Jörg A Auer

Localized insulin-like growth factor I delivery to enhance new bone formation

Insulin-like growth factor I (IGF I) exerts an important role during skeletal growth and bone formation. Therefore, its localized delivery appears attractive for the treatment of bone defects. To prolong IGF I delivery, we entrapped the protein into biodegradable poly(lactide-co-glycolide) microspheres (PLGA MS) and evaluated the potential of this delivery system for new bone formation in two defect models of ovine long bones, i.e., a 8-mm methaphyseal drill hole and a 10-mm segmental tibia defect. Administration of 100 microg of IGF I in PLGA MS resulted in new bone formation within 3 weeks in the drill hole and bridging of the segmental defect within 8 weeks. The observed increase of 12% newly formed bone in the drill hole defect after 3 weeks was substantial, compared to the measured morphometric bone-to-total area ratio of 31% bone in normal cancellous bone. Bone regeneration was further explored by measuring gene expression of typical markers for local mediators and growth factors by real-time polymerase chain reaction. Inflammation was reduced in presence of IGF I and this in vivo observation was corroborated in vitro by quantifying gene expression of inflammatory proteins and by assessing the activation of the NF-kappaB pathway, playing an important role in the regulation of inflammation. Administration of the IGF I delivery system downregulated inflammatory marker gene expression at the site of bone injury, induced new bone formation and reduced bone resorption, and resulted in bridging of 10-mm segmental tibial defects within 8 weeks.

Compositional changes of a dicalcium phosphate dihydrate cement after implantation in sheep

A hydraulic calcium phosphate cement having dicalcium phosphate dihydrate (DCPD) as end-product of the setting reaction was implanted in a cylindrical defect in the diaphysis of sheep for up to 6 months. The composition of the cement was investigated as a function of time. After setting, the cement composition consisted essentially of a mixture of DCPD and beta-tricalcium phosphate (beta-TCP). In the first few weeks of implantation, the edges of the cement samples became depleted in DCPD, suggesting a selective dissolution of DCPD, possibly due to low pH conditions. The cement resorption at this stage was high. After 8 weeks, the resorption rate slowed down. Simultaneously, a change of the color and density of the cement center was observed. These changes were due to the conversion of DCPD into a poorly crystalline apatite. Precipitation started after 6-8 weeks and progressed rapidly. At 9 weeks, the colored central zone reached its maximal size. The fraction of beta-TCP in the cement was constant at all time. Therefore, this study demonstrates that the resorption rate of DCPD cement is more pronounced as long as DCPD is not transformed in vivo.

Compensatory load redistribution of horses with induced weightbearing hindlimb lameness trotting on a treadmill

REASONS FOR PERFORMING STUDY The compensatory mechanisms of horses with weightbearing hindlimb lameness are still not fully understood. HYPOTHESIS That weightbearing, unilateral hindlimb lameness would not only alter stride characteristics to diminish structural stress in the affected limb but also induce compensatory load adjustments in the other supporting limbs. OBJECTIVE To document the load and time shifting mechanisms of horses with unilateral weightbearing hindlimb lameness. METHODS Reversible lameness was induced in 8 clinically sound horses by applying a solar pressure model. Three degrees of lameness (subtle, mild and moderate) were induced and compared with the nonlame (sound) control measurement. Vertical ground reaction forces were recorded for all 4 limbs simultaneously on an instrumented treadmill. RESULTS Compared to the sound situation, moderate hindlimb hoof lameness induced a decrease in stride duration (-3.3%) and stride impulse (-3.1%). Diagonal impulse decreased selectively in the lame diagonal stance (-7.7%). Within the diagonal limb pair, vertical impulse was shifted to the forelimb during the lame diagonal stance (+6.5%) and to the hindlimb during the sound diagonal stance (+3.2%). Peak vertical force and vertical impulse decreased in the lame limb (-15%), but only vertical impulse increased in the contralateral hindlimb (+5.7%). Stance duration was prolonged in both hindlimbs (+2.5%). Suspension duration was reduced to a greater extent after push-off of the lame diagonal limb pair (-21%) than after the sound diagonal limb pair (-9.2%). CONCLUSIONS Four compensatory mechanisms could be identified that served to reduce structural stress, i.e. peak vertical force on the affected limb: 1) reduction of the total vertical impulse per stride; 2) diagonal impulse decreased selectively in the lame diagonal; 3) impulse was shifted within the lame diagonal to the forelimb and in the sound diagonal to the hindlimb; and 4) the rate of loading and peak forces were reduced by prolonging the stance duration. POTENTIAL RELEVANCE Load shifting mechanisms are not only effective in diminishing peak forces in the affected limb, but also suppress compensatory overload in other limbs. Selected force and time parameters allow the unequivocal identification of the lame limb. Future studies have to examine how far these compensatory mechanisms may be generalised for other defined orthopaedic problems in the hindlimb.

Changes in subchondral bone in cartilage resurfacing—an experimental study in sheep using different types of osteochondral grafts

OBJECTIVE This article addresses the subchondral bone integrity in cartilage resurfacing by comparing fresh, untreated auto-, xeno-, and photooxidized osteochondral allo- and xenografts. Photooxidation was expected to improve mechanical stability of the osteochondral grafts through an improved linkage of the collagen fibers within the bone matrix. DESIGN Untreated auto- and xenografts and with photooxidation pretreated allo- and xenografts were surgically implanted in femoral condyles of sheep (n=40). After 2, 6, 12 and 18 months results were evaluated histologically using non-decalcified bone embedded in acrylic resin. Qualitative evaluation was performed with emphasis on bone matrix, biomechanical stability of graft anchorage, formation of cystic lesions, and bone resorption and formation. Quantitative evaluation of the total subchondral bone area was conducted histomorphometrically. Statistical analysis (factorial ANOVA test) was used to compare differences between groups with respect to the percentage of bone matrix and fibrous tissue per section. RESULTS Subchondral bone resorption was fastest in untreated, fresh autografts, followed by photooxidized allografts, untreated, fresh xenografts and last pretreated photooxidized xenografts. Cystic lesions were seen in all types of grafts, but were most pronounced at 6 months in autografts and least in photooxidized grafts. Cyst-like lesions had subsided substantially in the untreated auto- and photooxidized xenografts, if no graft dislocation occurred during the healing period. Mononuclear cell infiltration and an increase in the presence of multinuclear cells were observed at 2 months, mostly in untreated autografts, followed by photooxidized allo- and untreated xenografts. They were much higher in numbers compared to photooxidized grafts, at least in the early specimens at 2 months. Graft stability was linked to the rate of bone resorption. CONCLUSION Substantial resorption of the subchondral bone, involving the development of cyst-like lesions, lead to dislocation and finally to cartilage matrix degradation of the grafts. The process of photooxidation decreased the speed of bone resorption in osteochondral grafts and, thus, improved graft stability and cartilage survival. These results suggest that the remodeling of the subchondral bone of the host and the graft within the first 6 months is an important factor in graft stability and overall results of cartilage resurfacing.

Quantitative Real-Time PCR for Equine Cytokine mRNA in Nondecalcified Bone Tissue Embedded in Methyl Methacrylate

Abstract. Specific amplification and quantitation of nucleic acid sequences by the polymerase chain reaction (PCR) has been extensively used for the detection of viral infection and gene expression. Although successful amplification of DNA and RNA sequences extracted from paraffin embedded tissue have been described, there are presently no reports available regarding RNA analysis from bone and calcified tissues embedded in hydrophobic acrylic resin. Here we describe a general method for quantitation of specific mRNA sequences extracted from undecalcified bone sections, fixed in paraformaldehyde, and embedded in a hydrophobic acrylic resin. Total RNA was extracted from defined regions of single 50 μm sawed sections. These RNA preparations are suitable for quantitative PCR analysis of mRNA of different cytokines. In addition, the universally expressed housekeeping GAPDH mRNA proved to be useful as an amplification control and to correct for the degree of RNA degradation, which may vary considerably among samples. Reverse transcribed mRNA was amplified and quantitated in Real-Time PCR using a fluorescein labeled internal TaqMan® probe.

Spontaneous production of nitric oxide (NO), prostaglandin (PGE2) and neutral metalloproteinases (NMPs) in media of explant cultures of equine synovial membrane and articular cartilage from normal and osteoarthritic joints

Nitric oxide (NO), prostaglandin E2 (PGE2), and the activity of neutral metalloproteinases (NMPs) were measured in conditioned media of equine synovial membrane and articular cartilage explant cultures from horses with normal joints (n = 7) and from horses affected with moderate (n = 7) or severe osteoarthritis (n = 14) as judged by macroscopic appearance. Normal articular cartilage appeared glossy and bluish-white, was of normal thickness and showed no evidence of discolouration, fibrillation or other cartilage discontinuity. Slight discolouration and fibrillation or minor clefts of the cartilage were considered as moderate OA, whereas erosions of articular cartilage down to the subchondral bone were considered as cases of severe OA. Explant cultures of equine synovial membrane and articular cartilage released the local mediators, NO and PGE2, as well as detectable levels of NMP activity into culture media. Concentrations of NO were higher in articular cartilage explants compared to synovial membrane explants, whereas concentrations of PGE2 were higher in synovial membrane explants. The NMPs with collagenolytic activities were similar in both explant cultures, whereas gelatinolytic activities were higher in synovial membrane explant cultures and caseinolytic activities were generally higher in articular cartilage explant cultures. Furthermore it was shown that concentrations or enzyme activities increased according to the severity of disease of the joints. Concentrations for NO, collagenolytic and gelatinolytic NMPs were relatively stable, whereas PGE2 and caseinolytic NMP concentrations increased over time in culture.

Velocity-dependent changes of time, force and spatial parameters in Warmblood horses walking and trotting on a treadmill

REASONS FOR PERFORMING STUDY Gait analysis parameters are sensitive to alterations in velocity. For comparison of nonspeed-matched data, the velocity dependency needs to be known. OBJECTIVES To describe the changes in gait pattern and determine the relationships between stride duration, vertical impulse, contact time and peak vertical force within a range of walking and trotting speeds. METHODS Thirty-eight nonlame Warmblood horses were subjected to an incremental speed test. The spans of speed were adjusted individually to each horse and ranged from 1.1-2.1 m/s at walk and from 2.5-5.8 m/s at trot. Time, force and spatial parameters of each limb were measured with an instrumented treadmill and analysed with regression analysis using velocity as the independent variable. RESULTS At a slow walk the shape of the force curve was generally single-peaked in the fore- and trapezoidal in the hindlimbs. With increasing speed, the curves turned into the typical double-peaked shape with a higher second peak in the fore- and a higher first peak in the hindlimbs. With increasing velocity, stride duration, stance durations and limb impulses of the fore- and hindlimbs decreased in both gaits (r2 > 0.92). Increasing speed caused a weight shift to the forehand (walk: from 56 to 59%; trot: from 55 to 57%). Despite decreasing limb impulses, peak vertical forces increased in both gaits (r2 > 0.83). The suspension duration of the trot increased with faster velocities and reached a plateau of around 90 ms at the highest speeds. At a slow trot, the forelimbs impacted first and followed the hindlimbs at lift-off; with increasing speed, the horses tended to impact earlier with the hindlimbs. Contralateral symmetry indices of all parameters remained unchanged. CONCLUSIONS Subject velocity affects time, force and spatial parameters. Knowing the mathematical function of these interdependencies enables correction of nonspeed-matched data.

Performance parameters and post exercise heart rate recovery in Warmblood sports horses of different performance levels.

REASONS FOR PERFORMING STUDY Standardised exercise tests are used for fitness evaluation of sports horses. Standards are described for Thoroughbreds and Standardbreds; however, limited information is available for Warmbloods. OBJECTIVES To establish normative standards of performance parameters and heart rate recovery (HRR) in Warmblood riding horses of different levels of fitness using a submaximal incremental exercise test (SIET) performed on a treadmill. METHODS A SIET was carried out with 29 healthy and treadmill-accustomed Warmbloods: eleven 3-day event horses (TDE) and 18 horses from the National Equestrian Centre (NEC) competing in amateur jumping and/or dressage events. After a warm-up phase, horses performed 2 stages at trot and 3-5 stages at gallop at 6% incline. The first stage lasted 120 s, all others 90 s. Velocity (V) and heart rate (HR) were measured continuously and blood lactate concentration (LAC) at the end of each exercise stage. V at HR 150 and 200 beats/min (V(150), V(200)), V and HR at 2 and 4 mmol/l LAC (V(2), V(4) and HR(2), HR(4), respectively) were calculated and compared between discipline groups. For reference values, horses were divided on the basis of the V(4) -results in good (GP) and average performers (AP) (performance groups). Five minute passive HRR was compared between performance groups. Fifteen NEC horses were retested within 1-3 months. Groups were compared with t tests and P < 0.05 considered significant. RESULTS Three-day event horses had higher V(150), V(2) and V(4) values than NEC. GP had higher values in all performance parameters compared to AP. No differences were found between test and retest. GP mean recovery HR was different from that of AP from 120 s of recovery onwards. CONCLUSION Treadmill SIETs are suitable to objectify aerobic capacity in Warmblood riding horses. Normative standards were assessed for well and averagely-trained horses. The results can be referred to when diagnosing patients with exercise intolerance.

Long term in-vivo studies of a photo-oxidized bovine osteochondral transplant in sheep

BackgroundArticular cartilage has limited capacity to repair. Defects greater than 3 mm heal with formation of inferior fibrous cartilage. Therefore, many attempts have been made to find the ideal graft for larger cartilage lesions. Different grafts, such as untreated or cryopreserved osteochondral transplants, have been used with variable success.MethodsPhoto-oxidized osteochondral grafts were implanted in both femoral condyles of one ovine knee. Untreated xenogeneic and autogeneic grafts served as controls. Three groups of 8 sheep each were formed and they were sacrificed 6, 12 or 18 months after surgery.ResultsThe macroscopic evaluation of the condyle and graft showed a well-maintained cartilage surface in most grafts at all time points. However, the host cartilage matrix deteriorated considerably in all xenogeneic, most autogeneic and fewer of the photo-oxidized grafts at 12 and 18 months, respectively. The blue colour of the photo-oxidized grafts resulting from the process of photo-oxidation was visible in all grafts at 6 months, had diminished at 12 months and had completely disappeared at 18 months after surgery. Histologically a loss of matrix staining was almost never noticed in untreated xenografts, transiently at 6 months in photo-oxidized grafts and increased at 12 and 18 months. Fusion between graft and host cartilage could be seen in photo-oxidized grafts at 12 and 18 months, but was never seen in autografts and xenografts.ConclusionsThe photo-oxidation of osteochondral grafts and its use as transplant appears to have a beneficial effect on cartilage and bone remodelling. Osteochondral grafts pre-treated with photo-oxidation may be considered for articular cartilage replacement and therefore may delay artificial joint replacements in human patients.

Treatment of chronic proximal suspensory desmitis in horses using focused electrohydraulic shockwave therapy

The objective of the present clinical report was to investigate the short- and long-term outcomes of chronic proximal suspensory desmitis (PSD) treated with Extracorporeal Shockwave Treatment (ESWT). Fifty-two horses with chronic PSD in the forelimb (34 cases) or hindlimb (22 cases) were included in the study. Three horses had lesions in both hindlimbs and one in both forelimbs. The origin of the suspensory ligament was treated every three weeks for a total of three treatments using 2000 impulses applied by a focused ESWT device (Equitron) at an energy flux density of 0.15 mJ/ mm2. This treatment regime was followed by box rest and a controlled exercise program of 12 weeks duration. The horses were assessed 3, 6, 12, 24 weeks and one year after the first treatment. Of the 34 cases with forelimb PSD, 21 (61.8%) had returned to full work by six months after diagnosis and 19 cases (55.9%) were still in full work one year after ESWT. Of the 22 horses with hindlimb PSD, 9 (40.9%) had returned to full work by six months and 4 (18.2%) were still in full work one year after diagnosis. There was no association (chi-square test) between the outcome and the severity of the initial ultrasonographic and/or radiographic findings. Compared with the results of other clinical studies, these findings suggest that in horses with PSD of fore- and hindlimb, the prognosis for returning to full work six months after diagnosis can be improved when ESWT and a controlled exercise program are used. However, a high rate of recurrence occurred in the hindlimb despite treatment.