Nonselective matrix metalloproteinase but not tumor necrosis factor-α inhibition effectively preserves the early critical colon anastomotic integrity.

BACKGROUND: Increased matrix metalloproteinase (MMP) activity has been implicated in the pathogenesis of colorectal anastomotic leakage. Tumor necrosis factor-α (TNF-α) induces MMPs and may influence anastomosis repair. METHODS: We assessed the efficacies of the nonselective hydroxamate MMP inhibitor GM6001, the selective hydroxamate MMP inhibitor AG3340 and a TNF-α antagonist with respect to anastomotic breaking strength of left-sided colon anastomoses in male Sprague-Dawley rats. RESULTS: Systemic GM6001 treatment effectively blocked MMP activity and maintained the initial breaking strength day 0 of the anastomoses when administered subcutaneously as daily depositions (100 mg/kg) or continuously (10 mg/kg/day). In contrast, the anastomotic biomechanic strength was lowered by 55% (p < 0.001) in vehicle-treated rats on postoperative day 3. GM6001 treatment increased breaking strength by 88% (p < 0.0005) compared with vehicle-treated rats day 3 and reduced (p = 0.003) the occurrence of spontaneous anastomotic dehiscence. Histologically, the anastomotic wound was narrower (p < 0.05) in the longitudinal direction in GM6001-treated animals whereas GM6001 had no significant effect on inflammatory cell infiltration or epithelialization. AG3340 (10 mg/kg) increased (p < 0.012) breaking strength by 47% compared with vehicle on day 3 but did not significantly prevent the reduction of the initial breaking strength on day 0. Although the increased TNF-α levels in the wound were attenuated, the anastomotic breaking strength was not improved (p = 0.62) by the TNF-α (10 mg/kg) inhibitor given systemically. CONCLUSIONS: Pharmacological nonselective MMP inhibition ought to be explored as a prophylactic regimen to reduce anastomotic complications following colorectal resection. The involvement of TNF-α was insignificant in anastomotic wound healing in an experimental model.

Tobacco extract but not nicotine impairs the mechanical strength of fracture healing in rats.

The influence of nicotine and tobacco extract (without nicotine) alone and in combination on and mechanical strength of closed femoral fractures in rats was investigated. One hundred four male Sprague-Dawley rats were divided into four groups receiving: nicotine, tobacco extract, tobacco extract plus nicotine, and saline. One week prior to fracture, osmotic pumps were implanted subcutaneously in all animals to administer nicotine equivalent to the serum level of nicotine observed in a smoker consuming one to two packs of cigarettes daily. An equivalent volume of saline was administered to the control animals. Tobacco extract was administered orally. A closed transverse femoral diaphysial fracture was performed, and stabilized with an intramedullary pin. The fractures were mechanically tested after 21 days of healing. Tobacco extract alone decreased the mechanical strength. Ultimate torque and torque at yield point of the tobacco extract group were decreased by 21% (p=0.010) and 23% (p=0.056), respectively, compared with the vehicle (saline) group, and by 20% (p=0.023) and 26% (p=0.004), respectively, compared with the nicotine group. No difference was found between the tobacco extract and tobacco extract plus nicotine groups. An 18% (p=0.013) reduction in torque at yield point was observed in the tobacco extract plus nicotine group compared with the nicotine group. No differences in ultimate stiffness, energy absorption, and callus bone mineral content at the fracture line were found between any of the groups. Serum levels of nicotine were between 40-50 ng/mL in the group given nicotine alone and the group given tobacco extract plus nicotine (equivalent to serum levels observed in persons smoking one to two packs of cigarettes per day).

Growth hormone can reverse glucocorticoid-induced low bone turnover on cortical but not on cancellous bone surfaces in adult Wistar rats.

UNLABELLED: We evaluated the effect of glucocorticoids (GC) and growth hormone (GH) on cortical and cancellous bone turnover in adult rats using random vertical sections giving valid measurements of bone surfaces and bone formation parameters. GH administration could reverse GC-induced osteopenia and low bone turnover of cortical bone. However, GH could not reverse the GC-induced low bone turnover of cancellous bone. METHODS: Seventy female Wistar rats, 7 months of age, were divided into five groups: (1) start control, (2) saline, (3) GC 9 mg/kg/day (Solu Medrol), (4) GH 5 mg/kg/day, and (5) GC 9 mg/kg/day + GH 5 mg/kg/day, and injected for 3 months. The vertebral body was examined using dynamic histomorphometry and biomechanical tests. Nonparametric methods were used. RESULTS: Glucocorticoid administration induced a low bone turnover state of both the cortical and cancellous bone of the vertebral body, without altering the absolute amount of bone or the biomechanical competence of the vertebral body. GH administration induced a small increase in longitudinal bone growth and ventral modeling drift. This growth increased the total amount of cortical, endocortical, and cancellous bone in the vertebra. The biomechanical competence of a 3.5-mm-high cylinder of the central vertebral body was also increased due to an increase in the amount of cortical bone, whereas the total amount of cancellous bone in the cylinder was unaltered. The cancellous bone density (CBV) was, however, increased due to thicker trabeculae probably induced by an accelerated mineral appositional rate (MAR) induced by GH. GH also increased longitudinal and ventral modeling drifts in the GC-injected animals. GH increased the amount of cortical bone and also the amount of cancellous bone close to the epiphyseal growth plate, whereas the cancellous bone volume of the central vertebral cylinder was unaffected by GH administration in GC-injected animals. GH could also increase parameters of bone formation (bone mineralizing surface (MS) and MAR) on cortical bone surfaces in GC-injected animals, whereas parameters of bone formation [MS and bone formation rates (BFR)] on cancellous bone surfaces were even lower than those of animals injected with GC alone. CONCLUSION: GH can reverse GC-induced low bone turnover on cortical but not on cancellous bone surfaces.

Gene therapy with human osteoprotegerin decreases callus remodeling with limited effects on biomechanical properties.

Osteoprotegerin (OPG) is a naturally occurring protein, which prevents bone resorption by inhibition of osteoclastogenesis, function, and survival. Therefore, recombinant OPG may be an attractive drug in the treatment of chronic bone resorptive diseases such as osteoporosis. Gene therapy has the potential to achieve long-term treatment by delivering genes of anti-resorptive proteins to the recipient. The effects of OPG gene therapy on fracture healing have not been described previously. The influence of OPG gene therapy on callus formation, callus tissue structural strength, apparent material properties, and histology of tibia fractures in rats was investigated after 3 weeks and 8 weeks of healing. Intramuscular administration of adeno-associated virus (AAV) vector-mediated OPG resulted in increased levels of OPG in serum of approximately 100 ng/ml throughout the study period. Control animals with fractures received transduction with an AAV reporter gene construct (AAV-enhanced green fluorescent protein (eGFP)), and in this group serum OPG levels remained at baseline (<10 ng/ml). After 3 weeks of healing, AAV-OPG treatment reduced the number of osteoclasts in the callus tissue (33%, P < 0.001). However, AAV-OPG treatment did not influence callus dimensions, callus bone mineral content (BMC), fracture structural strength, or apparent callus tissue material properties. After 8 weeks of healing, AAV-OPG treatment reduced the number of osteoclasts in the callus tissue (31%, P < 0.001) compared with AAV-eGFP fractures. Furthermore, deposition of new woven bone at the fracture line of the original cortical bone was hampered (new woven bone present: in all AAV-eGFP animals, in 41% of AAV-OPG-treated animals, P < 0.001). AAV-OPG treatment also increased callus BMC (18%, P = 0.023) compared with AAV-eGFP fractures. AAV-OPG did not influence callus dimensions, structural strength of the fractures, or ultimate stress, whereas elastic modulus was reduced in the AAV-OPG groups (37%, P = 0.039). The experiment demonstrates that AAV-OPG gene therapy decreases the fracture remodeling, but this does not influence the structural strength of healing fractures.

Mechanical properties of the human lens capsule.

The human lens capsule has recently been the subject of much attention in an attempt to understand its physiological function in relation to the accommodative function, its functional reserve in the elderly population, and its potential in relation to cataract surgery. This overview presents our current knowledge of the mechanical properties of the human lens capsule, discussed on basis of its structure and its role in accommodation and cataract surgery.

Hypocalcemia and osteopathy in mice with kidney-specific megalin gene defect.

Megalin is an endocytic receptor highly expressed in the proximal tubules of the kidney. Recently, we demonstrated that this receptor is essential for the renal uptake and conversion of 25-OH vitamin D3 to 1,25-(OH)2 vitamin D3, a central step in vitamin D and bone metabolism. Unfortunately, the perinatal lethality of the conventional megalin knockout mouse model precluded the detailed analysis of the significance of megalin for calcium homeostasis and bone turnover in vivo. Here, we have generated a new mouse model with conditional inactivation of the megalin gene in the kidney by using Cre recombinase. Animals with a renal-specific receptor gene defect were viable and fertile. However, lack of receptor expression in the kidney results in plasma vitamin D deficiency, in hypocalcemia and in severe bone disease, characterized by a decrease in bone mineral content, an increase in osteoid surfaces, and a lack of mineralizing activity. These features are consistent with osteomalacia (softening of the bones) as a consequence of hypovitaminosis D and demonstrate the crucial importance of the megalin pathway for systemic calcium homeostasis and bone metabolism.

Simvastatin treatment partially prevents ovariectomy-induced bone loss while increasing cortical bone formation.

Statins are commonly prescribed drugs that inhibit hepatic cholesterol synthesis and thereby reduce serum cholesterol concentrations. Some of the statins are thought to possess bone anabolic properties. Effects of statin on tibia, femur, and vertebral cortical and cancellous bone were studied in ovariectomized (OVX) rats. Sixty Wistar female rats, 4 months old, were allocated into four groups: baseline control, sham + placebo group, OVX + placebo, OVX + simvastatin. Simvastatin, 20 mg/kg, or placebo was given twice daily by a gastric tube for 3 months. The rats were labeled with tetracycline at day 11 and calcein at day 4 before sacrifice. Concerning cortical bone, the tibial diaphysis bending strength was increased by 8% and the periosteal bone formation rate (BFR) at the mid-diaphysis increased by twofold in the OVX + simvastatin group compared with the OVX + placebo group, in harmony with increased serum osteocalcin concentrations. Simvastatin did not affect the endocortical bone formation. Concerning cancellous bone, the cancellous bone volumes in the proximal tibia and vertebral body were reduced in both OVX groups, but the reduction was less in the OVX + simvastatin group compared with the OVX + placebo group. This reduction in cancellous bone loss is in agreement with the 36% decreased activity of serum tartrate-resistant-acid-phosphatase 5b (TRAP-5b), indicating decreased osteoclast activity in the OVX + simvastatin group compared with the OVX + placebo group. In conclusion, simvastatin induces a moderate increase in cortical bone formation at the periosteal bone surface. The new cortical bone exhibits a normal lamellar structure, and simvastatin seems to respect the regional pattern of bone formation, bone resorption, and drift; for example, no periosteal bone formation is observed in the vertebral canal. Furthermore, simvastatin reduces the loss of cancellous bone induced by ovariectomy.

Mechanical properties of the human posterior lens capsule.

PURPOSE: To investigate mechanical properties of the human posterior lens capsule. METHODS: Twenty-five human donor eyes were obtained from an eye bank. The age of the donors ranged from 1 to 94 years. Test specimens were prepared as tissue rings from posterior lens capsules by means of excimer laser. Capsular thickness was measured microscopically as the difference in focus between microspherules placed on the outer and inner surfaces of the capsule. The capsular rings were slipped over two pins connected to a motorized micropositioner and a force transducer and stretched at a constant rate with continuous recording of load and deformation. Data for the posterior lens capsule were compared with previously published data for the anterior lens capsule. RESULTS: The thickness of the posterior lens capsule ranged from 4 to 9 micro m and showed no significant changes with age. Ultimate mechanical strength of the posterior lens capsule decreased significantly with age. Ultimate strain ranged from 101% to 34%, ultimate load ranged from 15.9 to 1.1 mN, ultimate stress ranged from 16.1 to 1.1 N/mm(2), ultimate elastic stiffness ranged from 52.1 to 5.7 mN, and ultimate elastic modulus ranged from 27.4 to 3.3 N/mm(2). The load-strain and the stress-strain relationships in the posterior lens capsule were nonlinear, and therefore elastic stiffness and elastic modulus varied as a function of strain. In the low-strain region (0%-10% strain), elastic stiffness and elastic modulus ranged between 0.3 to 2.4 mN and 0.3 to 2.3 N/mm(2), respectively, and seemed to increase during the first part of life until middle age. CONCLUSIONS: Mechanical strength of the posterior lens capsule was found to decrease markedly with age. The age-related loss of mechanical strength seemed to begin earlier in the posterior lens capsule than in the anterior lens capsule. In accommodative function range (low strains), the mechanical quality of the posterior lens capsule was similar to the anterior lens capsule, which indicates that the mechanical effectiveness of the lens capsule in situ varies proportionally with capsular thickness.

Parathyroid hormone induces formation of new cancellous bone with substantial mechanical strength at a site where it had disappeared in old rats.

OBJECTIVE: The present study addresses the question--can PTH induce formation of trabeculae in areas where cancellous bone has disappeared? Two-year-old male rats were chosen, because in this aged animal model the distal femurs have almost no cancellous bone, and the marrow cavity has reached a substantial dimension. DESIGN: The rats were injected for 56 days with either PTH(1-34), 15 nmol/kg/day (62.5 microg/kg/day), or vehicle. METHODS: Transverse specimens, 2-mm high, were cut from the distal femoral metaphysis. Marrow cavity diameters and cancellous bone trabeculae were analysed by a micro-computerized tomography scanner. The cancellous bone within the cortical and endocortical rim of each specimen was submitted to a biomechanical compression test. Furthermore, the cancellous bone was studied by dynamic tetracycline labelling and histomorphometry. RESULTS: In the vehicle-injected group the trabecular bone volume was 0% (0-1.4), median (range). All PTH-injected rats had trabeculae in the distal metaphysis and the trabecular bone volume (6.7% (2.3-12.0)) was markedly increased (P<0.003). The median trabecular thickness was increased (P<0.003) in the PTH-injected rats (118 microm (104-125)) compared with the vehicle group (0 microm (0-71)). The compressive stress was increased (P<0.003) in the PTH-injected group (0.7 MPa (0.1-2.1)) compared with the vehicle-injected group (0 MPa (0-0.4)). The histomorphometry revealed that only 3 animals of the 10 in the vehicle-injected group had trabeculae in the distal femoral metaphysis. All PTH-injected animals (12 of 12) had continuous trabecular bone network in the marrow cavity. CONCLUSION: Intermittent PTH treatment induced marked formation of new cancellous bone trabeculae with substantial mechanical strength, at a site where it had disappeared in old rats.

Intermittent parathyroid hormone treatment enhances guided bone regeneration in rat calvarial bone defects.

This study investigates the effects of intermittent parathyroid hormone (PTH(1-34)) treatment on bone regeneration and mechanical strength of critically sized rat calvarial bone defects covered with expanded membranes. A full-thickness bone defect (diameter 5 mm) was trephined in the central part of the parietal bones in 20-month-old female Wistar rats. The bone defects were covered with an exocranial and an endocranial expanded polytetrafluoroethylene membrane. The animals were killed 35 days after operation. 60 microg PTH(1-34)/kg was administered daily during the healing period, and control animals with calvarial bone defects were given vehicle. Mechanical testing was performed by a punch out testing procedure by placing a steel punch (diameter 3.5 mm) in the center of the healed defect. After mechanical testing, the newly formed tissue inside the defect was removed and the dry weight and ash weight were measured. PTH(1-34) increased dry weight by 48%, ash weight by 51%, and ash concentration by 26%. PTH(1-34) also augmented the mechanical strength of the new bone formed inside the defect by increasing ultimate stiffness by 87%. No differences in body weight were found between the vehicle-injected and the PTH-treated animals during the experiment. The experiment demonstrates that intermittent PTH(1-34) treatment increases bone deposition and enhances mechanical strength of healing rat calvarial defects covered with expanded polytetrafluoroethylene membranes.