Bone Regenerative Effect of Injectable Hypoxia Preconditioned Serum-Fibrin (HPS-F) in an Ex Vivo Bone Defect Model.

Biofunctionalized hydrogels are widely used in tissue engineering for bone repair. This study examines the bone regenerative effect of the blood-derived growth factor preparation of Hypoxia Preconditioned Serum (HPS) and its fibrin-hydrogel formulation (HPS-F) on drilled defects in embryonic day 19 chick femurs. Measurements of bone-related growth factors in HPS reveal significant elevations of Osteopontin, Osteoprotegerin, and soluble-RANKL compared with normal serum (NS) but no detection of BMP-2/7 or Osteocalcin. Growth factor releases from HPS-F are measurable for at least 7 days. Culturing drilled femurs organotypically on a liquid/gas interface with HPS media supplementation for 10 days demonstrates a 34.6% increase in bone volume and a 52.02% increase in bone mineral density (BMD) within the defect area, which are significantly higher than NS and a basal-media-control, as determined by microcomputed tomography. HPS-F-injected femur defects implanted on a chorioallantoic membrane (CAM) for 7 days exhibit an increase in bone mass of 123.5% and an increase in BMD of 215.2%, which are significantly higher than normal-serum-fibrin (NS-F) and no treatment. Histology reveals calcification, proteoglycan, and collagen fiber deposition in the defect area of HPS-F-treated femurs. Therefore, HPS-F may offer a promising and accessible therapeutic approach to accelerating bone regeneration by a single injection into the bone defect site.

Negative Pressure Wound Therapy in Free Muscle Flaps-Risk or Benefit?

BACKGROUND: Application of negative pressure wound therapy (NPWT) on free flaps not only reduces edema but also increases the pressure from outside. The impact of these opposite effects on flap perfusion remains elusive. This study evaluates the NPWT system's influence on macro- and microcirculation of free flaps and edema reduction to better assess the clinical value of this therapy in microsurgical reconstructions. METHODS: In this open-label, prospective cohort study, a total of 26 patients with free gracilis muscle flaps for distal lower extremity reconstruction were included. Flaps were covered with an NPWT (13 patients) or a conventional, fatty gauze dressing (13 patients) for 5 postoperative days (PODs). Changes in flap perfusion were analyzed by laser Doppler flowmetry, remission spectroscopy, and an implanted Doppler probe. Flap volume as a surrogate parameter for flap edema was evaluated by three-dimensional (3D) scans. RESULTS: No flap showed clinical evidence of circulatory disturbances. The groups showed significant differences in the dynamic of macrocirculatory blood flow velocity with an increase in the NPWT group and a decelerated flow in the control group from PODs 0 to 3 and PODs 3 to 5. No significant differences in microcirculation parameters were observed. 3D scans for estimation of edema development demonstrated significant differences in volume dynamics between the groups. Flap volume of the controls increased, while the volume in the NPWT group decreased during the first 5 PODs. The volume of NPWT-treated flaps decreased even further after NPWT removal from PODs 5 to 14 and significantly more than the flap volume in the control group. CONCLUSION: NPWT is a safe form of dressing for free muscle flaps that enhances blood flow and results in a sustainable edema reduction. The use of NPWT dressings for free flaps should therefore be considered not only as a pure wound covering but also as a supportive therapy for free tissue transfer.

In Vitro Comparison of Lymphangiogenic Potential of Hypoxia Preconditioned Serum (HPS) and Platelet-Rich Plasma (PRP).

Strategies for therapeutic lymphangiogenesis are gradually directed toward the use of growth factor preparations. In particular, blood-derived growth factor products, including Hypoxia Preconditioned Serum (HPS) and Platelet-rich Plasma (PRP), are both clinically employed for accelerating tissue repair and have received considerable attention in the field of regenerative medicine research. In this study, a comparative analysis of HPS and PRP was conducted to explore their lymphangiogenic potential. We found higher pro-lymphangiogenic growth factor concentrations of VEGF-C, PDGF-BB, and bFGF in HPS in comparison to normal serum (NS) and PRP. The proliferation and migration of lymphatic endothelial cells (LECs) were promoted considerably with both HPS and PRP, but the strongest effect was achieved with HPS-40% dilution. Tube formation of LECs showed the highest number of tubes, branching points, greater tube length, and cell-covered area with HPS-10%. Finally, the effects were double-validated using an ex vivo lymphatic ring assay, in which the highest number of sprouts and the greatest sprout length were achieved with HPS-10%. Our findings demonstrate the superior lymphangiogenic potential of a new generation blood-derived secretome obtained by hypoxic preconditioning of peripheral blood cells-a method that offers a novel alternative to PRP.

Hypoxia Preconditioned Serum (HPS) Promotes Proliferation and Chondrogenic Phenotype of Chondrocytes In Vitro.

Autologous chondrocyte implantation (ACI) for the treatment of articular cartilage defects remains challenging in terms of maintaining chondrogenic phenotype during in vitro chondrocyte expansion. Growth factor supplementation has been found supportive in improving ACI outcomes by promoting chondrocyte redifferentiation. Here, we analysed the chondrogenic growth factor concentrations in the human blood-derived secretome of Hypoxia Preconditioned Serum (HPS) and assessed the effect of HPS-10% and HPS-40% on human articular chondrocytes from osteoarthritic cartilage at different time points compared to normal fresh serum (NS-10% and NS-40%) and FCS-10% culture conditions. In HPS, the concentrations of TGF-beta1, IGF-1, bFGF, PDGF-BB and G-CSF were found to be higher than in NS. Chondrocyte proliferation was promoted with higher doses of HPS (HPS-40% vs. HPS-10%) and longer stimulation (4 vs. 2 days) compared to FCS-10%. On day 4, immunostaining of the HPS-10%-treated chondrocytes showed increased levels of collagen type II compared to the other conditions. The promotion of the chondrogenic phenotype was validated with quantitative real-time PCR for the expression of collagen type II (COL2A1), collagen type I (COL1A1), SOX9 and matrix metalloproteinase 13 (MMP13). We demonstrated the highest differentiation index (COL2A1/COL1A1) in HPS-10%-treated chondrocytes on day 4. In parallel, the expression of differentiation marker SOX9 was elevated on day 4, with HPS-10% higher than NS-10/40% and FCS-10%. The expression of the cartilage remodelling marker MMP13 was comparable across all culture conditions. These findings implicate the potential of HPS-10% to improve conventional FCS-based ACI culture protocols by promoting the proliferation and chondrogenic phenotype of chondrocytes during in vitro expansion.

Comparison of the Effect of Different Conditioning Media on the Angiogenic Potential of Hypoxia Preconditioned Blood-Derived Secretomes: Towards Engineering Next-Generation Autologous Growth Factor Cocktails.

Hypoxia Preconditioned Plasma (HPP) and Serum (HPS) are regenerative blood-derived growth factor compositions that have been extensively examined for their angiogenic and lymphangiogenic activity towards wound healing and tissue repair. Optimization of these secretomes' growth factor profile, through adjustments of the conditioning parameters, is a key step towards clinical application. In this study, the autologous liquid components (plasma/serum) of HPP and HPS were replaced with various conditioning media (NaCl, PBS, Glucose 5%, AIM V medium) and were analyzed in terms of key pro- (VEGF-A, EGF) and anti-angiogenic (TSP-1, PF-4) protein factors, as well as their ability to promote microvessel formation in vitro. We found that media substitution resulted in changes in the concentration of the aforementioned growth factors, and also influenced their ability to induce angiogenesis. While NaCl and PBS led to a lower concentration of all growth factors examined, and consequently an inferior tube formation response, replacement with Glucose 5% resulted in increased growth factor concentrations in anticoagulated blood-derived secretomes, likely due to stimulation of platelet factor release. Medium substitution with Glucose 5% and specialized peripheral blood cell-culture AIM V medium generated comparable tube formation to HPP and HPS controls. Altogether, our data suggest that medium replacement of plasma and serum may significantly influence the growth factor profile of hypoxia-preconditioned blood-derived secretomes and, therefore, their potential application as tools for promoting therapeutic angiogenesis.

The Donut Island Flap: Avoiding Stoma Relocation in Patients With Intractable Chronic Ileostomy Leakage.

INTRODUCTION: Severe skin scarring after multiple abdominal surgeries may lead to serious difficulties in stoma care, especially in patients with IBD. We demonstrate the technique of Donut Island Flap that we used in a female patient with colonic Crohn's disease that presented with intractable chronic ileostomy leakage. A relocation of the ileostomy was not possible because an alternative stoma site was not available anymore. TECHNIQUE: The scarred peristomal skin was radially excised up to a diameter of 10 cm. A pedicled anterolateral thigh perforator island flap was elevated from the right leg and was passed behind the rectus femoris muscle and through the inguinal tunnel into the defect. The ileostomy was passed through a small opening in the middle of the flap. The donor site at the thigh was closed primarily. RESULTS: No postoperative complications occurred. Three months after surgery, the ostomy care is providing no difficulties for the patient. CONCLUSION: The Donut Island Flap is a reliable and relatively simple technique to provide an adequate surrounding for ileostomy whose care is seriously impeded by severe skin scarring.

Ultrapulsed fractional ablative carbon dioxide laser treatment of hypertrophic burn scars: evaluation of an in-patient controlled, standardized treatment approach.

In this study, we aimed to quantify the effects of fractional ablative carbon dioxide laser therapy in the treatment of widespread hypertrophic burn scars. While many different pilot studies have described the potential of the technology and expert groups and current guidelines, alike, recommend its use, the level of evidence for the efficacy of fractional CO2-laser treatment for burn scars is currently very low. Ten patients (three male, seven female) with hypertrophic burn scars were treated with a single course of fractional CO2-laser therapy in an in-patient controlled setup, using a standardized treatment paradigm. Documentation was based on modern scar scales and questionnaires, like the Vancouver Scar Scale (VSS), Patient and Observer Scar Assessment Scale (POSAS), and Dermatology Life Quality Index (DLQI), as well as state of the art clinical measurements (PRIMOS, Cutometer). Over the course of 6 months after treatment, VSS and POSAS scores showed significant improvement in the rating of scar parameters, as did the quality of life rating according to the DLQI. In the treated scars, surface relief improved significantly, as S max decreased by 1893 µm (-36.92%) (p = 0.0273) and S z by 1615 µm (-36.37%) (p = 0.0488). Scar firmness in treated scars could be reduced by 30% after one treatment session, as R 0 improved by 0.0797 mm (+30.38%) (p = 0.0212). Fractional ablative CO2-laser treatment is a safe and efficacious option for the treatment of hypertrophic burn scars. While more treatment sessions are required for satisfying results, significant improvement is already apparent after a single course of treatment.

Less Is More? Impact of Single Venous Anastomosis on the Intrinsic Transit Time of Free Flaps.

Background The number of venous anastomoses advisable for a free flap continues to be controversial. Intrinsic transit time (ITT) is the time it takes dye during indocyanine green (ICG) microangiography to travel from the arterial to the venous anastomosis. ITT provides information on blood flow velocity and can predict postoperative circulatory complications. This study investigated the effect of the number of venous anastomoses on ITT. Methods The study enrolled 126 patients who had undergone microsurgical reconstruction and intraoperative ICG microangiography. Selection was limited to free gracilis and anterolateral thigh flaps as flaps with a single venous system. The retrospective assessment included reconstruction characteristics of the flaps, clinical outcome, ITT, and the number of venous anastomoses. Results The two groups were homogenous in terms of reconstruction characteristics. The single-venous anastomosis group (n = 75) had a reduced ITT (23.6 ± 11.7 vs. 43.8 ± 23.7 seconds; p < 0.001) compared with the double-anastomosis group (n = 51). A shorter ITT resulted in a significant reduction in the risk of reexploration for anastomotic thrombosis (OR 0.96; p = 0.024). Despite this, a higher reexploration rate tended to occur in the single-venous anastomosis group (9.3 vs. 7.8%; p = 1.0). Conclusion The results highlight the effect of shortening the ITT (thromboprotective blood flow acceleration) by using only one venous anastomosis. However, if the ITT is already at a low enough level with two veins, restriction to one vein does not appear to result in a reduced reexploration rate. For these flaps, the advantages of double-venous anastomosis prevail in terms of a backup drainage.

Perfusion Controlled Mobilization after Lower Extremity Free Flaps-Pushing the Limits of Time and Intensity.

Background The current standard to gradually adapt the fragile perfusion in lower extremity free flaps to an upright posture is the dangling maneuver. This type of flap training neither fits the orthostatic target load of an upright posture, nor does it assist in mobilizing the patients effectively. In this study, we quantitatively analyzed training effects of an early and full mobilization on flap perfusion. Methods A total of 15 patients with gracilis flaps for distal lower extremity reconstruction were included. Flap training was performed daily by mobilizing the patients on a tilt table into a fully upright posture for 5 minutes between the third and fifth postop days (PODs). Changes in micro- and macrocirculation were analyzed by laser Doppler flowmetry, remission spectroscopy, and an implanted Doppler probe. Results All flaps healed without complications. Yet, in three patients, the increased orthostatic load required an adjustment of the training duration due to a critical blood flow. The others showed an increasing compensation in the microcirculation. When tilting the patients, blood flow and oxygen saturation dropped significantly less on POD5 than on POD3. Furthermore, a significant increase of the blood flow was noted after an initial decrease during the mobilization on all days. An increasing compensation in the macrocirculation could not be determined. Conclusion Full mobilization of patients with lower extremity free flaps can be performed safely under perfusion monitoring, already starting on POD3. Additionally, monitoring allows a consideration of the individual orthostatic competence and therefore, exploitation of the maximum mobilization potential.

Impact of active thermoregulation on the microcirculation of free flaps.

BACKGROUND: While it is a known fact that warming increases blood flow in healthy tissue, little is known about the impact of active thermoregulation on the altered microcirculation of free flaps. The objective of the study was to identify the impact of postoperative active thermoregulation on free flap microcirculation. METHODS: Tissue temperature was assessed in 25 free perforator flaps using an implanted probe. Active thermoregulation was achieved using a water circulation based system. Changes in microcirculation were evaluated at the day of surgery and throughout the first three postoperative days after passive cooling (room temperature), passive warming (wound dressing), active warming (38 °C) and active cooling (15 °C) using laser Doppler flowmetry and remission spectroscopy. RESULTS: Active warming increased flap temperature by 7.7% to 36.4 °C ± 0.5 °C in comparison to the initial values of flaps without dressing (P < 0.001). As a result, the blood flow increased by 77.7% of the base value (P < 0.001). A significant correlation between all microcirculation parameters and tissue temperature was observed with a 5.52 AU blood flow increase per degree temperature increase (r = 0.7; P < 0.001). All microcirculation parameters showed a statistically significant increase after both passive and active warming, whereby active warming showed significantly higher values than passive warming. CONCLUSIONS: Active thermoregulation using water-based circulation is an effective and safe procedure to improve microcirculation in free flaps and is superior to conventional passive warming strategies.

Arteriovenous Loop-Independent Free Flap Reconstruction of Sternal Defects after Cardiac Surgery.

Background Sternal defects following deep wound infections are predominantly reconstructed using local and regional flaps. The lack of appropriate recipient vessels after cardiac surgery may explain the minor role of free flaps. To date, arteriovenous loops have been the leading solution to enable microsurgical closure of these defects. However, the related surgical effort and the risk of flap failure are increased. We reviewed our experiences with the right gastroepiploic vessels as alternative recipient vessels for free flap reconstructions. Methods Between September 2010 and July 2015, 12 patients suffering deep wound infection after cardiac surgery underwent sternal reconstruction with free flaps anastomosed to the right gastroepiploic vessels. Gracilis flaps (n = 8) and anterolateral thigh perforator flaps (n = 4) were used for sternal reconstruction. Recipient vessels were harvested by laparoscopic dissection in five patients. Half of the free flaps were variably combined with omental flow-through flaps. Results Healing of all flaps was uneventful with no partial or total flap loss. Simultaneous interdisciplinary harvesting of recipient vessels by laparoscopy significantly shortened mean operative time from 313 to 216 minutes (p = 0.018). One incisional hernia was observed in the laparotomy group. Revision of a gracilis donor site was necessary in another patient due to postoperative bleeding. No recurrent sternal infection occurred during a mean follow-up of 20 months (range, 3-59 months). Conclusions The concept of gastroepiploic recipient vessels allows reliable free flap reconstructions of sternal defects in such high-risk patients without the need for arteriovenous loops.

Hypoxia Preconditioned Serum (HPS)-Hydrogel Can Accelerate Dermal Wound Healing in Mice-An In Vivo Pilot Study.

The ability to use the body's resources to promote wound repair is increasingly becoming an interesting area of regenerative medicine research. Here, we tested the effect of topical application of blood-derived hypoxia preconditioned serum (HPS) on wound healing in a murine wound model. Alginate hydrogels loaded with two different HPS concentrations (10 and 40%) were applied topically on full-thickness wounds created on the back of immunocompromised mice. We achieved a significant dose-dependent wound area reduction after 5 days in HPS-treated groups compared with no treatment (NT). On average, both HPS-10% and HPS-40% -treated wounds healed 1.4 days faster than NT. Healed tissue samples were investigated on post-operative day 15 (POD 15) by immunohistology and showed an increase in lymphatic vessels (LYVE-1) up to 45% with HPS-40% application, while at this stage, vascularization (CD31) was comparable in the HPS-treated and NT groups. Furthermore, the expression of proliferation marker Ki67 was greater on POD 15 in the NT-group compared to HPS-treated groups, in accordance with the earlier completion of wound healing observed in the latter. Collagen deposition was similar in all groups, indicating lack of scar tissue hypertrophy as a result of HPS-hydrogel treatment. These findings show that topical HPS application is safe and can accelerate dermal wound healing in mice.

Hypoxia Preconditioned Serum (HPS) Promotes Osteoblast Proliferation, Migration and Matrix Deposition.

Interest in discovering new methods of employing natural growth factor preparations to promote bone fracture healing is becoming increasingly popular in the field of regenerative medicine. In this study, we were able to demonstrate the osteogenic potential of hypoxia preconditioned serum (HPS) on human osteoblasts in vitro. Human osteoblasts were stimulated with two HPS concentrations (10% and 40%) and subsequently analyzed at time points of days 2 and 4. In comparison to controls, a time- and dose-dependent (up to 14.2× higher) proliferation of osteoblasts was observed after 4 days of HPS-40% stimulation with lower lactate dehydrogenase (LDH)-levels detected than controls, indicating the absence of cytotoxic/stress effects of HPS on human osteoblasts. With regards to cell migration, it was found to be significantly faster with HPS-10% application after 72 h in comparison to controls. Further osteogenic response to HPS treatment was evaluated by employing culture supernatant analysis, which exhibited significant upregulation of OPG (Osteoprotegerin) with higher dosage (HPS-10% vs. HPS-40%) and longer duration (2 d vs. 4 d) of HPS stimulation. There was no detection of anti-osteogenic sRANKL (soluble Receptor Activator of NF-κB Ligand) after 4 days of HPS stimulation. In addition, ALP (alkaline phosphatase)-enzyme activity, was found to be upregulated, dose-dependently, after 4 days of HPS-40% application. When assessing ossification through Alizarin-Red staining, HPS dose-dependently achieved greater (up to 2.8× higher) extracellular deposition of calcium-phosphate with HPS-40% in comparison to controls. These findings indicate that HPS holds the potential to accelerate bone regeneration by osteogenic promotion of human osteoblasts.

Peripheral nerve reconstruction with collagen tubes filled with denatured autologous muscle tissue in the rat model.

Conventional nerve conduits lack cellular and extracellular guidance structures critical for bridging larger defects. In this study, an exogenous matrix for axonal regeneration was provided by pretreated muscle tissue. In 24 rats, 14-mm sciatic nerve segments were resected and surgically reconstructed using one of the following methods: autograft (AG); bovine type I collagen conduit; (MDM) collagen tube filled with modified denatured autologous muscle tissue. For 8 weeks, functional regeneration was evaluated by footprint and video gait analysis. Evaluation was complemented by electrophysiology, as well as qualitative and quantitative structural assessment of nerves and target muscles. Group AG was superior both structurally and functionally, showing higher axon counts, a more normal gait pattern, and less severe muscle atrophy. Fiber quality (fiber size and myelin thickness) was highest in group MDM, possibly related to the myelin-producing effect of muscular laminin. However, axon count was lowest in this group, and ultrastructural analysis of the denatured muscle tissue showed areas of incomplete denaturation that had acted as a mechanical barrier for regenerating axons. In light of these results, the often advocated use of muscular exogenous matrix for peripheral nerve reconstruction is reviewed in the literature, and its clinical application is critically discussed. In conclusion, combined muscle tubes may have a positive influence on nerve fiber maturation. However, muscle pretreatment is not without risks, and denaturation processes need to be further refined.

The intrinsic transit time of free microvascular flaps: clinical and prognostic implications.

BACKGROUND: Microscope-integrated indocyanine green near-infrared videoangiography (ICGA) is a new method for the intraoperative assessment of vascular flow through microvascular anastomoses. The intrinsic transit time (ITT) describes the time period from the dye appears at the arterial anastomosis (t(1)) till it reaches the suture line of the venous anastomosis (t(2)). As the transit time reflects blood flow velocity within the flap, prolonged ITT might correlate with low blood flow and a higher rate of postoperative thrombosis. We performed a clinical trial evaluating the association between intraoperative free flap transit time and early anastomotic complications in elective microsurgery. METHODS: One hundred consecutive patients undergoing elective microsurgical procedures underwent intraoperative ICG angiography (ICGA). In patients with anastomotic patency, angiograms were retrospectively reviewed and the intrinsic transit time was calculated. Postoperative outcome was registered and compared with the ITT. End points included early reexploration surgery and flap loss within the first 24 hours after surgery. RESULTS: Fourteen patients were excluded from the study due to technical anastomotic failure. The overall flap failure rate was 6% (5/86); the incidence of early re-exploration surgery was 10% (9/86). With a median of 31 seconds patients with an uneventful postoperative course showed significantly shorter ITTs than patients with flap loss or early postoperative reexploration (median: >120 seconds). An optimal cut-off value of ITT > 50 seconds was determined to be strongestly associated with a significantly increased risk of at least one positive end point. CONCLUSIONS: This study demonstrates a significant predictive value of the intrinsic flap transit time for the development of flap compromise and early re-exploration surgery.

Sensitivity and specificity of ICG angiography in free flap reexploration.

Microscope-integrated indocyanine green near-infrared videoangiography (ICGA) is a new imaging technique to assess vascular flow through diminutive vessels used in microvascular surgery. The purpose of this investigation was to evaluate the diagnostic accuracy of ICGA in detecting microvascular thrombosis in reexploration surgery. Patients undergoing emergent reexploration surgery after free tissue transfer were enrolled in this clinical study. After the patients had been returned to the operating room the pedicle vessels were exposed and a microangiography was performed. Independent of the result, the anastomoses were opened and surgically explored. Sensitivity and specificity was calculated, using the result of exploration surgery as a reference standard. Of 200 free flaps 20 (10%) underwent reexploration surgery. The most common surgical finding was microvascular thrombosis (55%). In one case vascular compromise was due to confusion of artery and vein with anastomosis of two veins. In 40% of patients an intact vascular pedicle was found. The sensitivity and specificity of ICGA to detect microvascular thrombosis was 100% and 86%, respectively. ICGA provides an excellent diagnostic accuracy for detecting microvascular thrombosis in reexploration surgery. Routine implementation of this technique may expedite a correct diagnosis and facilitate the surgical approach by preventing unnecessary surgical manipulation of intact anastomoses.

Effect of Hypoxia Preconditioned Secretomes on Lymphangiogenic and Angiogenic Sprouting: An in Vitro Analysis.

Hypoxia Preconditioned Plasma (HPP) and Serum (HPS) are two blood-derived autologous growth factor compositions that are being clinically employed as tools for promoting tissue regeneration, and have been extensively examined for their angiogenic activity. As yet, their ability to stimulate/support lymphangiogenesis remains unknown, although this is an important but often-neglected process in wound healing and tissue repair. Here we set out to characterize the potential of hypoxia preconditioned secretomes as promoters of angiogenic and lymphangiogenic sprouting in vitro. We first analysed HPP/HPS in terms of pro- (VEGF-C) and anti- (TSP-1, PF-4) angiogenic/lymphangiogenic growth factor concentration, before testing their ability to stimulate microvessel sprouting in the mouse aortic ring assay and lymphatic sprouting in the thoracic duct ring assay. The origin of lymphatic structures was validated with lymph-specific immunohistochemical staining (Anti-LYVE-1) and lymphatic vessel-associated protein (polydom) quantification in culture supernatants. HPP/HPS induced greater angiogenic and lymphatic sprouting compared to non-hypoxia preconditioned samples (normal plasma/serum), a response that was compatible with their higher VEGF-C concentration. These findings demonstrate that hypoxia preconditioned blood-derived secretomes have the ability to not only support sprouting angiogenesis, but also lymphangiogenesis, which underlines their multimodal regenerative potential.

Use of Oral Anticoagulation and Diabetes Do Not Inhibit the Angiogenic Potential of Hypoxia Preconditioned Blood-Derived Secretomes.

Patients suffering from tissue ischemia, who would greatly benefit from angiogenesis-promoting therapies such as hypoxia preconditioned blood-derived secretomes commonly receive oral anticoagulation (OA) and/or have diabetes mellitus (DM). In this study, we investigated the effect of OA administration on the in vitro angiogenic potential of hypoxia preconditioned plasma (HPP) and serum (HPS), prepared from nondiabetic/diabetic subjects who did not receive OA (n = 5) or were treated with acetylsalicylic acid (ASA, n = 8), ASA + clopidogrel (n = 10), or nonvitamin K antagonist oral anticoagulants (n = 7) for longer than six months. The effect of DM was differentially assessed by comparing HPP/HPS obtained from nondiabetic (n = 8) and diabetic (n = 16) subjects who had not received OA in the past six months. The concentration of key proangiogenic (vascular endothelial growth factor or VEGF) and antiangiogenic (thrombospondin-1 or TSP-1 and platelet factor-4 or PF-4) protein factors in HPP/HPS was analyzed via ELISA, while their ability to induce microvessel formations was examined in endothelial cell cultures. We found that OA use significantly reduced VEGF levels in HPP, but not HPS, compared to non-OA controls. While HPP and HPS TSP-1 levels remained largely unchanged as a result of OA usage, HPS PF-4 levels were significantly reduced in samples obtained from OA-treated subjects. Neither OA administration nor DM appeared to significantly reduce the ability of HPP or HPS to induce microvessel formations in vitro. These findings indicate that OA administration does not limit the angiogenic potential of hypoxia preconditioned blood-derived secretomes, and therefore, it does not prohibit the application of these therapies for supporting tissue vascularization and wound healing in healthy or diabetic subjects.

Sox2 controls Schwann cell self-organization through fibronectin fibrillogenesis.

The extracellular matrix is known to modulate cell adhesion and migration during tissue regeneration. However, the molecular mechanisms that fine-tune cells to extra-cellular matrix dynamics during regeneration of the peripheral nervous system remain poorly understood. Using the RSC96 Schwann cell line, we show that Sox2 directly controls fibronectin fibrillogenesis in Schwann cells in culture, to provide a highly oriented fibronectin matrix, which supports their organization and directional migration. We demonstrate that Sox2 regulates Schwann cell behaviour through the upregulation of multiple extracellular matrix and migration genes as well as the formation of focal adhesions during cell movement. We find that mouse primary sensory neurons and human induced pluripotent stem cell-derived motoneurons require the Sox2-dependent fibronectin matrix in order to migrate along the oriented Schwann cells. Direct loss of fibronectin in Schwann cells impairs their directional migration affecting the alignment of the axons in vitro. Furthermore, we show that Sox2 and fibronectin are co-expressed in proregenerative Schwann cells in vivo in a time-dependent manner during sciatic nerve regeneration. Taken together, our results provide new insights into the mechanisms by which Schwann cells regulate their own extracellular microenvironment in a Sox2-dependent manner to ensure the proper migration of neurons.

Comparative Evaluation of the Angiogenic Potential of Hypoxia Preconditioned Blood-Derived Secretomes and Platelet-Rich Plasma: An In Vitro Analysis.

Blood-derived factor preparations are being clinically employed as tools for promoting tissue repair and regeneration. Here we set out to characterize the in vitro angiogenic potential of two types of frequently used autologous blood-derived secretomes: platelet-rich plasma (PRP) and hypoxia preconditioned plasma (HPP)/serum (HPS). The concentration of key pro-angiogenic (VEGF) and anti-angiogenic (TSP-1, PF-4) protein factors in these secretomes was analyzed via ELISA, while their ability to induce microvessel formation and sprouting was examined in endothelial cell and aortic ring cultures, respectively. We found higher concentrations of VEGF in PRP and HPP/HPS compared to normal plasma and serum. This correlated with improved induction of microvessel formation by PRP and HPP/HPS. HPP had a significantly lower TSP-1 and PF-4 concentration than PRP and HPS. PRP and HPP/HPS appeared to induce similar levels of microvessel sprouting; however, the length of these sprouts was greater in HPP/HPS than in PRP cultures. A bell-shaped angiogenic response profile was observed with increasing HPP/HPS dilutions, with peak values significantly exceeding the PRP response. Our findings demonstrate that optimization of peripheral blood cell-derived angiogenic factor signalling through hypoxic preconditioning offers an improved alternative to simple platelet concentration and release of growth factors pre-stored in platelets.