Biomedical subjects

R Giardino

Publications and source records attributed to R Giardino.

At least 19 recordsLinked to original sources

PubMed2000

Autologous bone marrow stromal cells loaded onto porous hydroxyapatite ceramic accelerate bone repair in critical-size defects of sheep long bones.

The ability of marrow-derived osteoprogenitor cells to promote repair of critical-size tibial gaps upon autologous transplantation on a hydroxyapatite ceramic (HAC) carrier was tested in a sheep model. Conditions for in vitro expansion of sheep bone marrow stromal cells (BMSC) were established and the osteogenic potential of the expanded cells was validated. Ectopic implantation of sheep BMSC in immunocompromised mice led to extensive bone formation. When used to repair tibial gaps in sheep, cell-loaded implants (n = 2) conducted a far more extensive bone formation than did cell-free HAC cylinders (n = 2) over a 2-month period. In cell-loaded implants, bone formation was found to occur both within the internal macropore space and around the HAC cylinder while in control cell-free implants, bone formation was limited mostly to the outer surface and was not observed in most of the inner pores. As tested in an indentation assay, the stiffness of the complex HAC-bone material was found to be higher in cell-loaded implants compared to controls. Our pilot study on a limited number of large-sized animals suggests that the use of autologous BMSC in conjunction with HAC-based carriers results in faster bone repair compared to HAC alone. Potentially this combination could be used clinically in the treatment of extensive long bone defects.

Animals↗

PubMed2000

Isolation and characterization of osteoblast cultures from normal and osteopenic sheep for biomaterials evaluation.

Being very useful in the analysis of bone cell differentiation and activity, osteoblast cultures are also used in the in vitro biocompatibility study of new materials. The aim of this work was to evaluate sheep osteoblast cultures derived from normal and ovariectomized animals, and then to assess the in vitro biomaterial behavior on these cultures, taking into account the quality of bone where orthopedic devices are clinically used. For this purpose, we characterized sheep osteoblast cultures, isolated from iliac crest bone of normal (NB osteoblast culture) and osteopenic after ovariectomy (OB osteoblast culture) sheep. Moreover, we studied cell behavior when cultured on different biomaterials (titanium and two biological glasses, RKKP and AP40). Cell characterization at baseline demonstrated that both cultures (NB and OB) showed normal osteoblastic behavior. On the contrary, osteoblasts derived from osteopenic bone and cultivated on AP40 for 6 days revealed a different behavior in terms of both cell morphology and metabolic activity. Statistical analysis (one-way analysis of variance and Scheffé's post hoc multiple-comparison tests) revealed significant differences in Ca level (p<0.0005), MTT test (p<0.0005) and OC production (p<0.05). These in vitro tests demonstrated that sheep osteoblast cultures can be useful when determining biocompatibility and osteointegration of orthopedic materials, and also when evaluating for the presence of osteoporosis.

Animals↗

PubMed2000

Evaluation of pain and stress levels of animals used in experimental research.

This paper represents a contribution to researchers employing "in vivo" models in experimental surgery in order to obtain more reliable results in accordance with current legislation and Russel's "three R" statement: refinement, reduction, and replacement. After general consideration about the definition of pain and stress concerning laboratory animals, the authors suggest making an evaluation of the experimental protocol before approval by the local committee to allow assessment in terms of the costs/benefits of experimental research employing live animals.

Animals↗

PubMed2000

Guided regeneration with resorbable conduits in experimental peripheral nerve injuries.

Guided tissue regeneration is a new approach in the reconstructive surgery of peripheral nerves. Artificial conduits can be constructed from biodegradable polymers. Lactic/caproic acid copolymers and polyphospazenes are biocompatible materials with a slow resorption rate. Conduits made from either poly-[1-lactide-co-6-caprolatone] or poly-[bis-(ethylalanate)-phosphazene] were assessed for use as guides for nerve regeneration in experimental animals. Under general anesthesia and by using a microsurgery technique both sciatic nerves were exposed in 2 groups of 9 Wistar rats. On the right side, a 10 mm segment of the nerve was removed, and the defect was then repaired using a conduit. On the left side, the same defect was bridged using as an autograft the nerve segment, which had been removed from the right sciatic nerve. Histological and electron microscopy investigations were performed after 30, 90 and 180 days and showed the gradual degradation of both types of conduits without any evidence of local toxicity. The regeneration of the nerve fibers in the lumen was not significantly different from that shown by the autologous grafts. Likewise, no differences were found at 180 days in the functional recovery of the nerve (evoked muscle action potential). Both conduits were found to be effective for guided nerve regeneration. Poly-[1-lactide-co-6-caprolactone] tubes were easier to insert, while polyphosphazene conduits allowed the use of neurite-promoting factors.

Animals↗

PubMed2000

Discriminant capacity of quantitative ultrasound versus dual X-ray absorptiometry to determine cancellous bone loss in ovariectomized rats.

The capacity of dual x-ray absorptiometry and quantitative ultrasound to discriminate bone loss and to predict the mechanical and microarchitectural properties of cancellous bone in an animal model of osteopenia was evaluated. Thirty-five female Sprague-Dawley rats (10 months old) were randomized into three groups: baseline group, 10 rats killed at the beginning of the study; ovx group, 15 rats ovariectomized; and sham group, 10 rats sham operated. At the beginning and end of the study, all the animals underwent osteosonography to record the proximal tail (C3 vertebra) bone speed of sound. Sixteen weeks after surgery, the animals were euthanized and the L5-6 lumbar vertebrae of each rat were excised for densitometric, biomechanical (compression test), and histomorphometric studies. Significant differences were found among the groups for final speed of sound (p = 0.01). The L5 bone mineral density of the ovx group decreased by 12.1% (p = 0.049) and 12.6% (p = 0.035) compared, respectively, with baseline and sham groups. The biomechanical parameters of the ovx group decreased by 15-47% compared with the other groups, showing significant differences between the ovx and sham groups both for maximal stress (p = 0.026) and elastic modulus (p = 0.013). Histomorphometric parameters of the ovx group showed significant decreases in comparison with other groups. Logistic regression analysis showed that dual X-ray absorptiometry and quantitative ultrasound discriminate ovariectomized and healthy rats with a similar capacity, classifying correctly all rats used in the model in a range of 61-70%. This similar capacity seems to derive from two different capacities to detect bone changes. Dual X-ray absorptiometry, depending on bone mineralization and density, is able to detect modifications in bone stiffness and strength, confirmed also by the correlation with biomechanical data. On the contrary, quantitative ultrasound seems to depend more on cancellous bone microarchitecural changes because it is correlated to histomorphometric parameters.

Absorptiometry, Photon↗

PubMed2000

Coupled plasma filtration-adsorption in a rabbit model of endotoxic shock.

OBJECTIVE: To test the hypothesis that nonselective adsorption by a hydrophobic resin of cytokines and other proinflammatory mediators could improve 72-hr survival in a rabbit model of endotoxic shock. DESIGN: Prospective, randomized, controlled animal trial. SETTING: Animal care facility at a research institution. SUBJECTS: A total of 109 New Zealand white male rabbits. INTERVENTIONS: Anesthetized rabbits were cannulated with indwelling femoral arterial and venous lines. Septic shock was induced by a single intravenous injection of Escherichia coli lipopolysaccharide. The dose was experimentally assessed in 40 rabbits receiving 1.0, 0.5, 0.1, and 0.05 mg/kg body weight to determine LD80 at 72 hrs. Extracorporeal circulation consisted of plasma filtration coupled with passage of the plasma filtrate through a hydrophobic sorbent and reinfusion into the venous line. The extracorporeal treatment lasted for 3 hrs. Rabbits injected with endotoxin (0.05 mg/kg) were submitted to plasma filtration with (19 rabbits) or without (20 rabbits) sorbent adsorption. As controls, rabbits injected with vehicle alone were treated with plasma filtration (ten rabbits) or without (ten rabbits) sorbent adsorption. Ten rabbits were monitored under anesthesia to determine basal survival. MEASUREMENTS AND MAIN RESULTS: Plasma concentrations of endotoxin, bioactive tumor necrosis factor, resin-adsorbed platelet-activating factor, mean arterial pressure, base excess, and white cell count were assessed and a global severity score was established. At 72 hrs, cumulative survival was significantly (p = .0041) improved in septic rabbits treated with coupled plasma filtration-adsorption. Circulating tumor necrosis factor bioactivity remained similar in control and treated rabbits. Biologically significant amounts of platelet activating factor were eluted from the sorbent during the entire treatment time. The severity score inversely correlated with survival (p < .001). CONCLUSIONS: Coupled plasma filtration-adsorption improved survival in a rabbit model of endotoxic shock. Coupled plasma filtration-adsorption may be an extracorporeal treatment capable of removing structurally different inflammatory mediators associated with sepsis.

Animals↗

PubMed2000

In vitro pathological model of osteopenia to test orthopaedic biomaterials.

The association of in vitro tests and in vivo bone implants, has significantly improved the characterization of biomaterials for orthopaedic devices before their clinical use. However, neither cell cultures nor most animals models used for these tests entirely reflect the clinical conditions in which biomaterials are implanted. Pathological animal models are considered to substantially improve our knowledge of osteointegration of biomaterials; for this reason researchers increasingly use aged, osteopenic or arthritic animals in their experimental tests. The development of "pathological cell cultures" would also be of great importance for the study of biomaterials. It would allow a complete material evaluation, beginning with a biocompatibility test to a more finalized and specific preclinical evaluation. The present study, looks at the possibility of using cell culture methodology for the improvement of in vitro biomaterials characterization in the case of osteopenia. Cultures derived from normal (NB-OST) rats were compared to those of osteopenic (OB-OST) rats, by testing the osteoblasts against common parameters of characterization. Moreover, the reaction of these cultures to two biological glasses of known in vivo behavior (both in normal and osteopenic bone) by means of parameters on biocompatibility and bone formation index, was evaluated. Our results showed that there was no evidence of differences between the NB-OST and OB-OST cultures. After 6 days of culturing, the bioglass that did not osteointegrate in osteopenic animals, did not induce cytotoxicity in NB-OST, but a significative reduction of viability and Osteocalcin level in OB-OST was observed. We think that these data should stimulate researchers to develop further tests in order to improve preliminary in vitro comprehension on biomaterials.

Alkaline Phosphatase↗

PubMed2000

Biostimulation of human chondrocytes with Ga-Al-As diode laser: 'in vitro' research.

The aim of this study was to verify the effects of laser therapy performed with Ga-Al-As Diode Lasers (780 nm, 2500 mW) on human cartilage cells in vitro. The cartilage sample used for the biostimulation treatment was taken from the right knee of a 19-year-old patient. After the chondrocytes were isolated and suspended for cultivation, the cultures were incubated for 10 days. The cultures were divided into four groups. Groups I, II, III were subject to biostimulation with the following laser parameters: 300 J, 1 W, 100 Hz, 10 min. exposure, pulsating emission; 300 J, 1 W, 300 Hz, 10 min. exposure, pulsating emission; and 300 J, 1 W, 500 Hz, 10 min. exposure, pulsating emission, respectively. Group IV did not receive any treatment. The laser biostimulation was conducted for five consecutive days. At the end of the treatment, the Calcium, Alkaline Phosphate, MTT tests and proteoglycan were performed to assess cell metabolism and toxicity level. The data showed good results in terms of cell viability and levels of Ca and Alkaline Phosphate in the groups treated with laser biostimulation compared to the untreated group. The results obtained confirm our previous positive in vitro results that the Ga-Al-As Laser provides biostimulation without cell damage.

Adult↗

PubMed2000

Assessment of low-power laser biostimulation on chondral lesions: an "in vivo" experimental study.

The purpose of this study was to evaluate whether intraoperative laser biostimulation can enhance healing of cartilaginous lesions of the knee. Surgery was performed on eighteen rabbits: a bilateral chondral lesion of 1.25 +/- 0.2 mm in length and 0.8 +/- 0.2 mm in width was created in the femoral medial condyle with a scalpel. The lesion in the left knee of each animal was treated intraoperatively using the diode Ga-Al-As 780nm. laser (300 Joules/cm2, 1 Watt, 300 Hertz, 10 minutes), while the right knee was left untreated, as control group. The animals were divided into three groups, A, B and C, according to the survival time after surgery, two, six and twelve weeks, respectively. The explants from the femoral condyles, both treated employing laser energy and left untreated, were examined histologically. Results showed a progressive filling with fibrous tissue of the cartilaginous lesion treated with laser irradiation, while no changes in the original lesion of the untreated group were observed at the end of the study. Maybe, in this experimental research, underexposure to laser irradiation was the cause for the absence of the necessary conditions for biostimulation.

Animals↗

PubMed2000

Osteochondral lesion repair of the knee in the rabbit after low-power diode Ga-Al-As laser biostimulation: an experimental study.

The purpose of this study was to evaluate whether low-power laser biostimulation of the osteo-chondral lesions of the knee could by itself reduce repair healing time. Surgery was performed on eighteen rabbits; a bilateral osteo-chondral lesion of 2.5mm in diameter and 2mm depth was created in the femoral medial condyle with a drill. The left knee of each animal was treated intraoperatively using the diode Ga-Al-As laser (780nm) with the following parameters: 300 Joules/cm2, 1 Watts, 300 Hertz, 10 minutes; the right knee was left untreated, as control group. The animals were divided into three groups, A, B and C, according to the survival time after surgery, two, six and twelve weeks, respectively. The explants from the femoral condyles, either treated employing laser energy or left untreated, were examined histomorphometrically. Results after laser treatment showed faster healing of the lesion at week 2 (p=0.043) and an overall improvement in cellular morphology (p=0.044), while a more regular aspect of the osteocartilaginous tissue was observed at week 12 (p=0.004). A relationship between laser biostimulation properties and healing of the osteo-chondral defect has been demonstrated.

Aluminum↗

PubMed2000

The ovariectomised sheep as a model for testing biomaterials and prosthetic devices in osteopenic bone: a preliminary study on iliac crest biopsies.

A histomorphometric and ultrastructural evaluation on sheep iliac bone was performed. Six sheep were ovariectomised (OVX Group) and 6 were left intact (Sham-aged, Control Group). An iliac crest biopsy was performed randomly in 6 animals at the beginning of the study, then, in all the animals, after 12 and 24 months. A significant decrease in trabecular bone volume, trabecular thickness (p<0.0005) and cell volume (p<0.005) was observed in OVX animals. A modest decrease in trabecular number and osteoid thickness together with an increase in trabecular separation were observed in OVX animals at 12 and 24 months. The osteoid volume showed a significant difference (p<0.05) between the groups. In OVX animals, at 12 months, Scanning Electron Microscopy revealed an enlargement of the trabecular space and a progressive replacement of bone matrix with adipose tissue. These signs were accentuated at 24 months. In conclusion, OVX sheep showed a loss of trabecular bone starting at 12 months after ovariectomy. The developed osteopenic state may be considered as a useful tool when doing research on biomaterial osteointegration.

Analysis of Variance↗

PubMed2000

A resorbable biomaterial shaped as a tubular chamber and containing stem cells: a pilot study on artificial bone regeneration.

In a previous study, we showed how healing of non-union defects in rabbit radii can be achieved by means of a tubular resorbable chamber, in comparison with untreated defects. In the present study, we placed bone marrow stem cells inside the chamber. Bone marrow was obtained by percutaneous aspiration from the iliac crest in 9 adult New Zealand rabbits. Stem cells were separated by the centrifugation technique. In the same animals, a defect of 10 mm was created in both radii. On the left side, the defect was treated with the poly-DL-Lactide chamber, in which a suspension of autologous cells was injected; on the right side, only autologous cells were used. Radiological and histomorphometric data were compared within this study as well as with the results of our previous study. At 3, 6 and 9 months, there was no healing on the right side..On the left side, progressive bone formation with reunion of the stumps was observed in the chamber. We conclude that stem cells can accelerate bone healing when contained in the tubular chamber.

Animals↗

PubMed1999

Measurement of insertion torque of tapered external fixation pins: A comparison between two experimental models.

The aim of this study was to compare an in vitro versus an ex vivo experimental model to test the insertion torque of two different types of external fixation pins. A torque measuring machine was developed in order to perform accurate measurements. Forty tapered pins made of stainless steel were utilized. Half of the pins were plasma-spray coated with hydroxyapatite (HA) and the other half remained uncoated. For the in vitro model 20 cylinders were used that were made of synthetic polymer according to ASTM standards. For the ex vivo model 10 fresh femora harvested from adult sheep were used. All the pins were implanted after predrilling, and insertion torque was measured. Statistical analysis of the in vitro versus the ex vivo model showed significant differences in both coated (p < 0. 0005) and uncoated (p = 0.002) external fixation pins. These results may be due to the surface roughness that caused significant friction between the HA coating and the polyvinylchloride in the in vitro model. The significant difference between the in vitro and ex vivo results lead us to state that the in vitro model does not realistically simulate the behavior of external fixation pins implanted in bone.

Animals↗

PubMed1999

Reconstruction of extensive long-bone defects in sheep using porous hydroxyapatite sponges.

The capacity of hydroxyapatite (HA) implants to support large defect repair in weight-bearing long bones of large size animals was investigated. Diaphyseal resections 3.5 cm of the tibia were performed in five adult sheep. They were substituted with HA macroporous ceramic cylinders anatomically shaped, and an external fixator was assembled. The sheep were sacrificed at 20, 40, 60, 120, and 270 days after surgery, respectively. Histology and micro X-ray study of resected implants and adjacent tissues showed proper integration of ceramic with newly formed periosteal bone as early as 20 days after surgery. In one sheep, the external fixator was removed 5 months after surgery. The animal gained the ability to walk with no functional impairment until it was sacrificed 4 months later. At this time, extensive integration of ceramic with bone was detected radiographically and confirmed by a morphological study of the resected sample. Our data indicate that large defects in a weight-bearing long bone can be repaired to the extent necessary for full functional recovery in large animals. These data set the stage for further intervention on material properties as well as for preliminary attempts to use ceramic prostheses for reconstruction of large bone defects in humans.

Animals↗

PubMed1999

The mechanical properties of fluoride-treated bone in the ovariectomized rat.

The effect of fluoride therapy on the osteopenic bone of the ovariectomized rat was studied by comparing the densitometric and biomechanical data. Forty retired breeder female Sprague-Dawley rats aged 10 months were randomly divided into five groups. One group (Group A) was killed at the beginning of the study and was used as a baseline. Three groups were ovariectomized and one was sham-operated (Group B) and observed for the same period as a sham-aged group. A group of ovariectomized rats was used as a sham therapy control (Group C) and received only deionized drinking water; the other two groups (F1 and F2) received L-glutamine monofluorophosphate and calcium at a rate of 1:30 F/Ca at different doses by gavage (0.57 mg F/17 mg Ca per kg/day-Group F1; 0.21 mg F/6.30 mg Ca per kg/day-Group F2). Densitometric and biomechanical (compression and three-point bending test) assays, X-ray diffraction, and Fourier transformed infrared spectroscopy were performed on femoral specimens. Biomechanical data showed that the femoral heads of Group F2 required a significantly greater energy-to-failure than Group C (P < 0.05) as well as treated femoral diaphysis when compared with the others (P < 0.01). Significant increases in the elastic modules were observed in fluoride-treated groups (P < 0.001) when compared with other groups. Diffractometric and spectroscopic data showed the presence of fluorine-apatite in both treated groups with a high component of carbonates. Also, fluoride therapy causes an increase of bone stiffness due to the presence of fluoroapatite. It seems to produce two opposed properties in the osteopenic rat bone: a higher resistance to compression loading and a greater frailty to flexion loading.

Animals↗

PubMed1999

Xenogenic demineralized bone matrix: osteoinduction and influence of associated skeletal defects in heterotopic bone formation in rats.

Demineralized bone matrix (DBM) was ectopically implanted in 36 male Wistar rats. In 18 of the animals a bone defect in the femoral condyles was also created: the left was filled with DBM and the right was left empty as a control. The animals were killed after 2, 4 and 6 weeks and new bone was histologically evaluated, comparing ectopic bone formation with or without distant bone injury. Results showed: (1) osteoinductivity of xenogenic DBM, and (2) earlier mineralization of ectopically implanted DBM in the group with associated skeletal injury. Our results show that xenogenic bone matrix acts as an osteoinductive material and that skeletal injury improves osteogenesis at distant sites.

Animals↗

PubMed1999

Effects of essential amino acids and lactose on bony fractures and defects in rabbits: a preliminary histomorphometric study.

An experimental study was performed in order to test the possibility of improving bone repair with the administration of a drug (Calciofix, Farmaceutici Damor SpA, Naples, Italy) containing essential amino acids and lactose. Fifty rabbits were submitted to an open transversal fracture of the left fibula and to a right femoral condyle defect. They were left untreated or treated daily with the drug orally and were divided into subgroups depending on the experimental time: 15, 30, 40, 50, 60 days. Histomorphometric data showed a significantly faster healing rate in treated animals compared with untreated ones. Firstly, on day 30 there was a significantly larger amount of cartilage in the control bone callus (P < 0.01). On day 50 a significant difference existed between trabecular and lacunar percentages in the two subgroups (P < 0.0005). At 60 days no significant differences were observed, but bony trabeculae had become more oriented parallel to the long axis of the bone in treated animals. Secondly, after 15 days the defect area was significantly smaller in treated animals than in the untreated ones (P < 0.01). At 30 and 40 days, respectively, significant differences existed between the two subgroups in connective tissue and mature bone percentages (P < 0.01 and P < 0.001). Our results seem to demonstrate that the drug significantly accelerates the rate of bone formation in fractures and bone defects in rabbits.

Amino Acids↗

PubMed1999

In vitro and in vivo behaviour of Ca- and P-enriched anodized titanium.

The influence of different surface preparations on titanium biocompatibility and bone integration was evaluated. Commercially grade 2 titanium rods (diameter 2 mm, length: 3 mm), vacuum annealed and hydrofluoric acid etched was selected for its promising surface characteristics to achieve good direct osseointegration. Some rods were surface modified by Anodic Spark Discharge anodization and a thin layer (approximately 5 microm) of amorphous TiO2 containing Ca and P (Ti/AM) was obtained. Some of the Ti/AM specimens underwent a further hydrothermal treatment to produce a thin outermost layer (approximately 1 microm) of hydroxyapatite (Ti/AM/HA). Cytotoxicity tests (direct contact: ISO 10993-5) showed good cytocompatibility for all tested samples. Ti and tissue culture substrate + DMEM control, respectively, were associated with a significant higher proportion of attached cells than Ti/AM and Ti/AM/HA (P < 0.0005), but this was in the normal range of 10-20% of unattached cells for cytocompatible materials. Histomorphometric analysis conducted on samples inserted in the cancellous bone of distal femoral epiphysis of Sprague-Dawley rats gave the following results at 4 and 8 weeks: Affinity index (AI%) data proving the surface osteconductive properties of non-anodized acid etched Ti (AI-4 weeks: 67.1 +/- 17.0%; AI-8 weeks: 74.8 +/- 11.5%). Ti/AM samples showed the lowest values (AI-4 weeks: 45.8 +/- 15.9%; AI-8 weeks: 68.5 +/- 13.6%) while the best performances of the Ti/AM/HA samples (AI-4 weeks: 60.4 +/- 21.8%; AI-8 weeks: 79.5 + 9.37%) indicated that hydroxyapatite allowed a higher bone to implant contact respect to Ti only. Further investigations should be performed in order to better understand the mechanism of observed in vitro behaviour and to achieve information on long-term osseointegration process.

Analysis of Variance↗