A Composite Deferoxamine/Black Phosphorus Nanosheet/Gelatin Hydrogel Scaffold for Ischemic Tibial Bone Repair.

Introduction: Bone delay union is mostly caused by lack of blood supply. Although autografts, allografts and artificial bone have been widely used to treat bone delay union, the bone regeneration fails in the ischemic site accompanied by the bone donor site complications and disease transmission. Recently, there is a growing recognition of the importance of hydrogel scaffolds which are regarded as an eligible engineer tissue for bone repair. However, hydrogel is still limited in improving neovascularization. Methods: In this work, black phosphorus nanosheet and deferoxamine (BPN-DFO) were loaded in the gelatin hydrogel to overcome the high risk of bone delay union and systemically investigated the regeneration capability of BPN-DFO hydrogel in vitro and vivo. Results: The resulting BPN-DFO hydrogel scaffold showed superior swollen, degradation and release rate, as well as satisfied biocompatibility. BPN-DFO hydrogel shown the significant up-expression of mRNA related to bone regeneration and cell proliferation. In vivo, the proposed BPN-DFO hydrogel significantly improved osteogenesis and neovascularization in the ischemic tibial bone site of SD rats with acute femoral artery occlusion. Both macroscopic and histological evaluation of new regenerated bone showed newly formed blood vessel and collagen using BPN-DFO hydrogel. The immunohistochemistry and RT-PCR revealed that the bone regeneration could be improved via BMP/Runx2 pathway. Conclusion: The BPN-DFO hydrogel possesses potential tissue engineer material for ischemic bone defect treatment. However, furthermore studies are needed to testify the safety and efficacy of BPN-DFO hydrogel.

Associations of blood pressure and arterial stiffness with knee cartilage volume in patients with knee osteoarthritis.

OBJECTIVE: To describe the associations of blood pressure and arterial stiffness with knee cartilage volume in patients with knee OA. METHODS: A secondary analysis was performed on the data from participants in a randomized controlled trial that identified the effects of vitamin D supplementation on knee structures and symptoms among patients with symptomatic knee OA. Brachial and central blood pressure, arterial stiffness indicators and knee cartilage volume were measured at baseline and the 2 year follow-up. Associations were assessed using generalized estimating equations. RESULTS: Among 231 participants (average age 63.2 years), 48.9% were females. Higher supine systolic and diastolic pressures were significantly associated with lower tibial cartilage volume (systolic: lateral ß -6.23, medial ß -5.14, total ß -11.35 mm3/mmHg; diastolic: lateral ß -10.25, medial ß -11.29, total ß -21.50 mm3/mmHg). Higher supine systolic pressure was associated with lower femoral cartilage volume (lateral ß -17.35, total ß -28.31 mm3/mmHg). Central systolic pressure and arterial stiffness indicators (including pulse wave velocity, central pulse pressure and peripheral pulse pressure) were largely not associated with knee cartilage volume; however, higher augmentation index was associated with lower tibial and femoral cartilage volume (tibial: medial ß -8.24, total ß -19.13 mm3/%; femoral: lateral ß -23.70, medial ß -26.42, total ß -50.12 mm3/%). CONCLUSIONS: Blood pressure and arterial stiffness are associated with knee cartilage volume at several sites in knee OA patients. This supports that blood pressure and arterial stiffness may involve in the progression of knee OA.

Nanolayered hybrid mediates synergistic co-delivery of ligand and ligation activator for inducing stem cell differentiation and tissue healing.

Cellular behaviors, such as differentiation, are regulated by complex ligation processes involving cell surface receptors, which can be activated by various divalent metal cations. The design of nanoparticle for co-delivery of ligand and ligation activator can offer a novel strategy to synergistically stimulate ligation processes in vivo. Here, we present a novel layered double hydroxide (LDH)-based nanohybrid (MgFe-Ado-LDH), composed of layered MgFe hydroxide nanocarriers sandwiching the adenosine cargo molecule, maintained through an electrostatic balance, to co-deliver the adenosine (Ado) ligand from the interlayer spacing and the Mg2+ ion (ligation activator) through the dissolution of the MgFe nanocarrier itself. Our findings demonstrate that the MgFe-Ado-LDH nanohybrid promoted osteogenic differentiation of stem cells through the synergistic activation of adenosine A2b receptor (A2bR) by the dual delivery of adenosine and Mg2+ ions, outperforming direct supplementation of adenosine alone. Furthermore, the injection of the MgFe-Ado-LDH nanohybrid and stem cells embedded within hydrogels promoted the healing of rat tibial bone defects through the rapid formation of fully integrated neo-bone tissue through the activation of A2bR. The newly formed bone tissue displayed the key features of native bone, including calcification, mature tissue morphology, and vascularization. This study demonstrates a novel and effective strategy of bifunctional nanocarrier-mediated delivery of ligand (cargo molecule) and activation of its ligation to receptor by the nanocarrier itself for synergistically inducing stem cell differentiation and tissue healing in vivo, thus offering novel design of biomaterials for regenerative medicine.

Melatonin promotes angiogenesis during repair of bone defects: a radiological and histomorphometric study in rabbit tibiae.

OBJECTIVES: The pineal gland hormone, melatonin, is an immunomodulator and neuroendocrine hormone; it also stimulates monocyte, cytokine and fibroblast proliferations, which influence angiogenesis. The aim of this study was to investigate the effects of melatonin on angiogenesis during bone defect repair by means of radiological and histomorphometric evaluations of bone response to melatonin implants. MATERIALS AND METHODS: Twenty New Zealand rabbits weighing 3,900-4,500 g were used. Twenty melatonin implants were inserted in the proximal metaphyseal area of the animals' right tibia and 20 control areas were located in the left proximal metaphyseal area. Following implantation, the animals were sacrificed in groups of five, after 1, 2, 3 and 4 weeks, respectively. Anteroposterior and lateral radiographs were taken, and radiographic thermal imaging analysis was performed for all groups at different time stages following implant insertion. Samples were sectioned at 5 µm and stained using Hematoxylin-Eosin and Masson's trichrome, supplementing radiographic findings with histomorphometric analysis. RESULTS: After 4 weeks, radiological images showed complete repair of the bone defects. No healed or residual bone alterations attributable to the presence of the melatonin implant were observed. Histomorphometric analysis at 4 weeks showed the presence of a higher density newly formed bone. There were statistically significant differences in the length of cortical formation between the melatonin group and the control group during the first weeks of the study; there were also statistically significant differences in the number of vessels observed in the melatonin groups at the first two study stages. CONCLUSION AND CLINICAL RELEVANCE: Melatonin may have potential beneficial effects on bone defect repair.

Homocysteine mediated decrease in bone blood flow and remodeling: role of folic acid.

Deficiencies in folate lead to increased serum concentrations of homocysteine (Hcy), which is known as hyperhomocysteinemia (HHcy), is associated with bone disorders. Although, Hcy accumulates collagen in bone and contribute to decrease in bone strength. The mechanism of Hcy induced bone loss and remodeling is unclear. Therefore, the present study was aimed to determine the role of folic acid (FA) in genetically HHcy-associated decrease in bone blood flow and remodeling. Wild type (WT) and cystathionine-ß-synthase heterozygous (CBS+/-) mice were used in this study and supplemented with or without FA (300 mg/kg, Hcy reducing agent) in drinking water for 6 weeks. The tibial bone blood flow was measured by laser Doppler and ultrasonic flow probe method. The tibial bone density (BD) was assessed by dual energy X-ray absorptiometry. The bone homogenates were analyzed for oxidative stress, NOX-4 as oxidative marker and thioredoxin-1 (Trx-1) as anti-oxidant marker, bone remodeling (MMP-9) and bio-availability of nitric oxide (eNOS/iNOS/NO) by Western blot method. The results suggested that there was decrease in tibial blood flow in CBS+/- mice. The BD was also reduced in CBS+/- mice. There was an increase in NOX-4, iNOS, MMP-9 protein as well as MMP-9 activity in CBS+/- mice and decrease in Trx-1, eNOS protein levels, in part by decreasing NO bio-availability in CBS+/- mice. Interestingly, these effects were ameliorated by FA and suggested that FA supplementation may have therapeutic potential against genetically HHcy induced bone loss.

Effect of preconditioned hyperbaric oxygen and ozone on ischemia-reperfusion induced tourniquet in skeletal bone of rats.

BACKGROUND: The aim of the study was to investigate effect of I/R injury on bone tissue and protective role of hyperbaric oxygen precondition (HBO-PC) and ozone precondition (O(3)-PC) on I/R injury by using biochemical analysis. MATERIALS AND METHODS: Thirty-two rats were included in study. The animals were divided into four equal groups: sham operation, I/R, I/R+HBO and I/R+O(3). One session of 60 min, 3 ATA, 3-4 L/min, 100% oxygenation was defined as one dose of HBO. First dose of HBO was administrated 72 h before ischemia. Subsequent, one-dose of HBO administrated per 12 hours until ischemia time (total seven doses); 0.7 mg/kg ozone/oxygen mixture intraperitoneally was defined as one dose of ozone. First dose of O(3) was administered 72 h before ischemia (total four doses). I/R model was induced in anesthetized rats by unilateral (right) femoral artery clipping for 2 h followed by 22 h of reperfusion. The right tibia and were harvested. Tissue was assayed for levels of malondialdehyde (MDA) and protein carbonyl (PCO), activities of superoxide dismutase (SOD), and glutathione peroxidase (GSH-Px). RESULTS: MDA and PCO levels were increased in I/R group. SOD activity was increased; GSH-Px activity was decreased in I/R group. MDA and PCO levels were decreased, SOD and GSH-Px activities were increased in both HBO+I/R and O(3)+I/R groups. CONCLUSION: It has been shown that levels of MDA and PCO in bone were increased followed by 2 h of ischemia and 22 h of reperfusion period. Ozone-PC and HBO-PC has protective effect against skeletal bone I/R injury by decreasing levels of MDA and PCO, increasing activities of SOD and GSH-Px in rats.

[Effect of skeletal muscle contraction on bone capillary permeability: an experimental study in rabbits].

OBJECTIVE: To assess the effect of muscle contraction on bone capillary permeability. METHODS: Thirty female New Zealand white rabbits were randomly divided into the experimental and control groups (n=15). The bone chambers were implanted medially into the right tibiae of the rabbit, and in the experimental group, the rabbits were stimulated by transcutaneous electrical nerve stimulator (TENS) at 4 Hz (1 h/day, 6 days/week) starting from week 2. From week 3, intravital microscopic observation was carried out weekly till week 10, and the vasculature in the tibiae was observed under fluorescent microscope with FITC and RITC staining. All the images were analyzed by Metamorph program. RESULTS: The bone capillary permeability increased significantly in the experimental group. CONCLUSION: Skeletal muscle contraction can increase the capillary permeability.

Measurement of tibial endothelial cell function after cigarette smoking, cessation of smoking and hyperbaric oxygen therapy.

SUMMARY: Cigarette smoking is hazardous to a range of human tissues. For instance, cigarette smoke inhalation has been proven to delay bone healing. This study analysed the effects of cigarette smoking on tibial vascular endothelium and blood flow using the bone-chamber model. The effects of smoking cessation and hyperbaric oxygen (HBO) on the damage caused by smoking were also compared. 54 adult New Zealand rabbits were divided into three groups. Group 1: control, Group 2: 1 week smoking, and Group 3: 6 weeks' smoking. This study on rabbits confirmed that both short-term and long-term cigarette smoking is dangerous to the bony vascular endothelium of the tibia. The vasodilatation caused by nitric oxide production was significantly attenuated in Group 2 and 3's tibia. Long-term smoking damaged the vascular endothelium more severely than short-term smoking (P<.01). Cessation of smoking effectively reduces the adverse effects of smoking when the cessation time equals the smoking time. HBO also effectively reduces the adverse effects of smoking.

Induced angiogenesis with intramedullary direct current: experimental research.

The purpose of this study was to evaluate angiogenesis after the use of intramedullary direct electrical current in rabbit tibia. Thirty-two New Zealand rabbits were divided into four groups: group 1, false electrode group; group 2, hole group; group 3, control group; and group 4, intramedullary electrical stimulation group. One-half of the rabbits in each group were evaluated angiographically, pathologically, and scintigraphically on day 7, and the rest were evaluated on day 21. Results proved that electrical stimulation was not capable of the induction of angiogenesis in the subjects killed on day 7 and day 21. Furthermore, we found some fibrotic changes secondary to electrical stimulation on day 7 (P = 0.04) and day 21 (P = 0.01). However, an increase in new capillary vessels occurred in the false electrode group (P = 0.02). We found no useful effect of electrical stimulation in our study, a finding that is possibly due to our use of a method previously undocumented in the literature. We believe that this study can be the new baseline for further studies into the stimulation or inhibition of angiogenesis using intramedullary wire with or without electrical stimulation.

Prevention of venous thrombosis and thrombophlebitis in long-haul flights with pycnogenol.

The aim of this study was to evaluate the occurrence of deep venous thrombosis (DVT) and superficial vein thrombosis (SVT) and its prophylaxis with an oral anti-edema and antithrombotic agent (Pycnogenol, Horphag, Research Management SA, Geneva, Switzerland) in long-haul flights, in subjects at moderate to high-risk of DVT and SVT. The study pre-included 244 pre-selected subjects; 211 were included (33 were excluded for several reasons due to logistic problems) and 198 completed the study; 13 subjects were lost for follow-up at the end of the flight, all for non-medical problems (i.e., for difficult connections). All subjects were scanned within 90 minutes before the flight and within 2 hours after disembarking. Subjects were supplemented with 100 mg Pycnogenol per capsule. Treatment subjects received two capsules between 2 and 3 hours before flights with 250 mL of water; two capsules were taken 6 hours later with 250 mL of water and one capsule the next day. The control group received comparable placebo at the same intervals. The flight duration was on average 8 hours and 15 minutes (SD 55 min) (range, 7.45-12.33). In the control group there were five thrombotic events (one DVT and four superficial thromboses) while only nonthrombotic, localized phlebitis was observed in the Pycnogenol group (5.15% vs. no events; p<0.025). The ITT (intention to treat) analysis detects 13 failures in the control group (eight lost to follow up + five thrombotic events) of 105 subjects (12.4%) vs. five failures (4.7%; all lost, no thrombotic events) in the treatment group (p<0.025). No unwanted effects were observed. In conclusion, this study indicates that Pycnogenol treatment was effective in decreasing the number of thrombotic events (DVT and SVT) in moderate-to-high risk subjects, during long-haul flights.

The effects of low laser irradiation on angiogenesis in injured rat tibiae.

The influence of He-Ne laser radiation on the formation of new blood vessels in the bone marrow compartment of a regenerating area of the mid-cortical diaphysis of the tibiae of young adult rats was studied. A small hole was surgically made with a dentistry burr in the tibia and the injured area received a daily laser therapy over 7 or 14 days transcutaneously starting 24 h from surgery. Incident energy density dosages of 31.5 and 94.5 Jcm(-2) were applied during the period of the tibia wound healing investigated. Light microscopic examination of histological sections of the injured area and quantification of the newly-formed blood vessels were undertaken. Low-level energy treatment accelerated the deposition of bone matrix and histological characteristics compatible with an active recovery of the injured tissue. He-Ne laser therapy significantly increased the number of blood vessels after 7 days irradiation at an energy density of 94.5 Jcm(-2), but significantly decreased the number of vessels in the 14-day irradiated tibiae, independent of the dosage. These effects were attributed to laser treatment, since no significant increase in blood vessel number was detected between 8 and 15 non-irradiated control tibiae. Molecular mechanisms involved in low-level laser therapy of angiogenesis in post-traumatic bone regeneration needs further investigation.

Bone regeneration with calcium sulfate: evidence for increased angiogenesis in rabbits.

Autologous bone is the preferred bone graft material because it carries proteins as bone-enhancing substrates, minerals, and vital bone cells. Calcium sulfate (CS) is a well-tolerated, biodegradable, osteoconductive bone graft substitute and is a reasonable alternative to autogenous bone graft. Blood vessels are an important component of bone formation and maintenance. The process of vascular induction is called angiogenesis, and it plays a key role in all regenerative processes. Bone tissue differentiation is related to the local presence of blood vessels. One method to evaluate the presence of blood vessels in a tissue is to count the microvessels to evaluate microvessel density (MVD). The aim of the present study was to conduct a comparative evaluation of microvessel density in sites treated with CS and autologous bone in rabbits, with or without e-PTFE nonresorbable membranes (Gore-Tex, Flagstaff, Ariz). Nine New Zealand rabbits, each weighing about 2.5 kg, were used in this experiment. Three 6-mm wide defects were created in each tibial metaphysis. The defects were filled in a random way. The defects of group 1 (3 rabbits) were filled with CS granules (Surgiplaster, Classimplant, Rome, Italy) and covered with e-PTFE membranes. The defects in group 2 (3 rabbits) were filled with CS granules (Surgiplaster). The defects in group 3 (3 rabbits) were filled with autologous bone. A total of 54 defects were filled (18 with CS and e-PTFE membranes, 18 with CS alone, and 18 with autologous bone). No postoperative deaths or complications occurred. All nine animals were sacrificed at 4 weeks. MVD results were as follows: in the first group, 9.88 +/- 4.613; in the second group, 7.92 +/- 1.998; and in the third group, 5.56 +/- 1.895. P = .000 was highly significant. Statistically significant differences were found between groups 1 and 3, 1 and 2, and 2 and 3. The presence of more blood vessels in the sites treated with CS could help to explain the good results reported in the literature with the use of CS.

Effects of vitamin D analog, 22-oxa-1,25-dihydroxyvitamin D(3), on bone reconstruction by vascularized bone allograft.

We previously reported that vascularized bone allograft using immunosuppressants, such as cyclosporine A (CsA), is one approach for reconstruction of large bone defects in both experimental animals (Microsurgery 15:663; 1994) and clinically in humans (Lancet 347:970, 1996). Because immunosuppressive agents such as CsA induce significant side effects, including bone loss, other therapeutic agents supporting successful vascularized bone allografts have been sought after. We investigated the effects of 22-oxa-1,25-dihydroxyvitamin D(3) (OCT) on vascularized bone allograft, and compared its effects with CsA. Twelve-week-old DA rats with the major histocompatibility antigen (MHC) RT-1(a) were used as donors and age-matched Lewis rats with MHC RT-1(l) used as recipients. Allografted bones in rats treated with vehicle were rejected completely. Soft X-ray examination demonstrated that administration of OCT (0.5 microg/kg per day) for 12 weeks after bone graft induced bone union as effective as treatment for 12 weeks with CsA (10 mg/kg per day). Transplanted bones in OCT-treated rats showed higher bone mineral density than that in CsA-treated rats. Histologically, transplanted bones in OCT-treated rats at 12 weeks were nonvital, but these bones united with recipient vital bones. After cessation of 12 week treatment with OCT, new bone formation occurred around the grafted nonvital bones during a 9 month period. Transplanted bones in CsA-treated rats were vital and formed union with recipient bones, whereas cortical bones became thin when compared with nonvital bones in OCT-treated rats. Urinary deoxypyridinoline levels in rats treated with CsA were significantly higher than levels in rats treated with OCT, suggesting accelerated bone resorption in CsA-treated rats. These results suggest that OCT exerts an anabolic action on bone reconstruction by allogeneic bone transplantation.

Hyperbaric oxygen: a means of decreasing ischemic epiphyseal damage in a pediatric rabbit model.

The effect of hyperbaric oxygen on epiphyseal ischemia was evaluated using a pediatric rabbit model. Forty-five animals were compared in this study: 23 from a control pilot study and 22 hyperbaric exposed animals. In each animal the right distal femoral and proximal tibial epiphyses were isolated on a popliteal vascular pedicle. The left leg acted as the control. The growth difference between the rabbit's hindlimbs was the means of comparison throughout the groups established. Warm ischemia was induced by applying a vascular clamp to the right popliteal artery for 12 hours (20 animals) and 7 hours (17 animals). The remaining 8 animals underwent a sham operation without interruption of epiphyseal perfusion. On completion of the ischemic period hyperbaric oxygen therapy (HBOT) was performed on 12 12-hour (12h-HBOT) and 10 7-hour (7h-HBOT) animals at 2 atmospheres for 90 minutes twice per day for 4 postoperative days. The animals were killed on either postoperative day 14 or 90. Measurement of longitudinal bone growth was performed on the 90-day animals from serial radiographs at the time of surgery and then at 1 month, 2 months, and 3 months after surgery. There was no significant difference in longitudinal bone growth between the sham-operated and the 7h-HBOT animals at 1, 2, and 3 months. There was a statistically significant difference, however, between the normal growth of the 7h-HBOT group compared with the abnormal growth of the 7-hour, 12-hour, and 12h-HBOT animals. Histology was consistent, with the bone growth data demonstrating relative normalcy of the 7h-HBOT group epiphyseal plates versus severe architectural aberrance and necrosis of the 12h-HBOT group epiphyses. Our experimental data indicate that a clinical trial should be instituted using HBO for pediatric replantation patients when warm ischemia exceeds 7 hours. (J Hand Surg 2000; 25A:159-165.

Effects of infrared radiation on intraosseous blood flow and oxygen tension in the rat tibia.

In order to examine the effects of infrared radiation on intraosseous blood circulation, the intraosseous blood flow and oxygen tension were examined in rat tibiae. Infrared radiation was performed for 5 minutes over the ankle joint approximately 40 cm from the skin surface. The intraosseous blood flow was measured with a hydrogen washout technique. Before and after infrared radiation, the blood flow rates in the proximal metaphysis of the rat tibia were 8.7 +/- 2.5 ml/min/100g and 15.6 +/- 5.4 ml/min/100g, respectively, i.e. the intraosseous blood flow increased approximately 80% with the radiation. The intraosseous oxygen tension and temperature were measured with a Po2 sensor, altered to our own specifications. The former increased from 20.4 +/- 6.2 mmHg to 23.3 +/- 6.7 mmHg (about 15%), and the latter from 29.0 +/- 1.4 degrees C to 31.2 +/- 1.9 degrees C, respectively, during the 5 minutes infrared radiation. The results of the present study show that infrared radiation remarkably improves intraosseous blood circulation, and these effects continue for at least 30 minutes. It is, therefore, suggested that infrared radiation can be used as a measure of thermotherapy in some bones to improve intraosseous blood circulation.

The combined use of transluminal angioplasty and selective clot lysis.

14 cases of transluminal angioplasty and selective clot lysis are reported. One patient removed the catheter before successful lysis and one lost his leg after an almost complete lysis due to advanced necrosis. Treatment was successful in the other 12 cases with complete or partial thrombolysis. Remaining clots were either smashed by catheter and/or successfully treated by oral fibrinolytic therapy. The injected dose of streptokinase was 5,000 U/h after an initial dose of 20,000 U in one hand injection. The duration of infusions was 6-72 hours. A total dose of 60,000-520,000 U streptokinase was adopted. Intraarterial infusion therapy was followed by long-term low-grade fibrinolytic treatment with oral penthosan polysulphate. Four bleedings at the puncture site, one epistaxis and a bleeding at the site of a recently extracted tooth were seen. Only one infusion was stopped due to moderate bleeding and without any drawback.

Newer developments of extra-anatomic bypass.

We have analyzed our eight year experience with more than 200 instances of extra-anatomic bypass and have made certain observations. Extra-anatomic bypass provides an acceptable alternative to extensive direct intra-abdominal and intrathoracic vascular reconstructive procedures. This is particularly true in high risk patients and in the presence of infection. While axillobifemoral bypass is widely known and used, other types of extra-anatomic bypass are emphasized. These are axillary-axillary, axillopopliteal, axillotibial and axillofemoral bypass under local anesthesia. Technical factors, such as the type of graft, the course of the bypass and ancillary techniques to improve the long term patency, are also discussed herein.