The effects of immobilization on bone histomorphometry in rats

To determine whether immobilization acts directly on bone by alteration of mechanical loading or systemically, studies of the effects of immobilization were carried out on histomorphometry of diaphyses of tibiae and on subcutaneous implants of demineralized allogenic bone matrix of rats. The right hind leg of growing rats was denervated by severing the tibial nerve. A sham operation on the right hind leg was performed in control animals. Bone formation at the endosteal and periosteal surfaces was significantly lower in tibiae from limbs with severed nerves as compared to tibiae from the intact limbs of nerve-sectioned rats and from both limbs of sham-operated control rats. Bone formation was decreased at both 3 and 7 weeks after immobilization. The decreased formation resulted in significant reductions in cross-sectional area. At 3 weeks post denervation, the periosteal bone formation rate was lower in tibiae of intact limbs from denervated rats as compared to tibiae from intact limbs of sham-operated animals. This finding was attributed to reduced physical activity of the denervated rats. In the implants, nerve section did not alter the amount of implant matrix resorbed, the amount of bone matrix synthesized, or the amount of calcium in the implant. These findings support the hypothesis that inhibition of bone formation at the tibial diaphysis in response to immobilization resulted from altered mechanical loading and not from the production of substances acting systemically. Whereas the mean medullary area of tibiae was not altered by nerve section, it was decreased in tibiae of all groups compared to the values of basal controls, indicating that bone formation was greater than bone resorption.(ABSTRACT TRUNCATED AT 250 WORDS)     

Low dose PTH improves metaphyseal bone healing more when muscles are paralyzed

Stimulation of bone formation by PTH is related to mechanosensitivity. The response to PTH treatment in intact bone could therefore be blunted by unloading. We studied the effects of mechanical loading on the response to PTH treatment in bone healing. Most fractures occur in the metaphyses, therefor we used a model for metaphyseal bone injury.One hind leg of 20 male SD rats was unloaded via intramuscular botulinum toxin injections. Two weeks later, the proximal unloaded tibia had lost 78% of its trabecular contents. At this time-point, the rats received bilateral proximal tibiae screw implants. Ten of the 20 rats were given daily injections of 5 μg/kg PTH (1–34). After two weeks of healing, screw fixation was measured by pull-out, and microCT of the distal femur cancellous compartment was performed. Pull-out force provided an estimate for cancellous bone formation after trauma.PTH more than doubled the pull-out force in the unloaded limbs (from 14 to 30 N), but increased it by less than half in the loaded ones (from 30 to 44 N). In relative terms, PTH had a stronger effect on pull-out force in unloaded bone than in loaded bone (p = 0.03).The results suggest that PTH treatment for stimulation of bone healing does not require simultaneous mechanical stimulation.     

Alcohol alters whole body composition, inhibits bone formation, and increases bone marrow adiposity in rats

Summary  Chronic alcohol abuse is a risk factor for osteoporosis and sarcopenia, but the long-term effects of alcohol on the immature musculoskeletal system are less clear. The present investigation in growing rats was designed to determine the effects of alcohol consumption on body composition, muscle mass, and bone mass, architecture, and turnover. Introduction  Few studies have focused on the long-term effects of drinking on bone and muscle during skeletal maturation. Methods  Alcohol was included in the diet of 4-week-old male Sprague–Dawley rats (35% caloric intake) for 3 months. The controls were fed an isocaloric alcohol-free liquid diet ad libitum. A second study was performed in which the controls were pair-fed to the alcohol-fed animals. Results  Compared to ad libitum-fed age-matched controls, alcohol-fed rats weighed less and had lower lean mass, fat mass, and percent body fat. In addition, they had lower slow- and fast-twitch muscle mass, lower total body bone mineral content and bone mineral density, and lower cancellous bone volume in the lumbar vertebra and proximal tibia. The effects of alcohol consumption on body composition were reduced when compared to the pair-fed control diet, indicating that caloric restriction was a comorbidity factor. In contrast, the effects of alcohol to decrease bone formation and serum leptin and IGF-I levels and to increase bone marrow adiposity appeared independent of caloric restriction. Conclusions  The skeletal abnormalities in growing alcohol-fed rats were due to a combination of effects specific to alcohol consumption and alcohol-induced caloric restriction.     

Effect of zoledronate acid treatment on osseointegration and fixation of implants in autologous iliac bone grafts in ovariectomized rabbits

One main problem associated with alveolar bone augmentation in implant dentistry is resorption of grafted bone, which may be further compromised by systemic skeletal disorders such as osteoporosis. Zoledronate acid (ZOL) is the most potent bisphosphonate to treat osteoporosis and therefore it is hypothesized to be able to invert the negative effect of osteoporosis on osseointegration and fixation of dental implants in autologous bone grafts. In this study, 56 rabbits received bilateral ovariectomy (OVX) (40 rabbits) or sham operation (16 rabbits). Three months later, 8 animals from each group were sacrificed for bone mineral density (BMD) examination. Then the remaining animals underwent bilateral autologous iliac bone grafting with simultaneous implantation of titanium implants in tibiae and were divided into 5 groups (n = 8): Sham, OVX, Loc-ZOL (local treatment), Sys-ZOL (systemic treatment) and Loc + Sys-ZOL (local plus systemic) group. At 3 months after implantation, all animals were sacrificed and specimens were harvested for examinations. Both BMD and histological examinations of femurs showed osteoporotic changes after ovariectomy, while systemic treatment with ZOL restored mineralized bone. Micro-CT examination demonstrated that OVX group showed significant decrease of mineralized bone and implant-bone contact when compared with sham control, whereas both systemic and local treatments of ZOL significantly increased mineralized bone and implant-bone contact in ovariectomized animals. However, the best effects were observed in Loc + Sys-ZOL group (combined use of ZOL) and most of bone indices were similar to (IBCR, p > 0.05) or higher than (BV/TV, Conn.D and Tb.N) (p < 0.01) those of the sham group, except Tb.Th, which was still significantly lower (p < 0.01), and Tb.Sp, which was further decreased (p < 0.01). The aforementioned effects were also confirmed by histomorphometric analysis of bone indices on implant-bone contact and mineralized bone. In addition, biomechanical testing further supported the beneficial effect of ZOL treatment and maximal removal torque of titanium implants was observed in Loc + Sys-ZOL group. In conclusion, our study suggests that both systemic and local treatments with ZOL can invert negative effect of osteoporosis and promote osseointegration and fixation of dental implants in autologous bone grafts under osteoporotic condition. Combined systemic and local use of ZOL exerts best effects when compared to their single use.     

Effects of three different preservation methods on the mechanical properties of human and bovine cortical bone

Background and purposeIn the development of new strategies for fracture fixation, new methods have to be tested biomechanically under in vitro conditions before clinical trials can be performed. The gold standard for laboratory evaluations is fresh-frozen specimen. As the availability of fresh-frozen specimens is limited and since their use bears infectious risks, specimens treated with various chemical embalming fluids are also used. These preservation methods may alter the mechanical properties of the specimens used. Therefore, the aims of the present study were to determine the effects of three different preservation methods (formalin fixation (FO), Thiel-fixation (TH), and alcohol–glycerine fixation (AG)) on the elastic and postyield mechanical properties of cortical bone and to compare these properties to those of fresh-frozen (FF) specimens.Materials and methodsCylindrical cortical specimens (diameter 3 mm, length 60 mm) were obtained from human femurs (n = 48) and bovine tibiae (n = 40). Before specimen immersion in different fixation fluids, bone mineral density (BMD) as well as the initial Young's modulus was determined. The Young's modulus was determined in a nondestructive bending test, and measurements were repeated after 6 months of immersion in fixative solution. Subsequent to the nondestructive test, a destructive 3-point bending test was conducted to assess the postyield and fracture properties.ResultsThe BMD as well as the initial Young's modulus showed no significant differences between the four test groups. After 6 months in fixative solution, the Young's modulus was significantly lowered in human Thiel specimens and only showed minor changes in formalin- and alcohol–glycerine-treated specimens. The plastic energy absorption of human and bovine specimens was altered significantly. Formalin as well as alcohol–glycerine fixation yielded a significant decrease in plastic energy absorption, whereas Thiel fixation significantly increased the plastic energy absorption.Discussion/conclusionBecause of the significantly altered plastic mechanical properties of cortical bone, the use fresh-frozen bone specimens is recommended in biomechanical studies investigating failure loads of orthopaedic implants. The use of embalmed specimens should be restricted to pilot tests.     

Methotrexate effects on heterotopic bone in rats

We studied the effects of high-dose methotrexate on heterotopic bone formation induced by implants of demineralized bone matrix in the abdominal wall of growing rats. Methotrexate induced an arrest in normal weight gain of the animals, more pronounced the younger the animals were. The youngest animals had reduced ash weight and decreased isotope uptake in the tibiae and teeth. However, implants from these animals, given methotrexate 10 days before implantation of bone matrix, had a 33 per cent increase in ash content. When methotrexate was given at, or 10 days after, implantation, heterotopic bone formation was reduced by 40 and 22 per cent, respectively, whereas orthotopic bone was considerably less affected in these older animals. In a second experiment, no difference in elimination rates of 45Ca between methotrexate-treated and control rats in implants, teeth, or tibiae were found. It appears that a less detrimental effect of methotrexate on new bone formation can be expected if the drug is given before, or a substantial period after, surgery requiring bone formation for healing.     

Methotrexate effects on heterotopic bone in rats

We studied the effects of high-dose methotrexate on heterotopic bone formation induced by implants of demineralized bone matrix in the abdominal wall of-growing rats. Methotrexate induced an arrest in normal weight gain of the animals, more pronounced the younger the animals were. The youngest animals had reduced ash weight and decreased isotope uptake in the tibiae and teeth. However, implants from these animals, given methotrexate 10 days before implantation of bone matrix, had a 33 per cent increase in ash content. When methotrexate was given at, or 10 days after, implantation, heterotopic bone formation was reduced by 40 and 22 per cent, respectively, whereas orthotopic bone was considerably less affected in these older animals. In a second experiment, no difference in elimination rates of ⁴⁵Ca between methotrexate-treated and control rats in implants, teeth, or tibiae were found.

It appears that a less detrimental effect of methotrexate on new bone formation can be expected if the drug is given before, or a substantial period after, surgery requiring bone formation for healing.     

In vivo effects of modification of hydroxyapatite/collagen composites with and without chondroitin sulphate on bone remodeling in the sheep tibia

The addition of chondroitin sulphate (CS) to bone cements with calcium phosphate has lead to an enhancement of bone remodeling and an increase in new bone formation in small animals. The goal of this study was to verify the effect of CS in bone cements in a large animal model simulating a clinically relevant situation of a segmental cortical defect of a critical size on bone–implant interaction and bone remodeling. The influence of adding CS to hydroxyapatite/collagen (HA/Col) composites on host response was assessed in a standard sheep tibia model. A midshaft defect of 3 cm was created in the tibiae of 14 adult female sheep. The defect was filled with a HA/Col cement cylinder in seven animals and with a CS‐modified hydroxyapatite/collagen (HA/Col/CS) cement cylinder in seven animals. In all cases the tibia was stabilized with an interlocked universal tibial nail. The animals in each group were analyzed with X‐rays, CT scans, histology, immunohistochemistry, and enzymehistochemistry, as well as histomorphometric measurements. The X‐ray investigation showed a significantly earlier callus reaction around the HA/Col/CS implants compared to HA/Col alone. The amount of newly formed bone at the end point of the experiment was significantly larger around HA/Col/CS cylinders both in the CT scan and in the histomorphometric analysis. There were still TRAP‐positive osteoclasts around the HA/Col implants after 3 months. The number of osteopontin‐positive osteoblasts and the direct bone contact were significantly higher around HA/Col/CS implants. We conclude that addition of CS enhances bone remodeling and new bone formation around HA/Col composites. © 2008 Orthopaedic Research Society. Published by Wiley Periodicals, Inc. J Orthop Res 27:15–21, 2009     

Impaired osteoinduction in a rat model for chronic alcohol abuse

Alcohol abuse is a risk factor for bone fractures. Following a fracture, alcoholics have a higher risk for impaired fracture healing. However, the specific alcohol-induced defect(s) in bone healing are not known. Alcohol is a potent inhibitor of bone formation during bone growth and turnover. Thus, the purpose of this study was to determine the effects of alcohol consumption on induction of new bone formation. Demineralized allogeneic bone matrix (DABM) cylinders were used to model osteoinduction in a rat model for chronic alcohol abuse. DABM cylinders, prepared from femurs and tibiae of rats fed a normal diet, were implanted into sexually mature male rats adapted to alcohol (ethanol contributed 35% of caloric intake) or control liquid diets. Food intake in the control rats was restricted to match food intake of alcohol-fed animals. The implants were recovered 6 weeks later and analyzed by histology, muCT and chemical analysis. Histological evaluation revealed a robust osteoinductive response, resulting in mature bone ossicle formation, in DABM implants in rats fed the control diet. Alcohol consumption affected bone mass and architecture of the DABM implants but not volumetric density or mineral composition. Specifically, alcohol consumption resulted in significant decreases in DABM-induced bone volume, bone volume/mg original cylinder weight, connectivity density, trabecular number and thickness, ash weight and % ash weight. There were no changes in mineral (ash) density nor in the relative amounts of calcium, magnesium, iron, selenium and zinc (microg/mg ash), indicating that alcohol consumption did not impair mineralization. Taken together, these results show that alcohol abuse resulted in decreased bone formation within the DABM implant. We conclude that reduced osteoinduction may contribute to impaired bone healing in alcoholics.     

Relationships between bone geometry,volumetric bone mineral density and bone microarchitecture of the distal radius and tibia with alcohol consumption

PurposeChronic heavy alcohol consumption is associated with bone density loss and increased fracture risk, while low levels of alcohol consumption have been reported as beneficial in some studies. However, studies relating alcohol consumption to bone geometry, volumetric bone mineral density (vBMD) and bone microarchitecture, as assessed by high-resolution peripheral quantitative computed tomography (HR-pQCT), are lacking.MethodsHere we report an analysis from the Hertfordshire Cohort Study, in which we studied associations between HR-pQCT measures at the distal radius and tibia and alcohol consumption in 376 participants (198 men and 178 women) aged 72.1–81.4 years.ResultsA total of 30 (15.2%), 90 (45.5%) and 78 (39.4%) men drank minimal/none (< 1 unit/week), low (≥ 1 unit/week and < 11 units/week) and moderate/high (≥ 11 units/week) amounts of alcohol respectively. These figures were 74 (41.8%), 80 (45.2%) and 23 (13.0%) respectively in women for minimal/none (< 1 unit/week), low (≥ 1 unit/week and < 8 units/week) and moderate/high (≥ 8 units/week). At the distal radius, after adjustment for confounding factors (age, BMI, smoking status, dietary calcium intake, physical activity and socioeconomic status and years since menopause and HRT use for women), men that drank low alcohol had lower cortical thickness (p = 0.038), cortical vBMD (p = 0.033), and trabecular vBMD (p = 0.028) and higher trabecular separation (p = 0.043) than those that drank none/minimal alcohol. Similar differences were shown between minimal/none and moderate/high alcohol although these only reached statistical significance for the cortical parameters. Interestingly, after similar adjustment, women showed similar differences in the trabecular compartment between none/minimal alcohol and low alcohol at the distal tibia. However, women that drank moderate/high alcohol had significantly higher trabecular vBMD (p = 0.007), trabecular thickness (p = 0.026), and trabecular number (p = 0.042) and higher trabecular separation (p = 0.026) at the distal radius than those that drank low alcohol.ConclusionsOur results suggest that alcohol consumption (low and moderate/high) may have a detrimental impact on bone health in men in both the cortical and trabecular compartments at the distal radius with similar results in women in the trabecular compartment between none/minimal alcohol and low alcohol at the distal tibia suggesting that avoidance of alcohol may be beneficial for bone health.     

Effects of Nicotine on Bone and Calciotropic Hormones in Growing Female Rats

Limited research in young adults and immature animals suggests a detrimental effect of tobacco on bone during growth. This study investigated the effects of nicotine, the major alkaloid component of tobacco, on calciotropic hormone concentrations and bone status in growing female rats. One-month-old animals received either saline (n = 10), nicotine at 3.0 mg/kg/day (n = 10), or nicotine at 4.5 mg/kg/day (n = 10) administered subcutaneously via osmotic minipumps for either 2 or 3 months. Sera, femora, tibiae, and lumbar vertebrae (3–5) were collected at necropsy. The concentrations of serum calcium, phosphorus, 25-hydroxyvitamin D, 1,25-dihydroxyvitamin D, parathyroid hormone, calcitonin, and insulin-like growth factor-I were determined. Bone variables evaluated included mineral content and density (vertebrae and femora), cancellous and cortical histomorphometry (tibiae), and bone strength (vertebrae and femora). Statistically significant differences in serum mineral and hormone concentrations were not associated with nicotine dose or exposure time. No significant nicotine treatment effects were detected for bone mineral content and density, bone histomorphometry, or bone strength. We conclude that nicotine treatment for 2 or 3 months at serum concentrations in the upper range of those found in smokers has no detrimental effect on bone mass, volume, or strength in the growing rat. Received: 20 May 1999 / Accepted: 21 January 2000     

An experimental comparative study of hydroxyapatite and tricalcium-phosphate as bone substitutes

Hydroxyapatite (HAP) and beta tricalcium-phosphate (TCP) were implanted into the femoral distal metaphysis of 18 adult mongrel dogs. X-ray pictures were periodically taken following surgery, and the animals were sacrificed at 2, 4, 8, 12, 24 weeks, and 1, 2 years postoperatively. The undecalcified specimens were stained using Cole HE stain. During 2 years following surgery, both radiological and macroscopic examinations revealed no significant change in either HAP or TCP. In the undecalcified specimens, an apparent change in configuration was only noticed in the TCP, but there was no difference in the degree of new bone formation between HAP and TCP implants. At 2 years postoperatively, the Ca/P ratios of the HAP and TCP implants remained constant. The results of this study therefore suggest that TCP is a promising autograft extender.     

The affinity of bone to hydroxyapatite and alumina in experimentally induced osteoporosis

The authors studied how bone reacts to hydroxyapatite (HAP) and alumina (Al2O3) in osteoporosis experimentally induced in a rat animal model by resection of the sciatic nerve. Sixteen and 32 weeks after resection of the sciatic nerve, implants of HAP and Al2O3 were inserted into rat tibias. The reaction of bone to HAP was compared with its reaction to Al2O3, which is bioinert, as a control. The results were analyzed using the affinity index (the length of bone directly opposed to the implant without an intervening fibrous membrane/total length of the bone-implant interface x 100%). In the rats left for 16 weeks after resection of the sciatic nerve the index values 24 weeks after insertion of HAP and Al2O3 were 96.9 +/- 2.4% (N = 8) and 75.2 +/- 22.2% (N = 8), respectively. The index value of HAP was 97.2 +/- 2.6% (N = 12) and of Al2O3 was 86.9 +/- 15.4% (N = 12) at 24 weeks after insertion, when the rats were first left for 32 weeks, after sciatic nerve resection. Bone was demonstrated to have a superior affinity to HAP, compared with Al2O3 (P less than .05).     

Relative effects of wound healing and mechanical stimulus on early bone response to porous-coated implants

We hypothesized that early bone adaptation to well fixed porous-coated implants is influenced more by wound healing than by mechanical loading. To test this hypothesis, two groups of dogs with identical, hydraulically controlled porous-coated implants interference fit within distal femoral trabecular bone were used. One group had no load; the other had 35 N of load applied to the implants. At 5 weeks after surgery, the resulting adaptation of bone around the implants was quantified on a cellular basis by cytochemical analysis of type-I procollagen synthesis and on a structural basis using three-dimensional micro-computed tomography imaging. The percentage of trabecular surfaces covered by osteoblasts expressing type-I procollagen was significantly increased in bone surrounding the implant in both groups compared with contralateral control bone tissue. There was no difference between the groups with no load or 35 N of load. In addition, measures of trabecular bone structure did not differ significantly between the load and no-load groups. Taken together, these results suggest that wound healing plays a much greater role in the early response of bone to well fixed porous-coated implants than does mechanical stimulus.     

Bioactive and biodegradable silica biomaterial for bone regeneration

Biosilica, a biocompatible, natural inorganic polymer that is formed by an enzymatic, silicatein-mediated reaction in siliceous sponges to build up their inorganic skeleton, has been shown to be morphogenetically active and to induce mineralization of human osteoblast-like cells (SaOS-2) in vitro. In the present study, we prepared beads (microspheres) by encapsulation of β-tricalcium phosphate [β-TCP], either alone (control) or supplemented with silica or silicatein, into the biodegradable copolymer poly(d,l-lactide-co-glycolide) [PLGA]. Under the conditions used, ≈ 5% β-TCP, ≈ 9% silica, and 0.32 μg/mg of silicatein were entrapped into the PLGA microspheres (diameter ≈ 800 μm). Determination of the biocompatibility of the β-TCP microspheres, supplemented with silica or silicatein, revealed no toxicity in the MTT based cell viability assay using SaOS-2 cells. The adherence of SaOS-2 cells to the surface of silica-containing microspheres was higher than for microspheres, containing only β-TCP. In addition, the silica-containing β-TCP microspheres and even more pronounced, a 1:1 mixture of microspheres containing β-TCP and silica, and β-TCP and silicatein, were found to strongly enhance the mineral deposition by SaOS-2 cells. Using these microspheres, first animal experiments with silica/biosilica were performed in female, adult New Zealand White rabbits to study the effect of the inorganic polymer on bone regeneration in vivo. The microspheres were implanted into 5 mm thick holes, drilled into the femur of the animals, applying a bilateral comparison study design (3 test groups with 4–8 animals each). The control implant on one of the two hind legs contained microspheres with only β-TCP, while the test implant on the corresponding leg consisted either of microspheres containing β-TCP and silica, or a 1:1 mixture of microspheres, supplemented with β-TCP and silica, and β-TCP and silicatein. The results revealed that tissue/bone sections of silica containing implants and implants, composed of a 1:1 mixture of silica-containing microspheres and silicatein-containing microspheres, show an enhanced regeneration of bone tissue around the microspheres, compared to the control implants containing only β-TCP. The formation of new bone induced by the microspheres is also evident from measurements of the stiffness/reduced Young's modulus of the regenerated bone tissue. The reduced Young's modulus of the regenerating bone tissue around the implants was markedly higher for the silica-containing microspheres (1.1 MPa), and even more for the 1:1 mixture of the silica- and silicatein-containing microspheres (1.4 MPa), compared to the β-TCP microsphere controls (0.4 MPa). We propose that based on their morphogenetic activity on bone-forming cells in vitro and the results of the animal experiments presented here, silica/biosilica-based scaffolds are promising materials for bone repair/regeneration.     

Screw Fixation in Bone of Guinea Pigs Sensitized to Nickel and Cobalt

A screw was inserted transversely through the distal femora and proximal tibiae of guinea pigs sensitized to either Ni or Co. An equal number of nonsensitized animals were treated in the same way. The animals with Ni allergy received an ASIF stainless steel screw and the animals with Co allergy a Howmedica Vitallium screw. After 6 weeks no obvious differences were observed in the histological picture around the femur screws that could be ascribed to metal sensitivity. The mechanical strength of the bone adjacent to the tibial screws was tested by measuring maximum torque during continuous tightening of the screws at a rate of 2 revolutions/second. No significant differences in maximum torque resistance were found between sensitized and nonsensitized animals. The results indicate that sensitivity to Ni or Co does not compromise the mechanical fixation between metal and bone during the early period after the implantation.     

Apparent absence of local response to bone screws in guinea pigs with contact sensitivity

Guinea pigs sensitized to nickel, cobalt, or chromium were used to study if impaired fixation occurred as a result of allergy against metal implants in bone. Screws made from stainless-steel (ASTM F55, F138-139) or cobalt chromium (ASTM F75-76) were inserted in the right and left proximal tibiae, and the mechanical strength of the fixation to the bone was evaluated after 4 months. In addition, we studied the histology on decalcified sections of the implant bone interface from the distal femur where the same type of screws had been inserted. To see if changes in bone density occurred in the proximal tibia as a result of allergy, the amount of ash was determined. Although the animals maintained their contact sensitivity throughout the experimental period, there were no differences between allergic and control animals for any of the parameters studied. All screws were well fixed in the tibia at the end of the experiment, and histologically we did not find any significant differences between allergic and control animals by qualitative examination. The experiment supports our earlier results from a 6-week experiment and indicates that contact allergy may be unimportant for the fate of orthopedic implants.     

Primary cancellous bone formation with BMP and micro-chambered beads: Experimental study on sheep

ProblemThe physiological reconstruction of cancellous bone defects in surgery of the locomotor system is an unsatisfactorily solved problem.AimsThe aims of this study are to examine whether micro-chambered ß-tricalcium-phosphate (ß-TCP) beads provide a certain capillary force suctioning in blood and bone marrow thus forming a stable “negative”-replica of the bone marrow spaces. If so, a new approach for osteoconduction would yield primarily a scaffold of lamellar cancellous bone under load without a long-lasting remodeling process. Recombinant human bone morphogenetic protein (rhBMP) might even enhance all processes of defect healing, remodeling and ß-TCP resorption; gentamicin-loaded ε-caprolactone might protect the implant.Material and methodsTen sheep were operated on; the patella-groove model and the tibial head were used. A defect of 9.4 × 20 mm was created using wet-grinding-diamond instruments. Micro-chambered ß-TCP-beads of 4–6 mm with 0.35 mg rhBMP-7 + 0.1 g collagen per animal, or 1.5 g demineralized bone matrix (DBM) paste on the contra-lateral side were implanted. Both osteoinduction groups were compared with the defect in the tibial heads where plain micro-chambered ceramic beads were inserted. Added to the beads was 12.5 mg gentamicinsulphate in 12.5 mg ε-caprolactone-carrier. Outward diffusion was prevented using a 1-mm-thick press-fit inserted ceramic lid. The bone healing, remodeling and resorption of the ceramic in a right–left comparison of the patella groove and the tibial head was examined at 6 weeks, 2 and 3 months; one animal in reserve was followed for 14 months. The animals were perfusion-fixed, the vasculature micro-casted with an acrylate and nondemineralized processed, and with μ-CT and microscopically documented.ResultsA primary load-bearing spongiosa had developed around the beads, which shortened the remodeling process. The strong micro-chambered, resorbable ß-TCP-beads demonstrate high capillary strength, resorb blood and bone marrow, and represent a stable formative material which, as a carrier for the controlled local release of BMP, that accelerates bone healing, shortens resorption and remodeling compared with plain and DBM loaded implants.ConclusionMicro-chambered beads represent the bone-forming element, BMP yields a fast defect healing and enhanced remodeling of bone and resorption of ß-TCP compared to delayed and incomplete reconstruction and resorption of ß-TCP on the DBM-side, the plain implants reached nearly the same reconstruction, but far later compared with the BMP loaded implants.     

Epimedium-derived flavonoids promote osteoblastogenesis and suppress adipogenesis in bone marrow stromal cells while exerting an anabolic effect on osteoporotic bone

BackgroundEpimedium-derived flavonoids (EFs) have been reported to prevent bone loss in ovariectomized (OVX) rats and late postmenopausal women but the underlying mechanism of the anabolic effect is unknown.ObjectiveThis study aimed to investigate the effect of EFs on osteoporotic bone using histomorphometry and on osteoblastogenesis/adipogenesis of bone marrow stromal cells (BMSCs).Methods11-month-old female Wistar rats were divided into Sham, OVX, Sham + soluble vehicle (Sham + SV), OVX + SV and OVX + EFs (10 mg/kg/day) groups. 3 months after surgery, rats from the first two groups were euthanized to verify the establishment of OVX-induced osteoporosis. Other groups were orally treated with either daily SV or EFs for 4 months. At sacrifice, serum was analyzed for the levels of osteocalcin and TRACP 5b, BMD in the proximal femur was measured by pQCT. Static and dynamic bone histomorphometry was performed in proximal tibiae with microCT and undecalcified sections, respectively. The effect of EF treatment on differentiation of rat BMSCs was assessed by colony formation assays and gene expression analysis, respectively. Gene expression, ALP activity and adipocyte numbers were determined in differentiating human BMSCs after exposure to conditioned serum from SV- or EFs-treated OVX rats.ResultsThe serum level of osteocalcin was higher and TRACP 5b was lower in EFs versus SV-treated OVX rats. BMD, BV/TV, Tb.N and Conn.D in EFs-treated OVX rats were significantly greater than those of SV-treated OVX rats. Bone histomorphometric parameters OS/BS, MAR, and BFR/BS were significantly higher in EFs versus SV-treated OVX rats. EFs significantly increased osteogenesis and decreased adipogenesis of BMSCs, as evidenced by CFU-ALP and CFU-Adipo assays, respectively. The mRNA level of Runx2 and bone sailoprotein was significantly higher while PPARγ2 was significantly lower in BMSCs from EFs-treated versus SV-treated OVX rats. ALP activity and Runx2 mRNA was significantly higher while adipocyte number and PPARγ2 mRNA was significantly lower in human BMSCs after exposure to conditioned serum from EFs versus SV-treated OVX rats.ConclusionEFs exerted anabolic effect on osteoporotic bone by concomitantly promoting osteogenic and suppressing adipogenic differentiation of BMSCs.