Tensile properties in collagen-rich tissues of Quarter Horses with hereditary equine regional dermal asthenia (HERDA).

REASONS FOR PERFORMING STUDY: Hereditary equine regional dermal asthenia (HERDA) is an autosomal recessive disorder of Quarter Horses characterised by skin fragility. Horses with HERDA have a missense mutation in peptidyl-prolyl cis-trans isomerase B (PPIB), which encodes cyclophilin B and alters folding and post translational modifications of fibrillar collagen. OBJECTIVES: The study aimed to test the hypothesis that tendons, ligaments and great vessels, which, like skin, are rich in fibrillar collagen, will also have abnormal biomechanical properties in horses with HERDA. STUDY DESIGN: Ex vivo biomechanical study comparing horses with and without a diagnosis of HERDA. METHODS: Forelimb suspensory ligament, superficial and deep digital flexor tendons; withers, forelimb and abdominal skin; the main pulmonary artery and the aortic arch were harvested from 6 horses with HERDA and 6 control horses without the HERDA allele. Tissues were distracted to failure. Tensile strength (TS), elastic modulus (EM) and energy to failure (ETF) were compared. RESULTS: Horses with HERDA had significantly lower TS and EM in tendinoligamentous tissues and great vessels, respectively. The TS, EM and ETF were significantly lower in skin from horses with HERDA. Differences in TS and ETF were more extreme at the withers than at the forelimb or abdomen. CONCLUSIONS: Tendinoligamentous tissue, great vessels and skin are significantly weaker in horses with HERDA than in horses lacking the PPIB mutation, substantiating that diverse tissues with high fibrillar collagen content are abnormal in HERDA and that the HERDA phenotype is not limited to the integument.

In vitro biomechanical comparison of three methods for internal fixation of femoral neck fractures in dogs.

The in vitro biomechanical properties of three methods for internal fixation of femoral neck fractures were evaluated. Fifty cadaveric femura from Beagle dogs were used. Ten intact femora served as controls. In 40 femura, an osteotomy of the femoral neck was performed to simulate a transverse fracture. With the remaining 30 femura, three repair methods (two medium Orthofix pins, a 2.7 mm cortical bone screw placed in lag fashion and an anti- rotational Kirschner wire, or three divergent 1.1 mm Kirschner wires) were used to stabilize the osteotomies, and 10 osteotomies were stabilised per repair method. These 30 femura where then subject to monotonic loading to failure. Construct stiffness and load to failure were measured. In the remaining 10 femura, pressure sensitive film was placed at the osteotomy site prior to stabilization with either two Orthofix pins (n = 5) or a screw placed in lag fashion (n = 5) to determine the compressive pressure (MPa), compressive force (KN) and area of compression (cm²). There was no significant difference in the stiffness or load to failure for the three repair methods evaluated. There was no significant difference in the compressive pressure, compressive force or area of compression in osteotomies stabilized with Orthofix pins and 2.7 mm bone screws.

The effect of tibial tuberosity advancement and meniscal release on kinematics of the cranial cruciate ligament-deficient stifle during early, middle, and late stance.

OBJECTIVES: To evaluate the effect of tibial tuberosity advancement (TTA) and meniscal release on cranial-caudal and axial rotational displacement during early, middle and late stance phases in the canine cranial cruciate ligament- (CCL) deficient stifle. STUDY DESIGN: In vitro biomechanical study. METHODS: Eighteen pelvic limbs were evaluated for the effects of TTA on cranial-caudal displacement and axial rotation under a load equivalent to 30% bodyweight, and under the following treatment conditions: normal (intact CCL), CCL deficient, TTA-treated (CCL deficient + TTA), and meniscal release (TTA treated + meniscal release). The limbs were evaluated in the early, middle, and late stance phases using electromagnetic tracking sensors to determine cranial tibial displacement and tibial rotation relative to the femur. RESULTS: Transection of the CCL resulted in significant cranial tibial displacement during early, middle, and late stance (p < 0.0001) and significant internal rotation during early (p = 0.049) and middle stance (p = 0.0006). Performance of TTA successfully eliminated cranial tibial displacement in early, middle, and late stance (p <0.0001); however, the TTA was unsuccessful in normalizing axial rotation in middle stance (p = 0.030). Meniscal release had no effect on cranial-caudal or rotational displacement when performed in conjunction with the TTA. CLINICAL SIGNIFICANCE: Tibial tuberosity advancement effectively eliminates cranial tibial displacement during early, middle and late stance; however, TTA failed to provide rotational stability in mid-stance.

A cryogenic clamping technique that facilitates ultimate tensile strength determinations in tendons and ligaments.

OBJECTIVE: To describe the use of a cryogenic clamp of novel design for tensile strength testing of tendinous and ligamentous tissues with inherently high tensile strength. METHODS: Inexpensive, easily machined steel clamps were manufactured to facilitate rapid insertion into a standard wedge-screw grip apparatus installed on a testing system with a control system attached. The deep digital flexor tendon (DDFT) of six horses was trimmed to a uniform dumbbell shape and secured in clamps using partial submersion in liquid nitrogen for approximately 45 seconds and immediately tested. Approximate time between removal from liquid nitrogen and failure of tendon was four minutes. RESULTS: Failure was achieved in all tendons tested in a region approximating a midpoint between the clamps. Ultimate failure loads of up to 6745 N were achieved without slippage of the tissue from the grips. The ultimate tensile strength of the normal equine DDFT determined in this study was 111.82 ± 11.53 N/mm2, and the stress versus grip-to-grip elongation plots for our equine DDFT were representative of a standard non-linear elastic curve obtained in similar studies. CLINICAL SIGNIFICANCE: We present a low cost device for quantifying physical properties of specimens with high connective tissue concentrations and inherent high tensile strength. Results of this study indicate that this device provides a practical alternative to other more costly methods of adequately securing larger tendons and ligaments for tensile strength testing.

Changes to articular cartilage following remote application of radiofrequency energy and with or without Cosequin therapy.

OBJECTIVE: To determine the short- and long-term changes in the biomechanical properties and metabolic activity of articular cartilage following the remote application of bipolar radiofrequency (bRF) and monopolar radiofrequency (mRF) energy within the rabbit stifle joint. METHODS: The rabbits were randomly assigned to either Group-1 (normal rabbit food), or they were assigned to Group-2 (2% Cosequin in the diet). Each rabbit underwent bilateral stifle arthroscopy with either bRF or mRF applied to the infrapatellar fat pad for 45 seconds. Cartilage samples were collected at zero, four, and 14 weeks after surgery. Data were analyzed with a mixed model analysis of variance (ANOVA) for chondrocyte death, amount of GAG synthesis, and the equilibrium compressive modulus. RESULTS: A significant increase in histological damage was noted at weeks four and 14 compared to week zero. Most of the chondrocyte death noted with confocal laser microscopy (49 of 56 samples) was noted in the superficial region (outer 25%) of the articular cartilage. GAG synthesis was not significantly different between groups or devices at any time point. A significant difference was not noted in equilibrium compressive modulus throughout the study. CONCLUSIONS: Remote application of bRF and mRF energy lead to immediate chondrocyte death. Most of the damage was superficial hence the metabolic activity and biomechanical properties of the extracellular matrix were maintained throughout this study. Treatment with Cosequin did not prevent superficial chondrocyte death caused by the application of radiofrequency (RF) energy with in the joint.

A device for performing whole bone torsional testing in a single-axis linear motion testing machine.

OBJECTIVES: To design a device for the conversion of linear to rotational motion and to use it in order to determine torsional properties of canine tibiae in a universal tension/compression testing machine. METHODS: A fixture incorporating a cable and pulley at one end and a guide block and rail assembly at the other was used to test 10 whole canine tibiae to failure in torsion at 1 degrees/s. RESULTS: The device produced spiral fractures in all tibiae. Torsional strength and stiffness were 13.9 +/- 1.4 N m and 0.53 +/- 0.07 N m/degree, respectively. CLINICAL SIGNIFICANCE: This inexpensive device can be used to evaluate the mechanical properties of long bones after various interventions to improve fracture healing. The device can be adapted for use with any single-axis linear motion testing machine. Assessment of fracture healing often includes loading to failure in torsion (1-5). Torsional testing is typically performed using a biaxial servohydraulic testing machine or similar sophisticated electromechanical equipment. Due to its high cost, many researchers do not have such equipment at their disposal. However, uniaxial tension-compression testing machines are widely available. Therefore, a device that can be used to perform torsional testing in a tension-compression machine would be beneficial. In this study we present a custom fixture for converting the vertical linear motion of a universal testing machine to rotational motion and validate its performance in torsional testing of canine tibiae.

Characterization of chitosan films and effects on fibroblast cell attachment and proliferation.

Chitosan has been researched for implant and wound healing applications. However, there are inconsistencies in reports on the tissue and fibroblast responses to chitosan materials. These inconsistencies may be due to variations in chitosan material characteristics. The aim of this study was to correlate fibroblast responses with known chitosan material characteristics. To achieve this aim, chitosan was characterized for degree of deacetylation (DDA), molecular weight (MW), residual protein and ash contents, and then solution cast into films and characterized for hydrophilicity by water contact angle. The films were seeded with normal human dermal fibroblasts and the number of attached cells was evaluated for after 30 min. Cell proliferation was evaluated over 5 days. This study found no relationship between DDA, contact angle, cell attachment, and or proliferation. General trends were observed for increasing proliferation with increasing residual ash content and decreasing residual protein. These data indicate that chitosan characteristics other than DDA may be important to their biological performance.

The role of negative intra-articular pressure in the maintenance of shoulder joint stability in dogs.

The objective of this study was to evaluate the effect of negative intra-articular pressure on shoulder joint stability in canine cadavers. Cadaver forelimbs from 12 mature dogs were used. The forelimbs were placed in a testing frame and axially preloaded with 4 kg of weight. Shoulder joint stability was tested in flexion, extension, and neutral position before and after venting of the joint capsule. Humeral translation relative to the glenoid was induced by applying a 3 kg load in three different directions (cranial, lateral, and medial) and quantitatively measured by use of an electromagnetic motion tracking system. Peak translational data were compared in each joint position before and after venting of the joint capsule. After venting the shoulder joint capsule, a significant increase in translation was observed in the cranial direction with the joint in neutral position and in the medial direction with the joint in extension. The horizontal translations measured after venting of the joint capsule were likely not clinically relevant. Negative intra-articular pressure is not a major contributor to shoulder stability in dogs during weight-bearing.

Influence of cyclic hydrostatic pressure on fibrocartilaginous metaplasia of achilles tendon fibroblasts.

The goal of this study was to demonstrate whether cyclically imposed hydrostatic pressure, compressive in nature, could induce fibrocartilaginous metaplasia in a purely tendinous cell source in vitro. The effect of short-duration cyclic hydrostatic pressure on tendon fibroblasts (tenocytes) expanded from rat Achilles tendon was studied. Total RNA was isolated either immediately after loading or 24 h later. The mRNA expression of tendon and cartilage specific markers - Collagen types I and II, Sox9, and Aggrecan was quantified by real-time reverse transcription polymerase chain reaction over multiple biological samples (n=6). For immediately isolated RNA samples, there were statistically significant increases in mRNA expression of Aggrecan and Collagen type II, while Collagen type I significantly decreased. Noticeably, for RNA samples isolated 24 h later, there were further increases in mRNA expression of Aggrecan and Collagen type II, whereas Collagen type I increased roughly three-fold relative to the non-loaded control. These findings support the hypothesis that cyclic hydrostatic pressurization can induce fibrocartilaginous metaplasia in tenocytes by upregulation of cartilaginous gene expression. Also, it was demonstrated that changes in mRNA expression as a result of single 2 h pressurization persist even up to 24 h.

Cyclic hydrostatic compression stimulates chondroinduction of C3H/10T1/2 cells.

While the potential for intermittent hydrostatic pressure to promote cartilaginous matrix synthesis is well established, its potential to influence chondroinduction remains poorly understood. This study examined the effects of relatively short- and long-duration cyclic hydrostatic compression on the chondroinduction of C3H/10T1/2 murine embryonic fibroblasts by recombinant human bone morphogenetic protein-2 (rhBMP-2). Cells were seeded at high density into round bottom wells of a 96-well plate and supplemented with 25 ng/ml rhBMP-2. Experimental cultures were subjected to either 1,800 cycles/day or 7,200 cycles/day of 1 Hz sinusoidal hydrostatic compression to 5 MPa (applied 10 min on/10 min off) for 3 days. Non-pressurized control and experimental cultures were maintained in static culture for an additional 5 days. Cultures were then analyzed for alcian blue staining intensity, DNA and sulfated glycosaminoglycan (sGAG) content, and for the rate of collagen synthesis. Whereas cultures subjected to 1,800 pressure cycles exhibited no significant differences (statistical or qualitative) compared to controls, those subjected to 7,200 cycles stained more intensely with alcian blue, contained nearly twice as much sGAG, and displayed twice the rate of collagen synthesis as non-pressurized controls. This study demonstrates the potential for cyclic hydrostatic compression to stimulate chondrogenic differentiation of the C3H/10T1/2 cell line in a duration-dependent manner.

Contact angle, protein adsorption and osteoblast precursor cell attachment to chitosan coatings bonded to titanium.

Chitosan, a derivative of the bio-polysaccharide chitin, has shown promise as a bioactive material for implant, tissue engineering and drug-delivery applications. The aim of this study was to evaluate the contact angle, protein adsorption and osteoblast precursor cell attachment to chitosan coatings bonded to titanium. Rough ground titanium (Ti) coupons were solution cast and bonded to 91.2% de-acetylated chitosan (1 wt% chitosan in 0.2% acetic acid) coatings via silane reactions. Non-coated Ti was used as controls. Samples were sterilized by ethylene oxide gas prior to experiments. Contact angles on all surfaces were measured using water. 5 x 10(4) cells/ml of ATCC CRL 1486 human embryonic palatal mesenchyme (HEPM) cells, an osteoblast precursor cell line, were used for the cell attachment study. SEM evaluations were performed on cells attached to all surfaces. Contact angles and cell attachment on all surfaces were statistically analyzed using ANOVA. The chitosan-coated surfaces (76.4 +/- 5.1 degrees) exhibited a significantly greater contact angle compared to control Ti surfaces (32.2 +/- 6.1 degrees). Similarly, chitosan-coated surfaces exhibited significantly greater (P < 0.001) albumin adsorption, fibronectin adsorption and cell attachment, as compared to the control Ti surfaces. Coating chitosan on Ti surfaces decreased the wettability of the Ti, but increased protein adsorption and cell attachment. Increased protein absorption and cell attachment on the chitosan-coated Ti may be of benefit in enhancing osseointegration of implant devices.

Energetics of nanocrystalline TiO2.

The energetics of the TiO(2) polymorphs (rutile, anatase, and brookite) were studied by high temperature oxide melt drop solution calorimetry. Relative to bulk rutile, bulk brookite is 0.71 +/- 0.38 kJ/mol (6) and bulk anatase is 2.61 +/- 0.41 kJ/mol higher in enthalpy. The surface enthalpies of rutile, brookite, and anatase are 2.2 +/- 0.2 J/m(2), 1.0 +/- 0.2 J/m(2), and 0.4 +/- 0.1 J/m(2), respectively. The closely balanced energetics directly confirm the crossover in stability of nanophase polymorphs inferred by Zhang and Banfield (7). An amorphous sample with surface area of 34,600 m(2)/mol is 24.25 +/- 0.88 kJ/mol higher in enthalpy than bulk rutile.

Chondrocyte differentiation is modulated by frequency and duration of cyclic compressive loading.

As part of a program of research aimed at determining the role of mechanical forces in connective tissue differentiation, we have developed a model for investigating the effects of dynamic compressive loading on chondrocyte differentiation in vitro. In the current study, we examined the influence of cyclic compressive loading of chick limb bud mesenchymal cells to a constant peak stress of 9.25 kPa during each of the first 3 days in culture. Cells embedded in agarose gel were subjected to uniaxial, cyclic compression at 0.03, 0.15, or 0.33 Hz for 2 h. In addition, load durations of 12, 54, or 120 min were evaluated while holding frequency constant at 0.33 Hz. For a 2 h duration, there was no response to loading at 0.03 Hz. A significant increase in chondrocyte differentiation was associated with loading at 0.15 Hz, and an even greater increase with loading at 0.33 Hz. Holding frequency constant at 0.33 Hz, a loading duration of 12 min elicited no response, whereas chondrocyte differentiation was enhanced by loading for either 54 or 120 min. Although not statistically significant from the 120 min response, average cartilage nodule density and glycosaminoglycan synthesis rate were highest in the 54 min duration group. This result suggests that cells may be sensitive to the level of cumulative (nonrecoverable) compressive strain, as well as to the dynamic strain history.

Effect of compressive loading on chondrocyte differentiation in agarose cultures of chick limb-bud cells.

It is well established that mechanical loading is important to homeostasis of cartilage tissue, and growing evidence suggests that it influences cartilage differentiation as well. Whereas the effect of mechanical forces on chondrocyte biosynthesis and gene expression has been vigorously investigated, the effect of the mechanical environment on chondrocyte differentiation has received little attention. The long-term objective of this research is to investigate the regulatory role of mechanical loading in cell differentiation. The goal of this study was to determine if mechanical compression could modulate chondrocyte differentiation in vitro. Stage 23/24 chick limb-bud cells, embedded in agarose gel, were subjected to either static (constant 4.5-kPa stress) or cyclic (9.0-kPa peak stress at 0.33 Hz) loading in unconfined compression during the initial phase of commitment to a phenotypic lineage. Compared with nonloaded controls, cyclic compressive loading roughly doubled the number of cartilage nodules and the amount of sulfate incorporation on day 8, whereas static compression had little effect on these two measures. Neither compression protocol significantly affected overall cell viability or the proliferation of cells within nodules. Since limb-bud mesenchymal cells were seeded directly into agarose, an assessment of cartilage nodules in the agarose reflects the proportion of the original cells that had given rise to chondrocytes. Thus, the results indicate that about twice as many mesenchymal cells were induced to enter the chondrogenic pathway by cyclic mechanical compression. The coincidence of the increase in sulfate incorporation and nodule density indicates that the primary effect of mechanical compression on mesenchymal cells was on cellular differentiation and not on their subsequent metabolism. Further studies are needed to identify the primary chondrogenic signal associated with cyclic compressive loading and to determine the mechanism by which it influences commitment to or progression through the chondrogenic lineage, or both.

Facile Routes to NiF(6)(2)(-), AgF(4)(-), AuF(6)(-), and PtF(6)(-) Salts Using O(2)(+) as a Source of O(2)F in Anhydrous HF.

O(2)(+) salts dissolved in liquid anhydrous hydrogen fluoride (aHF) at 20 degrees C or below oxidize aHF solutions of PtF(6)(2)(-) to PtF(6)(-). The parent base of O(2)(+) salts in aHF (O(2)F((solv))) generated with alkali fluoride is long-lived below -50 degrees C. An aHF solution of O(2)F((solv)) oxidizes Au(III) to Au(V) below -50 degrees C (2O(2)F((solv)) + AuF(4)(-)((solv)) --> AuF(6)(-)((solv)) +2O(2(g))). In situ generation of O(2)F((solv)) (O(2)(+)((solv)) + F(-)((solv)) --> O(2)F((solv))) with AgF(2) or NiF(2) in suspension in the aHF made basic with alkali fluoride gives AgF(4)(-) and NiF(6)(2)(-)salts. Low solubility of AAsF(6)(A = Cs, K) in aHF provides for the metathetical preparation of (O(2))(2)PdF(6) solutions in aHF. Removal of aHF, even at -60 degrees C, results in some O(2) and F(2) loss, to a composition approaching (O(2))PdF(5).

A new optical system for the determination of deformations and strains: calibration characteristics and experimental results.

Many types of optical strain measurement systems have been used for the determination of deformations and strains in soft biological tissues. The purpose of this investigation is to report a new optical strain measurement system developed in our laboratory which offers distinct advantages over systems developed in the past. Our optical strain system has demonstrated excellent performance in calibration and experimental tests. Calibration tests illustrate the system's accuracy to 0.05% strain at 3.52% strain and 0.18% strain at 11.74% strain. Further, this system can measure strains to within 2% measurement error for strains in a 0-11.74% range when 100 microns increments of motion are used for calibration. The resolution of our system appears to be at least as good as the linear micrometer (2 microns) used as a calibrating standard. Errors in strain measurement due to whole specimen rotation or translation are quantified. Rotations about an in-plane axis perpendicular to the direction of strain and translations in/out of the plane of focus result in the largest sources of error. Finally, in an in vitro biomechanical study of the rabbit Achilles tendon, experimental failure strains are 4.3 +/- 0.9% using this system.

Revision arthroplasty facilitated by ultrasonic tool cement removal. An evaluation of whole bone strength in a canine model.

Ultrasonic driven tools have been developed to facilitate the removal of bone cement during revision arthroplasty. The effect on whole bone strength of cement removal by ultrasonic tools was examined in a canine femur model. Paired, fresh-frozen canine femora were divided into two groups. In group A, one femur from each pair was subjected to cement extraction with ultrasonic tools. In group B, one femur from each pair was subjected to manual cement extraction. Contralateral femora from each pair served as controls to determine the strength of intact femora. Torsional fractures were produced using a servocontrolled hydraulic testing machine (Minneapolis Testing System, Minneapolis, MN). Maximum torque, maximum angle, and energy capacity to failure were determined. Results were recorded as a reduction in percent value of the tested specimen versus the contralateral control. When comparing femora with cement removal by ultrasonic tools to the contralateral control femur, there were no statistical differences in ultimate torque (P = .83), maximum angle (P = .89), and energy capacity (P = .74) by analysis of variance. In addition, there were no significant differences between the group with ultrasonic tool cement removal and the group with manual tool removal. The authors conclude that in this canine model, removal of cement with ultrasonically driven tools has no adverse effects on whole bone strength.

Screw-augmented fixation of acetabular components. A mechanical model to determine optimal screw placement.

Sixteen embalmed hemipelves were used to determine the optimal acetabular screw placement to provide maximal screw pull-out strength in unicortical and bicortical screw fixation. The anterior column, superior ilium, posterior column, ischium, and pubis regions of the pelvis were tested using 6.5-mm titanium alloy screws and a hydraulic servo-controlled 1321 Instron testing machine. Force vs displacement data were acquired. Bicortical fixation was stronger than unicortical fixation in the four zones compared. This difference was significant in the superior ilium, posterior column, and ischium. The anterior column could not accept unicortical screws due to inadequate bone depth, which ranged between only 6 mm and 10 mm. Bicortical fixation was significantly greater in the superior ilium, posterior column, and ischium than in the anterior column or pubis. Unicortical fixation was greatest in the superior ilium. This information may aid decisions concerning the positioning of screws to augment acetabular component fixation.

The impact of supportive intervention after second trimester termination of pregnancy for fetal abnormality.

The reactions of women who had had a termination of pregnancy for fetal abnormality in the second trimester have been studied retrospectively using a semi-structured questionnaire. The severity of the grief reaction was measured and the outcome at 6 months was compared with the findings from a previous study in South Wales which had led to the introduction of skilled support from genetic fieldworkers and formal genetic counselling after the termination. Of the 69 women interviewed, 55 (80 per cent) experienced an acute grief reaction and 17 (25 per cent) had not resolved their grief 6 months after the termination, compared with 37 (77 per cent) and 22 (46 per cent) out of 48 respectively in the previous study. Fifty-seven (83 per cent) women had found the fieldworker's intervention useful or very useful, some describing her support as essential. An association between poor resolution of the grief reaction with increasing maternal age and with poor perceived support from partners was noted. Improved follow-up support and counselling have lessened the adverse emotional consequences and support should therefore be offered to all women undergoing termination for fetal malformation.

PIP: The reactions of women who had undergone a termination of pregnancy for fetal abnormality in the 2nd trimester have been studied retrospectively using a semistructured questionnaire. The severity of the grief reaction was measured and the outcome at 6 months compared with the findings from a previous study in South Wales which had led to the introduction of skilled support from genetic fieldworkers and formal genetic counseling after the termination. Of the 69 women interviewed, 55 (80%) experienced an acute grief reaction and 17 (25%) had not resolved their grief 6 months after the termination, compared with 37 (77%) and 22 (46%) of 48, respectively, in the previous study. 57 (83%) women has found the fieldworker's intervention useful or very useful, some describing the support received as essential. An association between poor resolution of the grief reaction with increasing maternal age and with poor perceived support from partners was noted. Improved followup support and counseling have reduced the adverse emotional consequences, and support should thus be offered to all women undergoing pregnancy termination for fetal malformations.