P. R. Weeren

Gelatin-methacrylamide hydrogels as potential biomaterials for fabrication of tissue-engineered cartilage constructs

Gelatin-methacrylamide (gelMA) hydrogels are shown to support chondrocyte viability and differentiation and give wide ranging mechanical properties depending on several cross-linking parameters. Polymer concentration, UV exposure time, and thermal gelation prior to UV exposure allow for control over hydrogel stiffness and swelling properties. GelMA solutions have a low viscosity at 37 °C, which is incompatible with most biofabrication approaches. However, incorporation of hyaluronic acid (HA) and/or co-deposition with thermoplastics allows gelMA to be used in biofabrication processes. These attributes may allow engineered constructs to match the natural functional variations in cartilage mechanical and geometrical properties.

Contact areas and pressure distribution on the proximal articular surface of the proximal phalanx under sagittal plane loading.

The objective of this study was to map topographically contact areas and pressure distributions on the proximal articular surface (PAS) of the proximal phalanx (PI) under various clinically relevant loading conditions. Left and right forelimbs of 13 mature horses were transected halfway down the radius and loaded in a position mimicking the weightbearing attitude close to the midstance phase. Five loads were used which corresponded with loads that can be expected in different gaits or during athletic performance (stance: 1800 N, walk: 3600 N, trot: 5400 N, gallop: 10,500 N and jumping: 12,000 N). Contact areas and pressure distributions at the PAS of PI were determined using a methylene blue dye staining technique and 2 pressure sensitive films (low pressure: range 2.5-10 MPa and medium pressure: range 10-50 MPa). The contact area of PI was positively correlated (r = 0.86; P<0.01) with the applied load. The contact area increased from 63% at 1800 N to 95% at 12,000 N and gradually shifted to include more of the edges of the articular surface, but especially the dorsal articular margin of PI. Pressure distribution patterns were similar under the different loading conditions. Pressure was less at the palmar margin and in the central depression and highest at the dorsal articular margin. With increasing load, the highest peak pressures were measured at sites of the dorsal articular margin that are not loaded in the standing or walking horse. The results of this study suggest that the frequent occurrence of osteochondral lesions at the dorsal articular margin of PI is caused by the combination of the intermittent character and the high absolute values of loads at this site as they occur during athletic performance.

Dynamic pressure measurements for the detailed study of hoof balance: the effect of trimming

REASONS FOR PERFORMING STUDY Studies on hoof balance have, so far, only assessed the effects of strong and relatively unrealistic interventions due to technical limitations of measuring equipment. For the assessment of more subtle interventions, other techniques are necessary. OBJECTIVES To test a sensitive pressure measurement system during locomotion and to set a standard for further studies by using the system to evaluate the effects of trimming. METHODS Eighteen horses were measured before and after trimming with an interval of 4 weeks. Trimming was standardised to a straight hoof-pastern axis. The horses trotted over a pressure/force measuring system, with a temporal resolution of 240 Hz and a spatial resolution of 0.39 cm2. RESULTS The preferred way of landing was lateral, asymmetrical in both front and hind feet. The duration of landing was shorter in forelimbs than in hindlimbs with an equal decreasing trimming effect. Horses had a fixed hoof-unrollment pattern; the centre of pressure (CoP) travelled towards a maximum lateral deviation and returned towards the dorsopalmar/plantar axis of the hoof. Trimming decreased the intra-individual left/right difference in maximum lateral displacement. CONCLUSIONS The technique used provided easily accurate data to quantify hoof balance characteristics and to measure short-term trimming effects. POTENTIAL RELEVANCE Determination of CoP patterns has added value in gait analysis and can improve our understanding of the effects of different interventions on hoof balance.

Inflammatory mediators and cartilage biomarkers in synovial fluid after a single inflammatory insult: a longitudinal experimental study.

IntroductionInflammation is an important feature of many joint diseases, and levels of cartilage biomarkers measured in synovial fluid may be influenced by local inflammatory status. Little is known about the magnitude and time course of inflammation-induced changes in cartilage tissue turnover as measured in vivo by synovial fluid markers. We aimed to study temporal changes in concentrations of inflammatory mediators, matrix metalloproteinase activity and cartilage biomarkers over 1 week in joints with experimentally induced inflammation.MethodsLocalized inflammation was induced in the intercarpal joint of six horses by sterile injection of 0.5 ng lipopolysaccharide, and synovial fluid was collected at post-injection hours (PIH) 0, 8, 24 and 168. Concentrations of inflammatory mediators (prostaglandin E2, substance P, and bradykinin), general matrix metalloproteinase activity and markers of collagen II turnover (CPII and C2C) as well as aggrecan turnover (CS846 and glycosaminoglycans) were measured with appropriate assays. One-way analysis of variance on repeated measures was used to analyze differences in synovial fluid marker levels over time.ResultsLipopolysaccharide-injection led to a sharp rise in prostaglandin E2 at PIH 8, while substance P, bradykinin and matrix metalloproteinase activity showed more sustained increases at PIH 8 and 24. Glycosaminoglycan release paralleled changes in the CS846 epitope, with an increase by PIH 8, a peak at PIH 24, and return to baseline by PIH 168. For type II collagen, a parallel time course between catabolic (C2C) and anabolic (CPII) markers was also observed, but the time course differed from that seen for proteoglycan markers: collagen II markers peaked later, at PIH 24, and were still elevated over baseline at PIH 168.ConclusionsA primary intra-articular inflammatory insult, characterized by local release of peptide and lipid mediators and matrix metalloproteinase activation, can alter synovial fluid levels of proteoglycan biomarkers as early as 8 hours post-induction, and can lead to sustained rises in collagen II biomarkers during at least 1 week after onset.

Collagenase-1 (MMP-1) activity in equine synovial fluid: influence of age, joint pathology, exercise and repeated arthrocentesis

REASONS FOR PERFORMING STUDY Matrix metalloproteinases (MMPs) are considered candidate biomarkers for both physiological and pathological tissue remodelling because of their key role in articular cartilage homeostasis. As disruption of the collagenous architecture is thought to be pivotal in chronic degenerative diseases such as osteoarthritis (OA), the collagenases form an interesting subset of the MMPs. The significance of any biomarker in synovial fluid (SF) can be assessed properly only when fluctuations in patterns induced by physiological processes such as development and growth, and by external influences and interventions such as exercise and repeated arthrocentesis, are known and taken into account. OBJECTIVES To investigate the activity of MMP-1 in equine SF at different stages of development and in joints affected by OA, and the influence of exercise and repeated arthrocentesis thereon. METHODS MMP-1 activity was determined in SF of normal joints of fetal, juvenile and mature horses, and in SF of horses suffering from OA, using an internally quenched fluorogenic peptide substrate. MMP-1 activity was also measured in SF from horses subjected to an exercise regimen and those subjected to repeated arthrocentesis. RESULTS An age-related decline in the SF levels of active MMP-1 was observed. MMP-1 activity was 15-fold higher in fetal than in juvenile animals, which showed significantly higher MMP-1 activity levels than mature horses. In SF of OA joints, MMP-1 activity was increased. Exercise did not affect MMP-1 activity in SF, but repeated arthrocentesis (within 60 h) increased MMP-1 activity significantly. CONCLUSIONS The high MMP-1 activity in SF of young individuals parallels the high metabolic activity occurring during rapid growth and differentiation at early age. The elevated MMP-1 activity in SF of OA joints probably reflects pathological matrix degradation, confirming the potential of MMP-1 to serve as a biochemical marker for early joint disease. Moderate exercise is not likely to influence the outcome of MMP-1 activity measurements in equine SF, but arthrocentesis should be taken into account as a possible confounding factor. POTENTIAL RELEVANCE Given the crucial role of the collagen matrix for tissue integrity, MMP-1 activity may be a useful tool in diagnostic, therapeutic or prognostic studies in horses suspected of OA. However, care should be taken to exclude fluctuations in MMP-1 activity induced by physiological processes such as development and growth, and by interventions such as repeated arthrocentesis.

Repeatability of back kinematics in horses during treadmill locomotion

We tested the hypothesis that repeatability of a standardised protocol for quantifying back kinematics is sufficiently high not to prevent its use in the clinical evaluation of horses with back problems. We investigated the extent to which differences between laboratories may affect the results when a standardised protocol is used. As a clinical tool, movement analysis techniques are helpful for the objective and quantitative assessment of kinematics. Knowledge about the repeatability of the kinematic data is very important. The present study investigates the repeatability of back kinematics in 10 sound horses over 5 successive days and in 2 laboratories (5 horses at each location). Measurements were performed on the treadmill during the walk and the trot. The between-stride, between-day and between-horse repeatability were determined. A high degree of between-stride and between-day repeatability was observed in the spatiotemporal parameters and in the time-angle diagrams of thoracic and lumbar vertebrae, the sacrum and the hindlimb during both the walk and trot. Much more variability was found between horses, with the highest degree of dissimilarity in the lateral bending rotation of the L1 vertebra. For range of motion values, the between-day coefficient of variability was <14% and the between-horse coefficient of variability was up to 4 times higher. Small differences were found in range of motion values between the 2 laboratories. It is concluded that an analysis of back kinematics in the horse can provide highly repeatable data, warranting clinical use.

Quantification and age‐related distribution of articular cartilage degeneration in the equine fetlock joint

REASONS FOR PERFORMING STUDY The equine fetlock joint has the largest number of traumatic and degenerative lesions of all joints of the appendicular skeleton. OBJECTIVE To gain insight into the distribution of cartilage degeneration across the articular surface in relation to age in order better to understand the dynamic nature and progression of osteoarthritis (OA). HYPOTHESIS That there would be a specific age-related distribution pattern of cartilage degeneration in the equine metacarpophalangeal joint. METHODS The proximal articular cartilage surfaces of the first phalanges (P1) of 73 slaughter horses (age range 0.4-23 years) with different stages of osteoarthritis were scored semiquantitatively on a 0 to 5 scale and also assessed quantitatively using the cartilage degeneration index (CDI(P1)), which ranges from 0 to 100%. Furthermore, CDI values were determined for special areas of interest; medial dorsal surface (CDI(mds)), lateral dorsal surface (CDI(lds)), medial central fovea (CDI(mcf)) and lateral central fovea (CDI(lcf)). Correlations were calculated for CDI(P1) values and CDI values at the specific areas of interest with macroscopic scores and with age. RESULTS There was a high correlation between the semiquantitative macroscopic score and the quantitative CDI(P1) values (r = 0.92; P < 0.001). A macroscopic score of 0 (i.e. no obvious cartilage degeneration) corresponded with a CDI(P1) mean +/- s.e. value of 25 +/- 2.8% and a macroscopic score of 5 (i.e. severe cartilage degeneration in localised areas) with a mean +/- s.e. value of 38.1 +/- 7.9%. There was a moderate but highly significant correlation between the CDI(P1) value and the age of the horses (r = 0.41; P < 0.001). Highest CDI values were calculated for the medial dorsal surface (from 10.6 +/- 2.8% at macroscopic Grade 0 to 63.1 +/- 8.4% at Grade 5). At the lateral dorsal surface, these values were 5.9 +/- 1.4% and 47.2 +/- 10.4%, respectively. The CDI(mcf) and CDI(lcf) were significantly lower (P < 0.05) than the CDI(mds) and CDI(lds) at all grades. The CDI(mcf) ranged from 1.0 +/- 2.9% at Grade 0 to 43.7 +/- 9.1% at Grade 5; laterally, these values were 1.5 +/- 2.6% and 15.2 +/- 6.2%, respectively. CONCLUSIONS CDI grading increased from lateral to medial and from central to dorsal. This specific distribution pattern confirms the heterogeneous nature of the OA process and strongly supports an important role for biomechanical loading, superimposed on age-related changes, in the spread of the disorder over the joint. POTENTIAL RELEVANCE Knowledge of the development of OA across the articular surface is essential for understanding the dynamic nature and progression of the disease and can form a basis for improvements in diagnostic and therapeutic approaches to degenerative joint disease.

Hoof growth between two shoeing sessions leads to a substantial increase of the moment about the distal, but not the proximal, interphalangeal joint

REASONS FOR PERFORMING STUDY There is little insight into the effects of routine farriery on the internal structures of the distal limb in sound horses. OBJECTIVES To measure the effect of change in hoof conformation during a shoeing interval on the moments about the proximal and distal interphalangeal joints (PIPJ, DIPJ) and to determine whether and how the horse compensates for this change in hoof conformation. METHODS Both front feet of 9 sound Warmblood horses were measured while standing on a pressure-force measuring system and radiographed in a lateromedial direction shortly after shoeing and 8 weeks later. From these data, ground reaction forces (GRF) and lever arms were measured in order to calculate joint moments. RESULTS After 8 weeks, the moment about the PIPJ did not increase significantly, but the moment about the DIPJ did so, indicating a compensatory mechanism for a change in hoof conformation in the DIPJ. CONCLUSIONS Standing horses compensate for hoof conformation change during an 8-week shoeing interval, which leads to increased DIPJ extension and consequently an increased loading of the deep digital flexor tendon. POTENTIAL RELEVANCE This study quantifies the effect of a shoeing interval on the internal structures of the foot and helps to determine an appropriate shoeing interval for individual horses in which the hoof with the lowest hoof angle is the best indicator. The exact determination of an optimal individual shoeing interval requires further study.

Differences in wound contraction between horses and ponies: the in vitro contraction capacity of fibroblasts.

The contribution of wound contraction to wound closure determines the speed of second intention wound healing and it has been shown that significant differences exist with regard to both contraction and inflammatory response between horses and ponies and between various areas of the body. In this study, the contraction capacity of fibroblasts from limbs and buttocks of 4 Dutch Warmblood horses and 4 Shetland ponies was studied in vitro, in order to determine whether differences in wound contraction are due to differences in the inherent contraction capacity of the fibroblasts or to differences in tissue environmental factors, such as the inflammatory response. Fibroblasts were harvested from subcutaneous tissue, cultured and then suspended in both floating and anchored collagen gels. Contraction capacity was assessed by measuring the decrease in area of the floating gels and by measuring the microforces generated in the anchored gels using a custom-built measuring device. In the floating gels, no difference existed in the contraction capacity of fibroblasts from horses and ponies, or from limbs and buttocks. In the anchored gels, no differences existed between horse and pony fibroblasts, but the fibroblasts from the limbs started to contract significantly sooner and produced significantly higher forces than those from the buttocks. It is concluded that the in vivo differences in wound contraction between horses and ponies and between different sites of the body are not caused by differences in the inherent contraction capacity of fibroblasts. The in vitro differences between fibroblasts from limbs and buttocks are thought to be due to the lower proliferation rate and the longer culture time of the fibroblasts originating from the limbs, because mature fibroblasts can develop higher contraction forces than immature fibroblasts. This means that tissue environmental factors, such as cytokine profiles during the inflammatory response, determine the extent of contraction during wound healing. Further research should be directed towards the role of the inflammatory response in wound healing.

The influence of repeated arthrocentesis and exercise on matrix metalloproteinase and tumour necrosis factor a activities in normal equine joints

REASONS FOR PERFORMING STUDY Matrix metalloproteinases (MMPs) and tumour necrosis factor alpha (TNF-alpha) may be useful as biomarkers of joint disease or inflammation. However, activity of both MMPs and TNF-alpha in synovial fluid (SF) may be influenced by nonpathological factors such as arthrocentesis or exercise. OBJECTIVE To investigate the influence of repeated arthrocentesis and exercise on MMP and TNF-alpha activities in SF from normal equine joints. METHODS SF was collected from the left metacarpophalangeal, radiocarpal and tarsocrural joints of 16 horses. Eight of these horses were subsequently subjected to an exercise programme on a treadmill and 8 were box-rested as controls. Arthrocentesis was repeated 14, 145, 17 and 24 days after the start of the exercise programme. General MMP and TNF-alpha activities were determined in SF. RESULTS Repeated arthrocentesis caused a gradual increase but the exercise regimen no significant increase in MMP activity. There was a significant increase in TNF-alpha activity in SF collected from horses 2 h after cessation of the exercise programme. CONCLUSIONS AND POTENTIAL RELEVANCE When using MMPs as biomarkers for joint disease, at least 14 days should elapse after previous arthrocentesis before subsequent SF collection. Moderate exercise does not increase MMP activity in SF from normal joints and it may be possible to ignore this as a source of error in evaluating MMP activity in diseased joints.