Functional outcomes of nerve allografts augmented with mesenchymal stem cells and surgical angiogenesis in a rat sciatic nerve defect model.

BACKGROUND: Motor function recovery following acellular nerve allograft (ANA) repair remains inferior to autologous nerve reconstruction. We investigated the functional recovery of ANAs after combined mesenchymal stem cell (MSC) delivery and surgical angiogenesis in a rat sciatic nerve defect model. METHODS: In 100 Lewis rats, unilateral sciatic nerve defects were reconstructed with (I) autografts, (II) ANAs, (III) ANAs wrapped with a superficial inferior epigastric artery fascial (SIEF) flap, combined with either (IV) undifferentiated MSCs or (V) Schwann cell-like differentiated MSCs. The tibialis anterior muscle area was evaluated during the survival period using ultrasonography. Functional recovery, histomorphometry, and immunofluorescence were assessed at 12 and 16 weeks. RESULTS: At 12 weeks, the addition of surgical angiogenesis and MSCs improved ankle contractures. The SIEF flap also significantly improved compound muscle action potential (CMAP) outcomes compared with ANAs. Autografts outperformed all groups in muscle force and weight. At 16 weeks, ankle contractures of ANAs remained inferior to autografts and SIEF, whereas the CMAP amplitude was comparable between groups. The muscle force of autografts remained superior to all other groups, and the muscle weight of ANAs remained inferior to autografts. No differences were found in histomorphometry outcomes between SIEF groups and ANAs. Vascularity, determined by CD34 staining, was significantly higher in SIEF groups compared with ANAs. CONCLUSIONS: The combination of surgical angiogenesis and MSCs did not result in a synergistic improvement in functional outcomes. In a short nerve gap model, the adipofascial flap may provide sufficient MSCs to ANAs without additional ex vivo MSC seeding.

The anatomic relationship of dorsal arterial perforators of the distal forearm, wrist, and dorsum of the hand with conclusions for the clinic.

BACKGROUND: Extended soft tissue defects of the fingers are often challenging to treat, and therefore, we performed cadaver dissections to elucidate the anatomic relationship between dorsal arterial perforators of the distal upper extremities to support the development of new local flaps. METHODS: Ten fixated cadaveric distal dorsal lower forearms were dissected to their arterial perforators down to 0.1 mm diameter in size with identification of their relationship. RESULTS: Dorsal distal fascia piercing perforators of the lower forearm come in two distinct rows, radial and ulnar, of the fourth extensor compartment. These were interconnected by subfascial axial arteries in line. The most proximal perforator is usually located 8-10 cm from the dorsal wrist, the most distal about 1-2 cm, and on average, only three subcutaneous bridging vessels connect both axial systems. The number of less reliable subcutaneous arterial connecting vessels between the dorsal wrist and forearm is also fairly limited to only one or two. More constant bridging arteries interconnect at the level of the dorsal retinaculum between the axial systems of the hand and lower forearm. At the level of the dorsum of the hand perforators reaching the skin, they interconnect in an arcuate fashion. This unique distribution pattern could be used for freestyle perforator propellor flaps of the hand, as we demonstrated in a case directly derived from our recent research. CONCLUSION: In our opinion, a super-extended perforator flap should be possible with the inclusion of the bridging arteries in flap design at the level of the dorsal retinaculum.

Surgical Angiogenesis of Decellularized Nerve Allografts Improves Early Functional Recovery in a Rat Sciatic Nerve Defect Model.

BACKGROUND: Surgical angiogenesis applied to nerve grafts has been suggested to enhance nerve regeneration after nerve injury. The authors hypothesized that surgical angiogenesis to decellularized nerve allografts would improve functional recovery in a rat sciatic nerve defect model. METHODS: Sixty Lewis rats were divided in three groups of 20 animals each. Unilateral sciatic nerve defects were repaired with (1) autografts, (2) decellularized allografts, and (3) decellularized allografts wrapped with a superficial inferior epigastric artery fascial flap to add surgical angiogenesis. Twelve and 16 weeks after surgery, nerve regeneration was assessed using functional, electrophysiologic, histologic, and immunofluorescence analyses. Ultrasonography was used during the survival period to noninvasively evaluate muscle atrophy and reinnervation by measuring cross-sectional muscle area. RESULTS: Surgical angiogenesis of allografts demonstrated significantly improved isometric tetanic force recovery at 12 weeks, compared to allograft alone, which normalized between groups at 16 weeks. Cross-sectional muscle areas showed no differences between groups. Electrophysiology showed superiority of autografts at both time points. No differences were found in histologic analysis, besides a significantly inferior N ratio in allografts at 12 weeks. Immunofluorescent expression of CD34, indicating vascularity, was significantly enhanced in the superficial inferior epigastric artery fascial group compared to allografts at 12 weeks, with highest expression at 16 weeks compared to all groups. CONCLUSION: Surgical angiogenesis with an adipofascial flap to the nerve allograft increases vascularity in the nerve graft, with subsequent improvement of early muscle force recovery, comparable to autografts.

Functional Outcomes of Nerve Allografts Seeded with Undifferentiated and Differentiated Mesenchymal Stem Cells in a Rat Sciatic Nerve Defect Model.

BACKGROUND: Mesenchymal stem cells have the potential to produce neurotrophic growth factors and establish a supportive microenvironment for neural regeneration. The purpose of this study was to determine the effect of undifferentiated and differentiated mesenchymal stem cells dynamically seeded onto decellularized nerve allografts on functional outcomes when used in peripheral nerve repair. METHODS: In 80 Lewis rats, a 10-mm sciatic nerve defect was reconstructed with (1) autograft, (2) decellularized allograft, (3) decellularized allograft seeded with undifferentiated mesenchymal stem cells, or (4) decellularized allograft seeded with mesenchymal stem cells differentiated into Schwann cell-like cells. Nerve regeneration was evaluated over time by cross-sectional tibial muscle ultrasound measurements, and at 12 and 16 weeks by isometric tetanic force measurements, compound muscle action potentials, muscle mass, histology, and immunofluorescence analyses. RESULTS: At 12 weeks, undifferentiated mesenchymal stem cells significantly improved isometric tetanic force measurement and compound muscle action potential outcomes compared to decellularized allograft alone, whereas differentiated mesenchymal stem cells significantly improved compound muscle action potential outcomes. The autografts outperformed both stem cell groups histologically at 12 weeks. At 16 weeks, functional outcomes normalized between groups. At both time points, the effect of undifferentiated versus differentiated mesenchymal stem cells was not significantly different. CONCLUSIONS: Undifferentiated and differentiated mesenchymal stem cells significantly improved functional outcomes of decellularized allografts at 12 weeks and were similar to autograft results in the majority of measurements. At 16 weeks, outcomes normalized as expected. Although differences between both cell types were not statistically significant, undifferentiated mesenchymal stem cells improved functional outcomes of decellularized nerve allografts to a greater extent and had practical benefits for clinical translation by limiting preparation time and costs.

Gene expression profiles of human adipose-derived mesenchymal stem cells dynamically seeded on clinically available processed nerve allografts and collagen nerve guides.

It was hypothesized that mesenchymal stem cells (MSCs) could provide necessary trophic factors when seeded onto the surfaces of commonly used nerve graft substitutes. We aimed to determine the gene expression of MSCs when influenced by Avance® Nerve Grafts or NeuraGen® Nerve Guides. Human adipose-derived MSCs were cultured and dynamically seeded onto 30 Avance® Nerve Grafts and 30 NeuraGen® Nerve Guides for 12 hours. At six time points after seeding, quantitative polymerase chain reaction analyses were performed for five samples per group. Neurotrophic [nerve growth factor (NGF), glial cell line-derived neurotrophic factor (GDNF), pleiotrophin (PTN), growth associated protein 43 (GAP43) and brain-derived neurotrophic factor (BDNF)], myelination [peripheral myelin protein 22 (PMP22) and myelin protein zero (MPZ)], angiogenic [platelet endothelial cell adhesion molecule 1 (PECAM1/CD31) and vascular endothelial cell growth factor alpha (VEGFA)], extracellular matrix (ECM) [collagen type alpha I (COL1A1), collagen type alpha III (COL3A1), Fibulin 1 (FBLN1) and laminin subunit beta 2 (LAMB2)] and cell surface marker cluster of differentiation 96 (CD96) gene expression was quantified. Unseeded Avance® Nerve Grafts and NeuraGen® Nerve Guides were used to evaluate the baseline gene expression, and unseeded MSCs provided the baseline gene expression of MSCs. The interaction of MSCs with the Avance® Nerve Grafts led to a short-term upregulation of neurotrophic (NGF, GDNF and BDNF), myelination (PMP22 and MPZ) and angiogenic genes (CD31 and VEGFA) and a long-term upregulation of BDNF, VEGFA and COL1A1. The interaction between MSCs and the NeuraGen® Nerve Guide led to short term upregulation of neurotrophic (NGF, GDNF and BDNF) myelination (PMP22 and MPZ), angiogenic (CD31 and VEGFA), ECM (COL1A1) and cell surface (CD96) genes and long-term upregulation of neurotrophic (GDNF and BDNF), angiogenic (CD31 and VEGFA), ECM genes (COL1A1, COL3A1, and FBLN1) and cell surface (CD96) genes. Analysis demonstrated MSCs seeded onto NeuraGen® Nerve Guides expressed significantly higher levels of neurotrophic (PTN), angiogenic (VEGFA) and ECM (COL3A1, FBLN1) genes in the long term period compared to MSCs seeded onto Avance® Nerve Grafts. Overall, the interaction between human MSCs and both nerve graft substitutes resulted in a significant upregulation of the expression of numerous genes important for nerve regeneration over time. The in vitro interaction of MSCs with the NeuraGen® Nerve Guide was more pronounced, particularly in the long term period (> 14 days after seeding). These results suggest that MSC-seeding has potential to be applied in a clinical setting, which needs to be confirmed in future in vitro and in vivo research.

Introducing human adipose-derived mesenchymal stem cells to AvanceⓇ nerve grafts and NeuraGenⓇ nerve guides.

BACKGROUND: When direct nerve coaptation is impossible after peripheral nerve injury, autografts, processed allografts, or conduits are used to bridge the nerve gap. The purpose of this study was to examine if human adipose-derived Mesenchymal Stromal/Stem Cells (MSCs) could be introduced to commercially available nerve graft substitutes and to determine cell distribution and the seeding efficiency of a dynamic seeding strategy. METHODS: MTS assays examined the viability of human MSCs after introduction to the AvanceⓇ Nerve Graft and the NeuraGenⓇ Nerve Guide. MSCs were dynamically seeded on nerve substitutes for either 6, 12, or 24 h. Cell counts, live/dead stains, Hoechst stains, and Scanning Electron Microscopy (SEM) revealed the seeding efficiency and the distribution of MSCs after seeding. RESULTS: The viability of MSCs was not affected by nerve substitutes. Dynamic seeding led to uniformly distributed MSCs over the surface of both nerve substitutes and revealed MSCs on the inner surface of the NeuraGenⓇ Nerve Guides. The maximal seeding efficiency of NeuraGenⓇ Nerve Guides (94%), obtained after 12 h was significantly higher than that of AvanceⓇ Nerve Grafts (66%) (p = 0.010). CONCLUSION: Human MSCs can be dynamically seeded on AvanceⓇ Nerve Grafts and NeuraGenⓇ Nerve Guides. The optimal seeding duration was 12 h. MSCs were distributed in a uniform fashion on exposed surfaces. This study demonstrates that human MSCs can be effectively and efficiently seeded onto commercially available nerve autograft substitutes in a timely fashion and sets the stage for the clinical application of MSC-seeded nerve graft substitutes clinically.

Adipose derived mesenchymal stem cells seeded onto a decellularized nerve allograft enhances angiogenesis in a rat sciatic nerve defect model.

PURPOSE: Adipose derived mesenchymal stem cells (MSCs) are hypothesized to supplement tissues with growth factors essential for regeneration and neovascularization. The purpose of this study was to determine the effect of MSCs with respect to neoangiogenesis when seeded onto a decellularized nerve allograft in a rat sciatic nerve defect model. METHODS: Allograft nerves were harvested from Sprague-Dawley rats and decellularized. MSCs were obtained from Lewis rats. 10 mm sciatic nerve defects in Lewis rats were reconstructed with reversed autograft nerves, decellularized allografts, decellularized allografts seeded with undifferentiated MSC or decellularized allografts seeded with differentiated MSCs. At 16 weeks, the vascular surface area and volume were evaluated. RESULTS: The vascular surface area in normal nerves (34.9 ± 5.7%), autografts (29.5 ± 8.7%), allografts seeded with differentiated (38.9 ± 7.0%) and undifferentiated MSCs (29.2 ± 3.4%) did not significantly differ from each other. Unseeded allografts (21.2 ± 6.2%) had a significantly lower vascular surface area percentage than normal nonoperated nerves (13.7%, p = .001) and allografts seeded with differentiated MSCs (17.8%, p = .001). Although the vascular surface area was significantly correlated to the vascular volume (r = .416; p = .008), no significant differences were found between groups concerning vascular volumes. The vascularization pattern in allografts seeded with MSCs consisted of an extensive nonaligned network of microvessels with a centripetal pattern, while the vessels in autografts and normal nerves were more longitudinally aligned with longitudinal inosculation patterns. CONCLUSIONS: Neoangiogenesis of decellularized allograft nerves was enhanced by stem cell seeding, in particular by differentiated MSCs. The pattern of vascularization was different between decellularized allograft nerves seeded with MSCs compared to autograft nerves.

Revascularization patterns of nerve allografts in a rat sciatic nerve defect model.

INTRODUCTION: The specific patterns of revascularization of allograft nerves after the addition of vascularization remain unknown. The aim of this study was to determine the revascularization patterns of optimized processed allografts (OPA) after surgically induced angiogenesis to the wound bed in a rat sciatic nerve model. MATERIALS AND METHODS: In 51 Lewis rats, sciatic nerve gaps were repaired with (i) autografts, (ii) OPA and (iii) OPA wrapped in a pedicled superficial inferior epigastric artery fascia flap (SIEF) to provide vascularization to the wound bed. At 2, 12, and 16 weeks, the vascular volume and vascular surface area in nerve samples were measured using micro CT and photography. Cross-sectional images were obtained and the number of vessels was quantified in the proximal, mid, and distal sections of the nerve samples. RESULTS: At 2 weeks, the vascular volume of SIEF nerves was comparable to control (P = 0.1). The vascular surface area in SIEF nerves was superior to other groups (P<0.05). At 12 weeks, vascularity in SIEF nerves was significantly higher than allografts (P<0.05) and superior compared to all other groups (P<0.0001) at 16 weeks. SIEF nerves had a significantly increased number of vessels compared to allografts alone in the proximal (P<0.05) and mid-section of the graft (P<0.05). CONCLUSIONS: Addition of surgical angiogenesis to the wound bed greatly improves revascularization. It was demonstrated that revascularization occurs primarily from proximal to distal (proximal inosculation) and not from both ends as previously believed and confirms the theory of centripetal revascularization.

Adhesion, distribution, and migration of differentiated and undifferentiated mesenchymal stem cells (MSCs) seeded on nerve allografts.

BACKGROUND: Although undifferentiated MSCs and MSCs differentiated into Schwann-like cells have been extensively compared in vitro and in vivo, studies on the ability and efficiency of differentiated MSCs for delivery into nerve allografts are lacking. As this is essential for their clinical potential, the purpose of this study was to determine the ability of MSCs differentiated into Schwann-like cells to be dynamically seeded on decellularized nerve allografts and to compare their seeding potential to that of undifferentiated MSCs. METHODS: Fifty-six sciatic nerve segments from Sprague Dawley rats were decellularized, and MSCs were harvested from Lewis rat adipose tissue. Control and differentiated MSCs were dynamically seeded on the surface of decellularized allografts. Cell viability, seeding efficiencies, cell adhesion, distribution, and migration were evaluated. RESULTS: The viability of both cell types was not influenced by the processed nerve allograft. Both cell types achieved maximal seeding efficiency after 12 h of dynamic seeding, albeit that differentiated MSCs had a significantly higher mean seeding efficiency than control MSCs. Dynamic seeding resulted in a uniform distribution of cells among the surface of the nerve allograft. No cells were located inside the nerve allograft after seeding. CONCLUSION: Differentiated MSCs can be dynamically seeded on the surface of a processed nerve allograft, in a similar fashion as undifferentiated MSCs. Schwann-like differentiated MSCs have a significantly higher seeding efficiency after 12 h of dynamic seeding. We conclude that differentiation of MSCs into Schwann-like cells may improve the seeding strategy and the ability of nerve allografts to support axon regeneration.

Gene expression profiles of differentiated and undifferentiated adipose derived mesenchymal stem cells dynamically seeded onto a processed nerve allograft.

BACKGROUND: Differentiation of mesenchymal stem cells (MSCs) into Schwann-like cells onto processed nerve allografts may support peripheral nerve repair. The purpose of this study was to understand the biological characteristics of undifferentiated and differentiated MSCs before and after seeding onto a processed nerve allograft by comparing gene expression profiles. METHODS: MSCs from Lewis rats were cultured in maintenance media or differentiated into Schwann-like cells. Both treatment groups were dynamically seeded onto decellularized nerve allografts derived from Sprague-Dawley rats. Gene expression was quantified by quantitative polymerase chain reaction (qPCR) analysis of representative biomarkers, including neurotrophic (GDNF, PTN, GAP43, PMP22), angiogenic (CD31, VEGF1), extracellular matrix (ECM) (COL1A1, COL3A1, FBLN1, LAMB2) or cell cycle (CAPS3, CCBN2) genes. Gene expression values were statistically evaluated using a 2-factor ANOVA with repeated measures. RESULTS: Baseline gene expression of undifferentiated and differentiated MSCs was significantly altered upon interaction with processed nerve allografts. Interaction between processed allografts and undifferentiated MSCs enhanced expression of neurotrophic (NGF, GDNF, PMP22), ECM (FBLN1, LAMB2) and regulatory cell cycle genes (CCNB2) during a 7-day time course. Interactions of differentiated MSCs with nerve allografts enhanced expression of neurotrophic (NGF, GDNF, GAP43), angiogenic (VEGF1), ECM (FBLN1) and regulatory cell cycle genes (CASP3, CCNB2) within one week. CONCLUSIONS: Dynamic seeding onto processed nerve allografts modulates temporal gene expression profiles of differentiated and undifferentiated MSCs. These changes in gene expressions may support the reparative functions of MSCs in supporting nerve regeneration in different stages of axonal growth.

New methods for objective angiogenesis evaluation of rat nerves using microcomputed tomography scanning and conventional photography.

INTRODUCTION: Nerve regeneration involves multiple processes, which enhance blood supply that can be promoted by growth factors. Currently, tools are lacking to visualize the vascularization patterns in transplanted nerves in vivo. The purpose of this study was to describe three-dimensional visualization of the vascular system in the rat sciatic nerve and to quantify angiogenesis of nerve reconstruction. MATERIALS AND METHODS: In 12 Lewis rats (weighing 250-300 g), 10 mm sciatic nerve gaps were repaired with ipsilateral reversed autologous nerve grafts. At 12 and 16 weeks of sacrifice, Microfil® contrast compound was injected in the aorta. Nerve autografts (N = 12) and contralateral untreated nerves (N = 12) were harvested and cleared while preserving the vasculature. The amount of vascularization was measured by quantifying the vascular surface area using conventional photography (two-dimensional) and the vascular volume was calculated with microcomputed tomography (three-dimensional). For each measurement, a vessel/nerve area ratio was calculated and expressed in percentages (vessel%). RESULTS: The vascular volume measured 3.53 ± 0.43% in autografts and 4.83 ± 0.45% vessels in controls at 12 weeks and 4.95 ± 0.44% and 6.19 ± 0.29% vessels at 16 weeks, respectively. The vascular surface area measured 25.04 ± 2.77% in autografts and 26.87 ± 2.13% vessels in controls at 12 weeks, and 28.11 ± 3.47% and 33.71 ± 2.60% vessels at 16 weeks, respectively. The correlation between both methods was statistically significant (p = .049). CONCLUSIONS: Both methods are considered to successfully reflect the degree of vascularization. Application of this technique could be used to visualize and objectively quantify angiogenesis of the transplanted nerve graft. Moreover, this simple method is easily reproducible and could be extrapolated to any other desired target organ ex vivo in small animals to investigate the vascular network.

Targeted stimulation of MSCs in peripheral nerve repair.

Mesenchymal stem cells (MSCs) have considerable translational potential in a wide variety of clinical disciplines and are the cellular foundation of individualized treatments of auto-immune, cardiac, neurologic and musculoskeletal diseases and disorders. While the cellular mechanisms by which MSCs exert their biological effects remain to be ascertained, it has been hypothesized that MSCs are supportive of local tissue repair through secretion of essential growth factors. Therapeutic applications of MSCs in peripheral nerve repair have recently been reported. This review focuses on how MSCs can promote nerve regeneration by conversion into Schwann-like cells, and discusses differentiation methods including delivery and dosing of naive or differentiated MSCs, as well as in vitro and in vivo outcomes. While MSC-based therapies for nerve repair are still in early stages of development, current progress in the field provides encouragement that MSCs may have utility in the treatment of patients with peripheral nerve injury.

Bacteremia and pneumonia in a tertiary PICU: an 11-year study.

OBJECTIVES: To describe trends in aetiology and resistance patterns and patient outcomes of bacteraemia and pneumonia in a PICU over an 11-year period. We also describe interventions aimed at reducing multi-resistant infections and other serious bacterial infections in the PICU. DESIGN AND SETTING: This cohort study involved data collected between January 2002 and December 2012 in the PICU of the Royal Children's Hospital in Melbourne, Australia. We documented all bacterial and fungal pathogens isolated from culture of blood or bronchial alveolar lavage fluid. Trends in aetiology and antibiotic resistance patterns were evaluated, as well as the case fatality rates of population-subgroups. PATIENTS, MEASUREMENT AND MAIN RESULTS: Overall, 881 patients (8.9%) had 1,480 serious bacterial or fungal infections and 1.2% of the PICU population suffered a multidrug-resistant infection. Twenty-six percent of 597 total deaths in the PICU during that time period were associated with a serious bacterial infection of the blood or lungs. Children in PICU with a serious infection in blood or lungs had a case fatality rate of 17.5% (95% CI, 15.0-20.2), compared with children overall in the PICU with a case fatality rate of 6.0% (95% CI, 5.5-6.5). The case fatality rate among children with multidrug-resistant sepsis was 25.6% (95% CI, 18.1-34.4) and among children with a persistent or recurrent infection in blood or lungs was 43.1% (95% CI, 30.8-56.0). Cases of multidrug-resistant bacteremia and bronchial alveolar lavage-proven pneumonia increased from 2002 to 2010 and significantly decreased from 2011, coinciding with improvements in antibiotic stewardship and infection control. CONCLUSIONS: Multiresistant bacterial sepsis and persistent or recurrent sepsis are major threats in pediatric intensive care and are associated with disproportionally high death rates. Our study describes a model for monitoring these serious infections and the effects of infection control interventions in the PICU.