Effect of Press-Fit Size on Insertion Mechanics and Cartilage Viability in Human and Ovine Osteochondral Grafts.

OBJECTIVE: The osteochondral allograft procedure uses grafts constructed larger than the recipient site to stabilize the graft, in what is known as the press-fit technique. This research aims to characterize the relationships between press-fit size, insertion forces, and cell viability in ovine and human osteochondral tissue. DESIGN: Human (4 donors) and ovine (5 animals) articular joints were used to harvest osteochondral grafts (4.55 mm diameter, N = 33 Human, N = 35 Ovine) and create recipient sites with grafts constructed to achieve varying degrees of press fit (0.025-0.240 mm). Donor grafts were inserted into recipient sites while insertion forces were measured followed by quantification of chondrocyte viability and histological staining to evaluate the extracellular matrix. RESULTS: Both human and ovine tissues exhibited similar mechanical and cellular responses to changes in press-fit. Insertion forces (Human: 3-169 MPa, Ovine: 36-314 MPa) and cell viability (Human: 16%-89% live, Ovine: 2%-76% live) were correlated to press-fit size for both human (force: r = 0.539, viability: r = -0.729) and ovine (force: r = 0.655, viability: r = -0.714) tissues. In both species, a press-fit above 0.14 mm resulted in reduced cell viability below a level acceptable for transplantation, increased insertion forces, and reduced linear correlation to press-fit size compared to samples with a press-fit below 0.14 mm. CONCLUSIONS: Increasing press-fit size required increased insertion forces and resulted in reduced cell viability. Ovine and human osteochondral tissues responded similarly to impact insertion and varying press-fit size, providing evidence for the use of the ovine model in allograft-related research.

Evaluation of equine articular cartilage degeneration after mechanical impact injury using cationic contrast-enhanced computed tomography.

OBJECTIVE: Cationic agent contrast-enhanced computed tomography (cationic CECT) characterizes articular cartilage ex vivo, however, its capacity to detect post-traumatic injury is unknown. The study objectives were to correlate cationic CECT attenuation with biochemical, mechanical and histological properties of cartilage and morphologic computed tomography (CT) measures of bone, and to determine the ability of cationic CECT to distinguish subtly damaged from normal cartilage in an in vivo equine model. DESIGN: Mechanical impact injury was initiated in equine femoropatellar joints in vivo to establish subtle cartilage degeneration with site-matched controls. Cationic CECT was performed in vivo (clinical) and postmortem (microCT). Articular cartilage was characterized by glycosaminoglycan (GAG) content, biochemical moduli and histological scores. Bone was characterized by volume density (BV/TV) and trabecular number (Tb.N.), thickness (Tb.Th.) and spacing (Tb.Sp.). RESULTS: Cationic CECT attenuation (microCT) of cartilage correlated with GAG (r = 0.74, P < 0.0001), compressive modulus (Eeq) (r = 0.79, P < 0.0001) and safranin-O histological score (r = -0.66, P < 0.0001) of cartilage, and correlated with BV/TV (r = 0.37, P = 0.0005), Tb.N. (r = 0.39, P = 0.0003), Tb.Th. (r = 0.28, P = 0.0095) and Tb.Sp. (r = -0.44, P < 0.0001) of bone. Mean [95% CI] cationic CECT attenuation at the impact site (2215 [1987, 2443] Hounsfield Units [HUs]) was lower than site-matched controls (2836 [2490, 3182] HUs, P = 0.036). Clinical cationic CECT attenuation correlated with GAG (r = 0.23, P = 0.049), Eeq (r = 0.26, P = 0.025) and safranin-O histology score (r = -0.32, P = 0.0046). CONCLUSIONS: Cationic CECT (microCT) reflects articular cartilage properties enabling segregation of subtly degenerated from healthy tissue and also reflects bone morphometric properties on CT. Cationic CECT is capable of characterizing articular cartilage in clinical scanners.

Engineering of hyaline cartilage with a calcified zone using bone marrow stromal cells.

OBJECTIVE: In healthy joints, a zone of calcified cartilage (ZCC) provides the mechanical integration between articular cartilage and subchondral bone. Recapitulation of this architectural feature should serve to resist the constant shear force from the movement of the joint and prevent the delamination of tissue-engineered cartilage. Previous approaches to create the ZCC at the cartilage-substrate interface have relied on strategic use of exogenous scaffolds and adhesives, which are susceptible to failure by degradation and wear. In contrast, we report a successful scaffold-free engineering of ZCC to integrate tissue-engineered cartilage and a porous biodegradable bone substitute, using sheep bone marrow stromal cells (BMSCs) as the cell source for both cartilaginous zones. DESIGN: BMSCs were predifferentiated to chondrocytes, harvested and then grown on a porous calcium polyphosphate substrate in the presence of triiodothyronine (T3). T3 was withdrawn, and additional predifferentiated chondrocytes were placed on top of the construct and grown for 21 days. RESULTS: This protocol yielded two distinct zones: hyaline cartilage that accumulated proteoglycans and collagen type II, and calcified cartilage adjacent to the substrate that additionally accumulated mineral and collagen type X. Constructs with the calcified interface had comparable compressive strength to native sheep osteochondral tissue and higher interfacial shear strength compared to control without a calcified zone. CONCLUSION: This protocol improves on the existing scaffold-free approaches to cartilage tissue engineering by incorporating a calcified zone. Since this protocol employs no xenogeneic material, it will be appropriate for use in preclinical large-animal studies.

Regional and depth variability of porcine meniscal mechanical properties through biaxial testing.

The menisci in the knee joint undergo complex loading in-vivo resulting in a multidirectional stress distribution. Extensive mechanical testing has been conducted to investigate the tissue properties of the knee meniscus, but the testing conditions do not replicate this complex loading regime. Biaxial testing involves loading tissue along two different directions simultaneously, which more accurately simulates physiologic loading conditions. The purpose of this study was to report mechanical properties of meniscal tissue resulting from biaxial testing, while simultaneously investigating regional variations in properties. Ten left, fresh porcine joints were obtained, and the medial and lateral menisci were harvested from each joint (twenty menisci total). Each menisci was divided into an anterior, middle and posterior region; and three slices (femoral, deep and tibial layers) were obtained from each region. Biaxial and constrained uniaxial testing was performed on each specimen, and Young's moduli were calculated from the resulting stress strain curves. Results illustrated significant differences in regional mechanical properties, with the medial anterior (Young's modulus (E)=11.14 ± 1.10 MPa), lateral anterior (E=11.54 ± 1.10 MPa) and lateral posterior (E=9.0 ± 1.2 MPa) regions exhibiting the highest properties compared to the medial central (E=5.0 ± 1.22 MPa), medial posterior (E=4.16 ± 1.13 MPa) and lateral central (E=5.6 ± 1.20 MPa) regions. Differences with depth were also significant on the lateral meniscus, with the femoral (E=12.7 ± 1.22 MPa) and tibial (E=8.6 ± 1.22 MPa) layers exhibiting the highest Young's moduli. This data may form the basis for future modeling of meniscal tissue, or may aid in the design of synthetic replacement alternatives.

Streaming potential-based arthroscopic device is sensitive to cartilage changes immediately post-impact in an equine cartilage injury model.

Models of post-traumatic osteoarthritis where early degenerative changes can be monitored are valuable for assessing potential therapeutic strategies. Current methods for evaluating cartilage mechanical properties may not capture the low-grade cartilage changes expected at these earlier time points following injury. In this study, an explant model of cartilage injury was used to determine whether streaming potential measurements by manual indentation could detect cartilage changes immediately following mechanical impact and to compare their sensitivity to biomechanical tests. Impacts were delivered ex vivo, at one of three stress levels, to specific positions on isolated adult equine trochlea. Cartilage properties were assessed by streaming potential measurements, made pre- and post-impact using a commercially available arthroscopic device, and by stress relaxation tests in unconfined compression geometry of isolated cartilage disks, providing the streaming potential integral (SPI), fibril modulus (Ef), matrix modulus (Em), and permeability (k). Histological sections were stained with Safranin-O and adjacent unstained sections examined in polarized light microscopy. Impacts were low, 17.3 ± 2.7 MPa (n = 15), medium, 27.8 ± 8.5 MPa (n = 13), or high, 48.7 ± 12.1 MPa (n = 16), and delivered using a custom-built spring-loaded device with a rise time of approximately 1 ms. SPI was significantly reduced after medium (p = 0.006) and high (p<0.001) impacts. Ef, representing collagen network stiffness, was significantly reduced in high impact samples only (p < 0.001 lateral trochlea, p = 0.042 medial trochlea), where permeability also increased (p = 0.003 lateral trochlea, p = 0.007 medial trochlea). Significant (p < 0.05, n = 68) moderate to strong correlations between SPI and Ef (r = 0.857), Em (r = 0.493), log(k) (r = -0.484), and cartilage thickness (r = -0.804) were detected. Effect sizes were higher for SPI than Ef, Em, and k, indicating greater sensitivity of electromechanical measurements to impact injury compared to purely biomechanical parameters. Histological changes due to impact were limited to the presence of superficial zone damage which increased with impact stress. Non-destructive streaming potential measurements were more sensitive to impact-related articular cartilage changes than biomechanical assessment of isolated samples using stress relaxation tests in unconfined compression geometry. Correlations between electromechanical and biomechanical methods further support the relationship between non-destructive electromechanical measurements and intrinsic cartilage properties.

A polarized light microscopy method for accurate and reliable grading of collagen organization in cartilage repair.

OBJECTIVES: Collagen organization, a feature that is critical for cartilage load bearing and durability, is not adequately assessed in cartilage repair tissue by present histological scoring systems. Our objectives were to develop a new polarized light microscopy (PLM) score for collagen organization and to test its reliability. DESIGN: This PLM score uses an ordinal scale of 0-5 to rate the extent that collagen network organization resembles that of young adult hyaline articular cartilage (score of 5) vs a totally disorganized tissue (score of 0). Inter-reader reliability was assessed using Intraclass Correlation Coefficients (ICC) for Agreement, calculated from scores of three trained readers who independently evaluated blinded sections obtained from normal (n=4), degraded (n=2) and repair (n=22) human cartilage biopsies. RESULTS: The PLM score succeeded in distinguishing normal, degraded and repair cartilages, where the latter displayed greater complexity in collagen structure. Excellent inter-reader reproducibility was found with ICCs for Agreement of 0.90 [ICC(2,1)] (lower boundary of the 95% confidence interval is 0.83) and 0.96 [ICC(2,3)] (lower boundary of the 95% confidence interval is 0.94), indicating the reliability of a single reader's scores and the mean of all three readers' scores, respectively. CONCLUSION: This PLM method offers a novel means for systematically evaluating collagen organization in repair cartilage. We propose that it be used to supplement current gold standard histological scoring systems for a more complete assessment of repair tissue quality.

Application of robotic technology in biomechanics to study joint laxity.

PRIMARY OBJECTIVE: To evaluate whether in vitro joint testing using a robot with six degrees of freedom is useful for evaluating changes in joint laxity as a result of chronic osteoarthritis (OA). RESEARCH DESIGN: Repeated measures. METHODS: Broyden's method of solving nonlinear systems of equations drove a hybrid method of load and position robotic control. Sheep stifles (knee joints) were loaded between 3 Nm of internal load through to 3 Nm of external load in 1 Nm increments. Kinematic and morphologic data from five healthy ovine stifles were compared to the chronic OA effects in four surgically destabilized stifles. RESULTS: Stifles with chronic OA showed increases in stiffness while range of motion decreased. Gross morphologic changes included osteophytes and cartilage fibrillation. DISCUSSION: Robotic testing proved useful for evaluating changes in joint mechanics as a result of chronic OA. We observed morphological changes and associated increases in joint stiffness and decreased laxity.

Contact mechanics of the ovine stifle during simulated early stance in gait. An in vitro study using robotics.

Ovine stifle joint contact pressures and contact areas were measured in vitro using a six degree-of-freedom (DOF) robotic system. The robot generated static joint loads of 1.875 times body weight (BW) compression, 0.15 BW medial shear and 0.625 BW cranial shear at 6.5 degrees of flexion for four specimens, simulating the early stance phase of gait (walking). This condition represents a period of intense loading and was implemented as a worst-case loading scenario for the joint at this gait. We determined that the medial and lateral compartments bore 5.5 +/- 0.9 MPa and 4.4 +/- 1.1 MPa of mean pressure, respectively, on 107.7 +/- 28.7 mm(2) and 60.8 +/- 56.3 mm(2) of area, respectively. The unique contribution of this study is that stifle contact pressures and areas were determined during loading which simulated physiological levels (early stance phase of gait). This information is important to our understanding of the stresses that must be borne by repair tissues/constructs that are implanted into human and animal tibio-femoral joints.

Mapping of donor and recipient site properties for osteochondral graft reconstruction of subchondral cystic lesions in the equine stifle joint.

REASONS FOR PERFORMING STUDY: To improve osteochondral graft reconstruction of subchondral cystic lesions in the medial and lateral femoral condyles by matching the material properties of donor and recipient sites. OBJECTIVES: To measure biomechanical and biochemical parameters that influence the function and healing of osteochondral grafts used to reconstruct subchondral cystic lesions. HYPOTHESIS: Suitable donor sites are available within the stifle joint for reconstructing the femoral condyles, despite considerable regional property variation. METHODS: Fifty-six osteochondral cores were harvested from 6 distal femurs for initial studies that determined subchondral bone modulus of elasticity and ultimate stress. In a second study, 28 osteochondral cores were harvested from 6 distal femurs to measure cartilage aggregate modulus, thickness and sulphated glycosaminoglycan (sGAG) content. Using micro-CT imaging, subchondral bone mineral density and bone volume fraction were also measured. In both studies 2-dimensional contour plots using a bicubic interpolation method and normalised data were generated to allow visual comparison of joint surface characteristics. Statistical comparisons between donor and recipient site raw data were made using an ANOVA for repeated measures with a post hoc Tukey test. RESULTS: Material properties of cartilage and bone vary considerably over the surface of the stifle joint but the central region of the medial condyle, where subchondral cystic lesions freqdently occur, typically demonstrated bone strength and modulus values of the highest observed. Cartilage thickness and aggregate modulus were highest in the medial femoral condyle and axial aspect of the lateral condyle. CONCLUSIONS: Material properties of the grafts from the trochlear groove and axial aspect of the lateral trochlear ridge were the closest match for those found in the medial condyle, whereas properties of the lateral condyle were most similar to those found in the trochlear groove and axial aspect of the medial trochlear ridge.

An investigation of 2 techniques for optimizing joint surface congruency using multiple cylindrical osteochondral autografts.

PURPOSE: To compare 2 techniques for optimizing joint congruency for miniature osteochondral autografting in the knee: intrinsic postoperative forces acting on overdrilled autografts protruding from the femur versus alignment by a surgeon at the time of grafting. TYPE OF STUDY: Controlled animal model experiment. METHODS: A full-thickness cartilage defect was created on the weight-bearing surface of the medial femoral condyle of 13 mature sheep. Three 4.5 x 10 mm cylindrical autografts were inserted into 14-mm deep recipient holes such that the grafts were held in place by side-wall friction alone. One treatment group received grafts that were delivered flush with the surrounding cartilage and the second group received grafts that were left 2-mm proud of the joint surface. RESULTS: Three months postoperatively, the proud grafts had been repositioned by weight bearing but perigraft fissuring and fibroplasia, and subchondral cavitations were serious complications. It is suspected that these complications were caused by excessive motion between the graft and recipient site in the proud grafts. CONCLUSIONS: Grafts should be delivered flush with the joint surface when performing osteochondral transfers to avoid graft micromotion and the consequent interference with graft integration and function.

Surgical repair of a ruptured cranial cruciate ligament in a dromedary camel.

A 14-year-old dromedary camel was examined because of lameness of the left hind limb of 3.5 months'duration. The camel injured the stifle joint when it slipped while reclining into sternal recumbency. Radiography did not reveal major abnormalities that could account for the lameness, but nuclear scintigraphy revealed increased radionuclide uptake in the left stifle joint. Intra-articular injection of anesthetic further localized the injury to the area of the left stifle joints, and arthroscopy of that joint revealed rupture of the cranial cruciate ligament. Surgical repair of the ligament was attempted, using an autogenous graft obtained from the tensor fascia lata muscle. Outcome for the camel initially was considered good on the basis of a substantial improvement in attitude, apparent increase in comfort, and decrease in lameness. The tendency for the camel to remain in sternal recumbency after surgery increased the number and severity of postoperative complications. The camel was doing well 9 months after surgery; however, it was euthanatized 16 months after surgery because of a 4-month episode of lameness. Necropsy revealed chronic osteoarthritis and bilateral rupture of the cranial cruciate ligaments.

Osteochondral dowel transplantation for repair of focal defects in the knee: an outcome study using an ovine model.

OBJECTIVE: A model system was developed to objectively assess the quality of articular cartilage after surgical reconstruction of focal defects in the median femoral condyle using osteochondral dowel grafts. STUDY DESIGN: The surgical technique was developed and customized to reproducibly minimize surgical trauma and graft instability in order to improve the survival of the transplanted cartilage and the long-term integrity of the joint surfaces. ANIMALS OR SAMPLE POPULATION: 24 adult female Suffolk-Romanoff crossbred sheep. METHODS: Biomechanical creep testing, paravital staining for chondrocyte viability, histological analysis, and gross morphological analysis were performed at 3, 6, and 12 months postoperatively to compare fresh autografted osteochondral dowels with allografts that had been subject to a freezing protocol known to kill chondrocytes. The latter was used to investigate the time course of cartilage degeneration after injury. These two groups were also compared with normal unoperated control tissue. RESULTS: Biomechanical behavior, chondrocyte survival, and cartilage histology differed significantly between fresh grafts and those that had been frozen. CONCLUSIONS: Indentation testing and paravital staining were able to identify degenerative changes earlier than other methods of assessment. The technique developed here reproducibly and reliably transplanted osteochondral dowel grafts while minimizing the confounding effects of surgical trauma and graft instability. CLINICAL RELEVANCE: The technique provides both a promising surgical technique for the repair of focal defects of the medial femoral condyle and a sensitive model for the future study of cryopreservation strategies for articular cartilage.

Development of a laboratory animal model of postoperative small intestinal adhesion formation in the rabbit.

In order to establish a model of postoperative intestinal adhesions that would simulate the problem experienced in horses, New Zealand White rabbits were utilized to compare two models of adhesion formation that had been successful in the horse, an ischemic strangulating obstruction (ISO) model and a serosal scarification model. An untreated control group was compared with animals subjected to 1, 2, 3 and 4 h periods of ISO, and to serosal scarification. At postmortem examination 14 d postoperatively, the number of rabbits in each group with adhesions was recorded. Serosal scarification was significantly more consistent at producing adhesions than ISO (Fisher's exact test, P = 0.0022). The 3 h of ISO group was significantly different from the control group: however, compared to the serosal scarification group, fewer animals had adhesions and one animal died of complications associated with the experimental procedure. Based on these results, serosal scarification was selected as the best model for utilization in further studies of adhesion prevention.

Utilization of the serosal scarification model of postoperative intestinal adhesion formation to investigate potential adhesion-preventing substances in the rabbit.

A rabbit serosal scarification model was utilized to compare the ability of four drugs, previously administered peri-operatively to horses undergoing exploratory celiotomy, to prevent the development of postoperative intestinal adhesions. The substances compared were 32% Dextran 70 (7 mL/kg), 1% sodium carboxymethylcellulose (7 mL/kg), trimethoprim-sulfadiazine (30 mg/kg), and flunixin meglumine (1 mg/kg). The first two were administered intra-abdominally following surgery, while the latter two were administered systemically in the peri-operative period. Fibrous adhesions were evident in all animals in the untreated serosal scarification group. No significant difference in the number of animals with adhesions was found between the untreated control group and any treatment group, nor among the treatment groups. Microscopic examination of adhesions collected at postmortem examination revealed fibers consistent with cotton, surrounded by a giant-cell reaction and ongoing acute inflammation. The source of the fibers was likely the cotton laparotomy sponges used to scarify the intestinal surface, since the pattern in the granuloma and sponge fibers appeared similar under polarized light. Though consistent intestinal adhesion formation was produced in the rabbit, the presence of foreign body granulomas may prevent consideration of this model for future research. The drugs tested were ineffective in preventing the formation of postoperative small intestinal adhesions in this model.

Geometrical properties of the ovine tibia: a suitable animal model to study the pin-bone interface in fracture fixation?

To determine whether dimensional scaling (relative to the human) is necessary for screwed pins used in externally applied fracture fixation studies on sheep, geometrical data were determined for six ovine tibiae. Each tibia was potted relative to a lengthwise reference axis and sectioned at 5 per cent length intervals over its central 80 per cent. Enlarged (280 per cent) images of each cross-section were digitized at 1 mm increments around the periphery of the periosteal and endosteal surfaces, the data were digitally filtered, and geometrical properties were computed to include cross-sectional area A, maximum and minimum second moments of area (Imax and Imin), polar second moment of area J, and effective polar second moment of area J(eff). Proportional scaling of geometrical properties with respect to bone length (L2 for A, and L4 for second moments of area) significantly (p < 0.000001) decreased the coefficient of variation in data by an average 36 per cent. From 30-90 per cent distal, J(eff) for the ovine tibia is smaller but within 7 per cent of J--in stark contrast with the human tibia, where J(eff) has been reported as 70-80 per cent of J over the same tibial length. While previous ovine studies involving external fixator pins have employed the same diameter of pin as has been used in humans (that is 5 or 6 mm), a 'first-order' approximation of the data for A, Imax, Imin and J(eff) suggests these pins should be scaled down to 4 mm and 4.75 mm respectively for use on the ovine tibia over the range 25-80 per cent distal along its length.

Heterotopic transfer of fresh and cryopreserved autogenous articular cartilage in the horse.

Two 10 mm thick osteochondral grafts were harvested from the lateral aspect of the lateral trochlear ridge of the left talus in each of 10 anesthetized horses. The grafts were frozen in a 7.5% DMSO solution and stored in liquid nitrogen. The horses were anesthetized again on day 14 and the thawed grafts were press-fitted into drill holes in the trochlear ridges of the right stifle. A fresh graft was transferred from the right hock to the left stifle. To control for the effects of surgery, another fresh graft was transferred from the right stifle to the left stifle. The result was two grafts in each femoropatellar joint. Fresh and frozen osteoarticular autografts appeared to maintain a durable weight-bearing surface for 3 months; however, the fresh grafts were clearly superior. Frozen grafts had fewer living chondrocytes, decreased safranin-O staining, and decreased SO435 uptake. Graft stability and articular surface congruency were determining factors in the outcome of all grafts. Since the availability of osteochondral autografts is limited, further work on the use of preserved allogeneic osteochondral tissue is warranted.

Incorporation of fresh and cryopreserved bone in osteochondral autografts in the horse.

The structural integrity of subchondral bone in fresh and frozen osteochondral autografts was investigated at month 3 in 10 horses. Two osteochondral autografts were harvested from the lateral aspect of the lateral trochlear ridge of the left talus in each of 10 anesthetized horses. Grafts were frozen in 7.5% DMSO. After 14 days, the thawed grafts were press-fitted into drill holes in the trochlear ridges of the right stifles. A fresh graft from the right hock was implanted in each left stifle. To control for the effects of surgery, a fresh graft was transferred from the right stifle to the left stifle. The end result was two grafts in each femoropatellar joint. Fresh and frozen bone grafts maintained a structurally intact support for the cartilage surface. Graft stability and surface congruency were determining factors in the outcome of the grafts. Incorporation of both types of graft was complete at month 3, but remodeling of the fresh grafts was more active.

Diagnosis of equine stifle joint disorders: three cases.

The clinical, radiographic, arthrographic, arthroscopic, and pathological findings of three horses with femorotibial joint injuries are presented. Overall diagnostic accuracy is improved when clinical signs, arthrography, and arthroscopy are combined. Treatment of these injuries remains limited. One horse was euthanized, and two were treated by stall rest after diagnosis. Of the two surviving horses, one became a successful breeding animal, and the other was unable to perform as a pleasure horse.

Cartilage healing: A review with emphasis on the equine model.

Articular cartilage is a remarkably resilient tissue capable of withstanding considerable stress and repeated loading. Since this tissue has no blood vessels, nerve elements, or lymphatics, it is not surprising that it has a limited capacity for repair when damaged. In the horse, cartilage damage occurs as an occupational hazard. Furthermore, developmental defects such as osteochondrosis can lead to osteochondritis dissecans. Resultant cartilage flaps, fissures, and poorly organized subchondral bone produce disruption of joint surfaces.Veterinarians are often called upon to intervene when damaged cartilage has healed incompletely. Basic understanding of the physiology and repair mechanisms of cartilage is paramount to successfully managing such injuries. This literature review gives a brief overview of recently published clinical and experimental studies on the healing of cartilage. The discussion centers on the equine model.