma. In HO, mesenchymal stem cells (MSCs) are inappropriately activated to undergo osteogenic differentiation. Through the correlation of in vitro assays and in vivo studies (dorsal scald burn with Achilles tenotomy), we have shown that burn injury enhances the osteogenic potential of MSCs and causes ectopic endochondral heterotopic bone formation and functional contractures through bone morphogenetic protein-mediated canonical SMAD signaling. We further demonstrated a prevention strategy for HO through adenosine triphosphate (ATP) hydrolysis at the burn site using apyrase. Burn site apyrase treatment decreased ATP, increased adenosine 3',5'-monophosphate, and decreased phosphorylation of SMAD1/5/8 in MSCs in vitro. This ATP hydrolysis also decreased HO formation and mitigated functional impairment in vivo. Similarly, selective inhibition of SMAD1/5/8 phosphorylation with LDN-193189 decreased HO formation and increased range of motion at the injury site in our burn model in vivo. Our results suggest that burn injury-exacerbated HO formation can be treated through therapeutics that target burn site ATP hydrolysis and modulation of SMAD1/5/8 phosphorylation.

Grasp frequency and usage in daily household and machine shop tasks.

In this paper, we present results from a study of prehensile human hand use during the daily work activities of four subjects: two housekeepers and two machinists. Subjects wore a head-mounted camera that recorded their hand usage during their daily work activities in their typical place of work. For each subject, 7.45 hours of video was analyzed, recording the type of grasp being used and its duration. From this data, we extracted overall grasp frequency, duration distributions for each grasp, and common transitions between grasps. The results show that for 80 percent of the study duration the housekeepers used just five grasps and the machinists used 10. The grasping patterns for the different subjects were compared, and the overall top 10 grasps are discussed in detail. The results of this study not only lend insight into how people use their hands during daily tasks, but can also inform the design of effective robotic and prosthetic hands.

Early detection of burn induced heterotopic ossification using transcutaneous Raman spectroscopy.

INTRODUCTION: Heterotopic ossification (HO), or the abnormal formation of bone in soft tissue, occurs in over 60% of major burn injuries and blast traumas. A significant need exists to improve the current diagnostic modalities for HO which are inadequate to diagnose and intervene on HO at early time-points. Raman spectroscopy has been used in previous studies to report on changes in bone composition during bone development but has not yet been applied to burn induced HO. In this study, we validate transcutaneous, in-vivo Raman spectroscopy as a methodology for early diagnosis of HO in mice following a burn injury. METHODS: An Achilles tenotomy model was used to study HO formation. Following tenotomy, mice were divided into burn and sham groups with exposure of 30% surface area on the dorsum to 60° water or 30° water for 18s respectively. In-vivo, transcutaneous Raman spectroscopy was performed at early time points (5 days, 2 and 3 weeks) and a late time point (3 months) on both the tenotomized and non-injured leg. These same samples were then dissected down to the bone and ex-vivo Raman measurements were performed on the excised tissue. Bone formation was verified with Micro CT and histology at corresponding time-points. RESULTS: Our Raman probe allowed non-invasive, transcutaneous evaluation of heterotopic bone formation. Raman data showed significantly increased bone mineral signaling in the tenotomy compared to control leg at 5 days post injury, with the difference increasing over time whereas Micro CT did not demonstrate heterotopic bone until three weeks. Ex-vivo Raman measurements showed significant differences in the amount of HO in the burn compared to sham groups and also showed differences in the spectra of new, ectopic bone compared to pre-existing cortical bone. CONCLUSIONS: Burn injury increases the likelihood of developing HO when combined with traumatic injury. In our in-vivo mouse model, Raman spectroscopy allowed for detection of HO formation as early as 5 days post injury. Changes in bone mineral and matrix composition of the new bone were also evidenced in the Raman spectra which could facilitate early identification of HO and allow more timely therapy decisions for HO patients.