Bioactive glass granules as extender of autogenous bone grafting in cementless intercalary implant of the canine femur.

BACKGROUND AND AIMS: Ceramic bone graft substitutes have a potential to be used as replacement of allogeneic bone grafting and, under optimal distribution of particle size, they may even provide mechanical support. The current study examined the efficacy of bioactive glass granules as an extender of autogenous bone grafting in a segmental bone replacement model of the canine femur. MATERIAL AND METHOD: A 16 mm long segment of the femur shaft was bilaterally replaced with an intercalary titanium implant in eight animals. The implant had cementless grooved proximal and distal stems. In one leg, the peri-implant space was packed with composite graft consisting of a mixture of bioactive glass granules and autogenous bone graft in proportion of 50:50. In the opposite leg, the peri-implant space was treated with autogenous bone graft alone. After surgery, unlimited functional loading was allowed. The outcome was evaluated at three months. RESULTS: Eight out of sixteen autografted implants and seven out of sixteen composite-grafted implants were radiographically incorporated and clinically stable at three months. In the paired comparison, the proximal components of composite-grafted implants showed lower maximum load under torsional testing (p = 0.068), less new bone in the longitudinal grooves of the stems (p = 0.036) and lower affinity of new bone to implant surface (p = 0.046). The distal components of the two sides showed a similar trend for less new bone in the grooves and lower bone affinity of new bone in the distal composite-grafted components. CONCLUSIONS: The current study suggests that supplementation of periprosthetic bone graft with bioactive ceramic particles may not help to promote healing of cementless implants under high dynamic loading conditions.

Orbital floor reconstruction with poly-L/D-lactide implants: clinical, radiological and immunohistochemical study in sheep.

In this study the reconstruction capacity of orbital wall in sheep was evaluated when poly-L/D-lactide (PLDLA96) implants were used for large blow-out defects in 18 sheep. The contralateral side, where the defects healed spontaneously, served as controls. The follow-up was 12, 16, 22 and 36 weeks. Healing was evaluated clinically, radiologically, histologically and immunohistochemically. Physiochemical properties of the implants were also studied. At first, the implants were surrounded by elastic capsules, which gradually ossified. At 36 weeks, 60% were still visible and deformed but surrounded by bone. Light microscopy revealed a low grade inflammatory reaction. Expression of Tn-c and cFn was intense throughout the study. Shear strength decreased gradually and was not measurable after 16 weeks. Crystallinity increased steadily from 1.5 to 29.30% and molecular weight decreased from 49,000 to 4186. In CT, the final bony defect was smaller in the reconstructed sides than in the controls. Based on this study it can be concluded that PLDLA96 implant provokes a local inflammation, which does not prevent bone healing. The deformation of the implant, however, indicates that this PLDLA96 plate is not suitable for orbital floor reconstruction.

Cloning and analysis of the cDNA encoding the rod G-protein transduction alpha, beta1 and gamma1 subunits from the canine retina.

The canine (Canis familiaris) retinal rod transducin (G(T)) alpha, beta1 and gamma1 subunits were sequenced. Cloning of the cDNAs was accomplished by polymerase chain reaction (PCR) using degenerate and wild type retinal cDNA libraries as templates. The deduced amino acid sequences were highly similar to rod transducins from other species: G(T alpha) differed by 5 amino acids from the corresponding human sequence, whereas beta1 and gamma1 were identical to human sequences. The coding sequence of rod transducin was evaluated as a possible cause for the recessively inherited retinal rod-cone degeneration: there were no nucleotide differences between the wild type and retinal degenerate strains.

Cloning of the cDNA encoding rod photoreceptor cGMP-phosphodiesterase alpha and gamma subunits from the retinal degenerate Labrador retriever dog.

The nucleotide (nt) sequence of the cDNA encoding the retinal rod cyclic 3'5'-GMP phosphodiesterase (PDE) alpha and gamma subunits from two strains of dogs-(i) Labrador Retrievers homozygous for autosomally recessively inherited rod-cone degeneration and (ii) the wild-type Beagle-are reported. Cloning of these subunits was accomplished by polymerase chain reaction using retinal cDNA libraries as templates. The nt sequence of alpha PDE predicts a 861-amino-acid polypeptide which is 97.7 per cent and 96.9 per cent identical to the bovine and human counterparts, respectively. PDE gamma encodes an 87-amino-acid polypeptide differing from bovine and murine gamma subunits by only one amino acid. Since no differences were found between these two strains of dogs, the cause of the Labrador Retriever's degeneration remains to be determined.

Morphological and biomechanical difference in healing in segmental tibial defects implanted with Biocoral or tricalcium phosphate cylinders.

To evaluate the effects of two bioceramics on bone regeneration during repair of segmental bone defects, Biocoral and tricalcium phosphate cylinders were implanted in osteotomized sheep tibial defects 16 mm in length and followed up for 16 weeks. In comparison with the TCP-implanted defect, a significant increment in area and density of external callus was quantified radiomorphometrically at 3 weeks, and a marked increase in maximal torque capacity, maximal angle of deformation and absorption of energy was demonstrated mechanically in the Biocoral-implanted tibia at 16 weeks after implantation. Better bone integration with the substratum was microscopically observed in Biocoral cylinders. With good osteointegration and biomechanical-performance, Biocoral seems to be superior to TCP in repair of segmental defects in weight-bearing limbs.

Impaired retinal function in young labrador retriever dogs heterozygous for late onset rod-cone degeneration.

Xenon-flash d.c.-electroretinograms were recorded from dark adapted, rod-cone degenerate homozygote affected (n = 6), heterozygote carrier (n = 3) and control retinas (n = 4) at 3 and 4 months of age, starting at 0.6 log units below control PII threshold. One log unit higher stimuli were necessary to evoke PII in heterozygote and affected retinas compared to controls. Unique to the heterozygotes, double peaked PII responses that were evoked by -2 log relative units intensity stimulation were significantly (P = 0.028) lower in amplitude than those of controls. PII amplitudes of homozygotes were significantly (P = 0.005) lower in amplitude than those of controls at both ages examined in response to -2 and 0 log relative intensity stimulation. No differences were found in scotopic threshold response amplitudes or times to peak between the three groups. Homozygote affected PII times to peak were significantly (P = 0.005) shorter in relation to controls at -2 log units. Findings suggest that heterozygotes exhibit an impaired retinal function which can be demonstrated at 3 and 4 months in this mutant.

Stabilization of an inserted tricalcium phosphate spacer enhances the healing of a segmental tibial defect in sheep.

The effect of inserting a tricalcium phosphate (TCP) spacer stabilized by a rigid or non-rigid fixation technique on the healing of segmental tibial defects of critical size was established. The osteotomized tibiae, 11 with and 8 without TCP spacers, were fixed by an external circular device in 11 mature sheep and by plates in 8 mature sheep, respectively. Healing was evaluated roentgenographically 16 weeks after the operation. Compared with the defects without TCP spacers, enhanced stability and healing were observed in the defects with TCP spacers under an identical external fixation. Furthermore, a significantly higher incidence of healing was obtained with plate fixation than with external device fixation in the TCP-implanted defects (P < 0.04). An abundant bridging callus was roentgenograpically demonstrated in most of the healed defects, but none in the unhealed defects. The TCP spacer with its mechanical integrity enhances the stability of external fixation, and the stable immobilization provided by rigid fixation is essential for osteoconduction of an inserted TCP spacer in the healing of segmental diaphyseal defects in sheep.

The use of a coral composite implant containing bone morphogenetic protein to repair a segmental tibial defect in sheep.

A composite implant consisting of a coral cylinder, moose bone morphogenetic protein and type IV collagen was used to repair a segmental tibial defect in sheep. Healing, related variance in mechanical strength and immune responses were evaluated. In comparison with a coral control, a larger amount of newly formed external callus was observed in the composite group at 6 weeks. The maximal torque capacity, maximal angular deformation at failure and bone stiffness of a healed osteotomised tibia recovered 113%, 117% and 120% in the coral controls and 67%, 92% and 79% in the composite implants against the corresponding contralateral tibia at 16 weeks respectively. A significantly elevated anti-BMP antibody was detectable in the composite group at 3 and 6 weeks. Augmented bone formation at an early stage and weakened torsional performance at a later stage in the composite implants may indicate the phase-specific osteoinduction and the immune response of xenogenic BMP with time.

The introns of the canine rod opsin gene show higher sequence homology to the human than to the rodent introns.

Using genomic DNA from late-onset retinal degenerate and wild type Labrador Retrievers as templates and canine exon-specific oligonucleotides as primers in polymerase chain reaction, all four introns of opsin were cloned and sequenced. Dot-matrix comparisons were made for human, murine and canine introns. Selected sequences containing either intronic or coding sequences were aligned and used for phylogenetic relationship analysis. The opsin gene introns are conserved between the human, the mouse and the dog with regards to number and length. In addition there is an astonishingly high degree of sequence homology between the second and fourth introns. Introns 2(1277 bp in dog) and 4 (863 bp in dog) are 72% and 71% homologous to the human introns, and 57% and 52% homologous to the mouse introns, respectively. The coding sequence (CDS) of the dog shows 93% homology to human CDS and 88% homology to mouse CDS. A phylogenetic analysis of the intronic sequences 2 and 4 confirms the higher relatedness between dog and human than between mouse and human opsin genes. As there are good reasons to believe that the primate and rodent lineages are closer to each other than to the Canis familiaris, there must be some functional constraints on the evolution of human and dog opsins.

Inflammatory cells in experimental intervertebral disc injury.

STUDY DESIGN: Inflammatory cells were located by immunocytochemistry in areas of experimental intervertebral disc injury in pigs. OBJECTIVES: To study the occurrence of T lymphocytes and macrophages 1 week, 1 month, and 3 months after partial-thickness transverse scalpel injuries in pig lumbar discs. SUMMARY OF BACKGROUND DATA: Inflammatory cells and mediators recently have been observed in disc herniation tissue that was removed at disc prolapse surgery. The prevalence of inflammatory cell infiltrates in such clinical disc tissue material also has been studied. There are no studies, however, that have analyzed, using immunocytochemical methodology, the occurrence of, types of, and time dependence of inflammatory cells in an experimental disc injury model. The role of inflammation in intervertebral disc injury and repair has not been determined. METHODS: Transverse scalpel injuries 5-mm long and 4-mm deep were cut in the anterolateral anulus of L5-L6 and L4-L5 discs in 16 pigs. The cuts in the center of the anulus did not reach the nucleus pulposus and never produced a disc prolapse. In every pig, two non-adjacent lumbar discs (L1-L2 and L2-L3) were used as controls. Four discs per animal were studied in parallel by two different complementary immunohistochemical staining protocols. T lymphocytes and macrophages were located immunohistochemically using CD3 and CD68 antibodies, respectively. Discs were removed for analysis from four pigs at 1 week, from six pigs at 1 month, and from six pigs at 3 months. Inflammatory cells were categorized by two independent observers as being entirely absent (-), only few scattered cells (+), and at least one larger cellular infiltrate (+2). RESULTS: In none of the discs could extensive inflammatory cell infiltration be observed. T lymphocytes were present in significantly more sections cut from injured discs than in sections cut from control discs. The difference was highly significant particularly at 1 week and 1 month after disc removal. Only the 1-month-after-injury sections from injured discs exhibited significantly more macrophages than those from control discs. CONCLUSIONS: The results suggest the presence of only modest inflammatory cell infiltration in experimental intervertebral disc injury at all follow-up times. The inflammatory response in partial-thickness anterior experimental intervertebral disc injury, in the absence of disc prolapse, seems to be dominated by a T lymphocyte response. The macrophage response is apparently strongest at 1 month after such injury. These findings differ from what has been observed in herniated disc tissue.

Enhanced healing of segmental tibial defects in sheep by a composite bone substitute composed of tricalcium phosphate cylinder, bone morphogenetic protein, and type IV collagen.

Diaphyseal segmental defects in the tibia of 18 sheep were used to evaluate the healing potential of a composite bone substitute device (CBS) composed of a tricalcium phosphate cylinder (TCP), naturally occurring sheep bone morphogenetic protein (sBMP), and type IV collagen. A total of 100 mg of sBMP and 20 mg of type IV collagen in the high-dose group (CBSH), and 13 mg of sBMP and 2.5 mg of type IV collagen in the low-dose group (CBSL) were adsorbed to TCP cylinders, respectively. TCP cylinders impregnated with type IV collagen alone (TCPC) were used as control. A significantly larger area and more highly integrated intensity of newly formed external callus between CBSH and CBSL or TCPC group were quantified by computerized image analyzer at both 3 and 6 weeks. A torsion test showed that the maximal torque capacity, maximal angular deformation, and bone stiffness of healed osteotomized tibia with implants recovered 117-125% in CBSH, 72-109% in CBSL, and 63-80% in TCPC, compared with the corresponding contralateral tibia at 16 weeks. A healing superiority of the segmental bone defects replaced by the implants was demonstrated in the CBSH group. Thus, the composite bone substitute device defined in this study was shown to possess osteoinductivity, osteoconductivity, and mechanical strength.

Xenogeneic moose (Alces alces) bone morphogenetic protein (mBMP)-induced repair of critical-size skull defects in sheep.

A standardized skull defect in adult sheep was used to test the healing capacity of xenogeneic, partially purified, moose-derived bone morphogenetic protein (mBMP) extracted from the fresh long bones of moose (Alces alces) calves. An amount of 52 mg of mBMP mixed with 13 mg of purified type IV collagen (5:1) (mBMP/COL) in gelatin capsules was implanted into six 22-mm-diameter skull defects in adult sheep for comparison with six defects implanted with fresh autogenous bone marrow (BM) and six other controls implanted with a gelatin capsule containing 13 mg of type IV collagen (C). The amount of new bone formed was quantified from radiographs by computerized image analysis and histology. The healing percentage in the mBMP/COL group was significantly higher (93.18 +/- 4.51%) than in the BM (33.17 +/- 20.05%) or C group (31.32 +/- 17.41%) at 16 weeks after implantation. The difference between BM and C was not statistically significant. The level of anti-BMP antibody in the serum showed a significant increase in the group implanted with mBMP, but returned to normal after 6 weeks. The experiment demonstrated that xenogeneic mBMP possesses a strong osteoinductive capacity and weak immunogenicity.

Propofol influences the electroretinogram to a lesser degree than thiopentone.

BACKGROUND: The Electroretinogram (ERG) is used clinically to assess the function of retina. Anaesthetic agents are known to affect ERG, and as anaesthesia is often needed in children and uncooperative patients, knowledge about its effects is of clinical importance. Barbiturates selectively depress ERG components, and we compared thiopentone with propofol to assess if the latter preserved retinal function better. METHODS: Ten pigs, average weight 17 kg (SD +/- 2 kg) were anaesthetized randomly with propofol 10 mg kg-1 or thiopentone 30 mg kg-1. Anaesthesia was maintained by 65% nitrous oxide in oxygen and continuous infusion of the induction agent, i.e. 10 mg kg-1 h-1 of propofol, or 10 mg kg-1 h-1 for the first hour, then 5 mg kg-1 h-1 of thiopentone, with doses being based on pilot studies. After an interval of one week the programme was repeated using the other agent. After 40 minutes dark-adaptation, responses to single flashes of graded intensities from a xenon flashlamp were recorded at five-minute intervals. The a- and b-wave amplitudes and implicit times (time to peak), and a-wave slopes were determined. RESULTS: The b-wave implicit time was significantly shorter during propofol anaesthesia than when using thiopentone. The effect was most pronounced at the lowest intensities (P < 0.01). No statistically significant differences were found in the amplitudes of the b-waves. The a-wave appeared at lower stimulus intensity (P < 0.05) and the a-wave slopes were significantly steeper (P < 0.01) during propofol anaesthesia. CONCLUSION: Propofol accordingly appeared to preserve the photoreceptor response better than thiopentone, and may therefore be considered to be more suitable for ERG recordings than thiopentone.

Elevation of cGMP with normal expression and activity of rod cGMP-PDE in photoreceptor degenerate labrador retrievers.

Cyclic guanosine 3',5'-monophosphate (cGMP) levels were determined in retinas from a strain of Labrador Retrievers with inherited retinal dystrophy manifesting at early stages of retinal differentiation. The cGMP contents of dystrophic retinas of dogs from 1 to 4 months of age (n = 7) were significantly higher (p = 0.001) than in age-matched controls of the same breed (n = 11). Ultrastructure along the vertical retinal meridian was studied in developing retinas and findings were related to those of age-matched wild-type controls of the same breed. Slow central to peripheral progression of degeneration was observed in affected dogs. No differences were found in total cGMP-phosphodiesterase (PDE) activity, in PDE subunit composition as determined by Western blotting of 2-month-old homozygote affected retinas, or in the amino acid sequence deduced from the nucleotide sequence of the PDE beta-subunit as compared to controls. This model of photoreceptor degeneration thus is the first case of an apparent abnormality of cGMP metabolism that is not associated with a defect in the PDE catalytic subunits, and it is also the first reported model not associated with severe developmental abnormalities and rapid degeneration.

Microscopic evaluation of bone-implant contact between hydroxyapatite, bioactive glass and tricalcium phosphate implanted in sheep diaphyseal defects.

In order to compare morphological discrepancies in bone-implant contact in vertebrates, cylinders of hydroxyapatite, bioactive glass and tricalcium phosphate were implanted in segmental defects of the tibia in sheep. Three types of visible bone-implant contact were observed microscopically at 4 months after implantation. The trabecular web-like bone-implant contact noted in tricalcium phosphate seemed superior to the disseminated patchy bone-implant contact in bioactive glass and the buttressed bone-implant contact in hydroxyapatite with respect to both bone ingrowth and bioresorption of the implant. Differences of physicochemical properties on the surface among the three kinds of bioceramic implants probably give rise to different types of bone-implant contact.

The late positive retinal potential in dogs.

The late positive potential of the mammalian electroretinogram has been called the 'PI' or the 'c-wave' potential. It is unusual among retinal potentials because its peak implicit time increases in response to increasing stimulus intensity and because it cannot be demonstrated consistently in small samples of normal humans or normal dogs. We recorded wideband (DC-1 kHz) responses from 34 normal Beagles or dogs of similar size. Of the 34, 11 produced a late positive potential set that satisfied the criteria for c-waves. Multiple aspartate injections always increased c-wave amplitude and stimulus-response linearity in all 'producers'. Non-producers were never converted to producer status by aspartate blocking of the inner retina. Interaction of late positive and negative potentials and the possible influence of normal individual variations in the trans-epithelial potential are discussed. Individual mammal c-wave production is controlled by outer retinal phenomena which vary between individuals.

Early morphometry of a retinal dystrophy in Labrador retrievers.

Retinal morphometry was assessed in 7 dogs from a colony of Labrador Retrievers with dystrophic retinas at 1,2,3,4 and 18 months of age. Rod outer segment length and outer nuclear layer width were measured in the central, midperipheral and peripheral retina at six locations along the vertical meridian. Early striking regional differences in onset and rate of progression were characteristic for this inherited retinal degeneration. Notably, some areas of the retina developed fully and normally before degenerating. The central parts of the vertical meridians showed slightly disorganized rod outer segments already at 1 month of age and they were significantly shorter than those of control animals at 3 and 4 months (p < 0.01 and p < 0.001, respectively). The rod outer segments of the midperipheral and peripheral regions were, however, comparable to control animals as late as at 4 months of age. At 18 months the rod outer segments of dystrophic animals were significantly shorter in all retinal regions (p < 0.0005). At the age the outer nuclear layer of the dystrophic animals had become significantly thinner than that of control animals in all retinal regions (p < 0.001), indicating a clear visual cell loss. It is reasonable to characterize this as a retinal degeneration having a relatively slow progression, which enhances its relevance to conditions of clinical significance.

Pigment epithelial function in canine retina.

The lateral distribution and temporal changes in the eye standing potential of 15 dogs with normal eyes (as determined by use of an ophthalmoscope and electroretinography) were measured by use of noninvasive methods. The standing potential was converted to an alternating potential by controlled eye movement. The light peak occurred 6 minutes after a stimulus intensity increase of 4 log units. The ratios of the highest measured voltage after the light step divided by the voltage measured immediately before the light step ranged from 1.27 to 2.07 (mean 1.74 +/- SEM, 0.064). The responses typically decayed slowly after the light peak. The potential after the light peak did not return to prelight step values during the observation period. The field potential of the standing potential decreased nonlinearly in temporal direction from the outer canthus.

Ultrastructural and ERG findings in progressive rod-cone dystrophy in a litter of Labrador retrievers.

Early ultrastructural findings of a progressive photoreceptor dystrophy and corresponding ERG findings are reported in 3 Labrador Retrievers from a litter of 7 pups bred from 2 dogs clinically and electroretinographically affected with generalized progressive retinal dystrophy. The pups were euthanized at 5, 11 and 15 months post partum. The most prominent ultrastructural finding was photoreceptor dystrophy. At 5 months the outer nuclear layer (ONL) consisted of 8-10 layers and seemed reduced in thickness, pyknotic nuclei were seen in this layer. The receptor outer segments (OS) were short and swollen. Some disorientation of OS discs occurred. In the 11-months specimen 7-8 ONL layers were identified. Overall thinning of the neuro-retina had occurred and fewer receptors compared to the 5-months specimen were present. By 15 months the ONL was further reduced to about 4 layers. Enlarged internuclear spaces were present in the ONL as well as around inner segments (IS). Phagocytic cells were frequent among remains of OS. The pigment epithelium appeared normal. The dark adapted ERG b-wave amplitudes and photopic 30 Hz flicker responses were low in comparison to controls of the same breed, and decreased with age. The condition represents a progressive rod-cone dystrophy which shares similarities with primary receptor dystrophy in man such as retinitis pigmentosa.

A DC electroretinography method for the recording of human a-, b- and c-waves.

In the clinical ERG the c-wave is not usually recorded due to methodological problems. Because of the potential importance of the c-wave recording in assessing the function of the pigment epithelium in several retinal diseases, we describe a DC ERG method which is convenient for the patient and suitable also for clinical practice. The light stimuli are provided by a Ganzfeld stimulator and the potentials are recorded with a disposable corneal wick electrode. The method allows the recording of the c-wave from co-operative subjects as well as to study the a- and b-wave properties.