Effects of low-intensity pulsed ultrasound on new trabecular bone during bone-tendon junction healing in a rabbit model: a synchrotron radiation micro-CT study.

This study was designed to evaluate the effects of low-intensity pulsed ultrasound on bone regeneration during the bone-tendon junction healing process and to explore the application of synchrotron radiation micro computed tomography in three dimensional visualization of the bone-tendon junction to evaluate the microarchitecture of new trabecular bone. Twenty four mature New Zealand rabbits underwent partial patellectomy to establish a bone-tendon junction injury model at the patella-patellar tendon complex. Animals were then divided into low-intensity pulsed ultrasound treatment (20 min/day, 7 times/week) and placebo control groups, and were euthanized at week 8 and 16 postoperatively (n = 6 for each group and time point). The patella-patellar tendon specimens were harvested for radiographic, histological and synchrotron radiation micro computed tomography detection. The area of the newly formed bone in the ultrasound group was significantly greater than that of control group at postoperative week 8 and 16. The high resolution three dimensional visualization images of the bone-tendon junction were acquired by synchrotron radiation micro computed tomography. Low-intensity pulsed ultrasound treatment promoted dense and irregular woven bone formation at week 8 with greater bone volume fraction, number and thickness of new trabecular bone but with lower separation. At week 16, ultrasound group specimens contained mature lamellar bone with higher bone volume fraction and thicker trabeculae than that of control group; however, there was no significant difference in separation and number of the new trabecular bone. This study confirms that low-intensity pulsed ultrasound treatment is able to promote bone formation and remodeling of new trabecular bone during the bone-tendon junction healing process in a rabbit model, and the synchrotron radiation micro computed tomography could be applied for three dimensional visualization to quantitatively evaluate the microarchitecture of new bone in bone-tendon junction.

Experimental remodellation of extracorporeal irradiated autogenous and allogenic patellar grafts.

BACKGROUND: Irradiated autografts have been used to aid the recovery of bone defects, and the results are well documented. Recently, bone allografts with tendinous attachments have been used to restore the function of joints. Similar reconstructions can be performed with irradiated autografts. However, little information is available on the biology of extracorporeal irradiated tendon autografts. QUESTIONS/PURPOSES: An experimental rabbit model was used to investigate the quality of healing and remodeling of the irradiated autogenous patellar tendon graft after 3 and 12 weeks using immunohistochemical and morphometric analyses. METHODS: New Zealand rabbits (n = 40) were randomly divided into autograft and allograft groups. The right knees of all animals served as the control (sham). The patellar tendon graft of the control right knee was reimplanted into its original location without any processing, while the patellar tendon of the left knee in the autograft group was reimplanted into the original location after 50 Gy irradiation. In the allograft group, the patellar tendon was sutured to the knee of another rabbit following 50 Gy irradiation. Five rabbits from each group were sacrificed and examined histologically. RESULTS: There were significant differences in the number of fibroblasts after 12 weeks between allograft and sham groups (P = 0.002). On the other hand, there were no differences between the allograft and autograft groups at the 12th week (P = 0.139). The difference in fibroblast numbers between autograft and allograft groups was statistically significant after the 3rd week (P < 0.05). Collagen fibril thickness was different between both the allograft and sham groups (P = 0.002) and the allograft and autograft groups at the 12th week (P = 0.000). Collagen fibrils were thicker in the sham and autograft groups compared with the allograft group at the 3rd week of evaluation (P < 0.05). The Ki67 index was significantly different between the allograft and sham groups at the 12th week (P < 0.032), while there was no difference between the allograft and autograft groups (P > 0.05). At the 3rd week, Ki67 reactivity was higher in the allograft group compared with the other two groups (P < 0.05).

Effect of low dose and moderate dose gamma irradiation on the mechanical properties of bone and soft tissue allografts.

The increased use of allograft tissue for musculoskeletal repair has brought more focus to the safety of allogenic tissue and the efficacy of various sterilization techniques. Gamma irradiation is an effective method for providing terminal sterilization to biological tissue, but it is also reported to have deleterious effects on tissue mechanics in a dose-dependent manner. At irradiation ranges up to 25 kGy, a clear relationship between mechanical strength and dose has yet to be established. The aim of this study was to investigate the mechanical properties of bone and soft tissue allografts, irradiated on dry ice at a low absorbed dose (18.3-21.8 kGy) and a moderate absorbed dose (24.0-28.5 kGy), using conventional compressive and tensile testing, respectively. Bone grafts consisted of Cloward dowels and iliac crest wedges, while soft tissue grafts consisted of patellar tendons, anterior tibialis tendons, semitendinosus tendons, and fascia lata. There were no statistical differences in mechanical strength or modulus of elasticity for any graft irradiated at a low absorbed dose, compared to control groups. Also, bone allografts and two soft tissue allografts (anterior tibialis and semitendinosus tendon) that were irradiated at a moderate dose demonstrated similar strength and modulus of elasticity values to control groups. The results of this study support the use of low dose and moderate dose gamma irradiation of bone grafts. For soft tissue grafts, the results support the use of low dose irradiation.

The effects of radiofrequency energy probe speed and application force on chondrocyte viability.

OBJECTIVE: To determine the thermal effects of monopolar radiofrequency energy (mRFE) on bovine articular cartilage when it was moved at different speeds and using varying application forces. METHODS: Thirty-six fresh osteochondral sections divided into two groups (18 sections/group) were used in this study. The first group was tested at three speed rates of mRFE probe (1 mm/sec, 5 mm/sec and 10 mm/sec) at a constant force (50 g) applied to the probe tip. In the second group, three application forces of the probe tip were tested (25 g, 50 g and 75 g) at a constant speed (5 mm/sec) (n = 6/test). All tests were performed using a custom-built jig to control the mRFE (Vulcan EAS) probe during a 20-mm pass on each section. After treatment, viability of osteochondral sections was determined by confocal laser microscopy (CLM) combined with vital cell staining. RESULTS: There were not any significant differences in cartilage thickness of tested osteochondral sections among the different speeds or forces. During the mRFE probe treatments at different speeds, CLM demonstrated that probe application at the speed of 1 mm/sec caused significantly greater chondrocyte death than at the speeds of 5 and 10 mm/sec, whereas there were no significant differences in chondrocyte death among the variable application forces (p > 0.05). DISCUSSION: This in vitro study demonstrated that RFE thermal penetration correlated most closely with probe application speed than application force for this mRFE probe. CLINICAL RELEVANCE: Improper use of mRFE may cause thermal injury on articular cartilage.

Determination of factors influencing tissue effect of thermal chondroplasty: an ex vivo investigation.

PURPOSE: Scientific investigation of thermal chondroplasty using radiofrequency energy (RFE) is confounded by multiple factors associated with the technique. Our purpose was to determine the relative importance of the following factors on tissue effect (depth of tissue debridement plus depth of underlying cell death) of thermal chondroplasty: probe design, generator power setting, speed, force, and number of passes of the probe over treated tissue. We hypothesized the relative importance of these factors would be (from most to least important) power, passes, speed, force, and design. METHODS: Bovine patellae were treated using monopolar RFE. Sample size was based on a 2-level, half-factorial design. Low and high extremes of the factors tested were power setting (50 W v 110 W), passes (1 v 5), speed (3 mm/sec v 10 mm/sec), force (0.15 N v and 0.59 N), and probe design (electrode protrusion 25 microm v 125 microm). Samples were incubated with cell viability stain and examined using confocal laser microscopy to determine tissue effect. Data were analyzed using multiple regression. RESULTS: All factors that were tested significantly influenced tissue effect (P < .05). Power setting had the greatest effect, followed by design, speed, passes, and force. The following interactions of factors were also significant: design and force, power and passes. The optimal configuration resulting in least tissue effect was a power setting of 50 W, electrode protrusion of 25 microm, speed of 10 mm/sec, 1 pass, and 0.15 N of applied force during treatment, which resulted in a predicted tissue effect of 99 +/- 15 microm. CONCLUSIONS: The least tissue effect of thermal chondroplasty was achieved with lower power using a probe with minimal electrode protrusion while performing a rapid, single, lower force pass of the probe over treated tissue. CLINICAL RELEVANCE: Power and probe design have the greatest influence among the factors tested; selecting these parameters preoperatively could control tissue effect.

Low intensity pulsed ultrasound increases the matrix hardness of the healing tissues at bone-tendon insertion-a partial patellectomy model in rabbits.

BACKGROUND: This study evaluated the low intensity pulsed ultrasound enhancement on matrix hardness of the healing tissues at the bone-tendon junction. METHODS: Sixteen 18 week-old mature female rabbits were used. An established transverse partial patellectomy was performed at the distal one-third of the patella. Animals were then divided according to their body weight into ultrasound group (n = 8) with daily treatment of low intensity pulsed ultrasound and control group (n = 8) without ultrasound treatment. Animals were euthanized at week 8 and 16 postoperatively to evaluate the radiographic new bone formation and the Vickers hardness of the matrix of the healing tissues at the bone-tendon junction. FINDINGS: (1) Comparing with the control group, the anterior-posterior area of the new bone in the ultrasound treated group was found on average to be 3.0 and 3.1 times greater at week 8 and 16, respectively (P < 0.01). (2) The Vickers hardness of the new bone in ultrasound group was 11.3% (P < 0.05) significantly lower at week 8 but 20.0% (P < 0.05) significantly higher at week 16 as compared with that of the control group. (3) The Vickers hardness of the newly regenerated fibrocartilage zone, healing tendon, and cartilaginous metaplasia in ultrasound group was found higher than the control group at both week 8 and 16, but the difference was significant at week 16 only, being 44.1% (P < 0.05), 20.1% (P < 0.01), and 46.4% (P < 0.01) higher, respectively. INTERPRETATION: The preliminary findings suggested for the first time that low intensity pulsed ultrasound treatment resulted in the enhancement of the matrix hardness in new bone, fibrocartilage, cartilaginous metaplasia, and healing tendon at the healing bone-tendon junction. These findings can be extrapolated into clinical practice, i.e. the more rapid healing induced by low intensity pulsed ultrasound, the earlier mobilization of the affected joint. The beneficial effects on prevention of the musculoskeletal deterioration resulting from the prolonged immobilization would be therefore expected.

Radiofrequency energy-induced heating of bovine articular cartilage: evaluation of a new temperature-controlled, bipolar radiofrequency system used at different settings.

This in vitro investigation determined temperature changes associated with radiofrequency energy-induced heating of bovine articular cartilage using a newly developed, temperature-controlled, bipolar radiofrequency system at different settings. Cartilage tissue samples were placed in a saline bath maintained at room temperature. Radiofrequency energy was applied using a temperature-controlled, bipolar radiofrequency system at four different settings. Fluoroptic thermometry recorded temperatures at the radiofrequency electrode-tissue interface at 1-second intervals before, during delivery (1-5 seconds), and after delivery (1-3 seconds) of radiofrequency energy. Ten data acquisitions were obtained at each equipment setting. There were statistically significant (P<.05) increases in cartilage tissue temperatures associated with the different temperature settings. The temperature at the radiofrequency electrode-tissue interface was relatively close to the equipment's set temperature. These data provide basic information for the temperature-controlled, bipolar radiofrequency system applied to articular cartilage and may be useful in guiding electrosurgical equipment for thermal-assisted chondroplasty.

Radiofrequency energy induced heating of bovine articular cartilage: comparison between temperature-controlled, monopolar, and bipolar systems.

This in vitro investigation characterized temperature changes associated with radiofrequency (RF) energy induced heating of bovine articular cartilage using temperature-controlled, monopolar (Vulcan RF system and Vulcan, TAC-S Electrothermal Probe) and bipolar (VAPR II RF system and VAPR TC RF electrode) electrosurgical equipment. The RF generators were used at the same setting (set temperature 70 degrees C; 30 W). The cartilage tissue sample was placed in a saline bath maintained at room temperature. Temperatures were recorded using fluoroptic thermometry at the RF electrode-tissue interface at 1-s intervals before, during deliver of RF energy (1- and 2-s), and after (1- to 3-s). For both electrosurgical systems the mean RF electrode-tissue interface temperatures were significantly ( P<0.05) higher than the mean baseline value during delivery of RF energy (monopolar, highest mean temperature, 65.7 degrees C; bipolar, highest mean temperature, 54.1 degrees C). In general, during and after the deliver of RF energy, the monopolar RF system produced tissue temperatures that were significantly ( P<0.05) higher than those produced by the bipolar RF system. Neither electrosurgical system exceeded the set temperature of 70 degrees C. These findings provide basic tissue temperature characteristics for the newly developed, temperature-controlled RF devices applied to articular cartilage.

The effect of radiofrequency energy on the ultrastructure of joint capsular collagen.

This study evaluated the effect of radiofrequency energy on the histological and ultrastructural appearance of joint capsular collagen. Femoropatellar joint capsular specimens from adult sheep were treated with one of three treatment temperatures (45 degrees C, 65 degrees C, and 85 degrees C) with a radiofrequency generator or served as control in a randomized block design. Twenty-four specimens (n = 6) were processed for histological examination as well as ultrastructural analysis using transmission electron microscopy. A computer-based area determination program was used to calculate the area affected in histological samples. Histological changes consisted of thermal tissue damage characterized by collagen fiber fusion and fibroblastic nuclear pyknosis at all application temperatures with clear demarcations between treated and untreated tissue. Mean tissue affected ranged from 50.4% for 85 degrees C to 22.5% for 45 degrees C. There was a strong correlation between treatment temperature and percent area affected (P < .001, R2 = .65). Ultrastructural alterations included a general increase in cross-sectional fibril diameter and loss of fibril size variation with increasing treatment temperature. Longitudinal sections of collagen fibrils showed increased fibril diameter and the loss of cross-striations in the treated groups. Thermally induced ultrastructural collagen fibril alteration is likely the predominant mechanism of tissue shrinkage caused by application of radiofrequency energy.

Dose-dependent response of gamma irradiation on mechanical properties and related biochemical composition of goat bone-patellar tendon-bone allografts.

We studied the effects of gamma irradiation on the dimensions, mechanical and material properties, and mature hydroxypyridinium crosslink density of collagen in goat patellar tendon-bone specimens. Left and right patellar tendon-bone units were removed from 10 adult female goats and were bisected longitudinally. Each tendon half was frozen, and then the left halves were exposed to 4, 6, or 8 Mrad (40,000, 60,000, or 80,000 Gy) of gamma irradiation. The contralateral tendon halves served as controls (no irradiation). Each specimen then was loaded to failure in tension, and its soft-tissue midsubstance was processed to measure collagen content and hydroxypyridinium crosslink density. Dose-dependent reductions in the mechanical properties were found, including 46% (p < 0.01) and 18% (p < 0.05) reductions in maximum force and stiffness, respectively, at 4 Mrad. Similar reductions were noted in material properties, including 37% (p < 0.005) and 8% (p > 0.05) reductions in maximum stress and modulus, respectively, at 4 Mrad. These results are consistent with our previous report involving 2 and 3 Mrad (20,000 and 30,000 Gy) of exposure. We also found significant decreases in hydroxypyridinium crosslink density with 6 Mrad of irradiation (p < 0.05). However, since only one biomechanical parameter (modulus) correlated significantly with only one biochemical measure (hydroxypyridinium crosslink density) (p < 0.05), other possible mechanisms also are being explored to more fully explain these dose-dependent changes.

The effects of test environment and cyclic stretching on the failure properties of human patellar tendons.

There is a need to document the mechanical properties of patellar tendon allografts used for reconstructive surgery of the damaged anterior cruciate ligament, especially the effects of irradiation sterilization. The purpose of this study was to investigate the influences of in vitro test environment and low-level cyclic stretching prior to failure tests on nonirradiated and irradiated human graft tissues. Bilateral patellar tendons were split and each half processed accordingly. Some graft tissues were stretched cyclically at 2.5 mm deformation before failure. Experiments were performed in a 37 degrees C saline bath or with tissues moistened with a drip of the same. The irradiated grafts relaxed less and generated less slack length in the drip environment than the nonirradiated controls. Cyclic stretching did not alter failure characteristics of either graft tissue. While no significant differences in the tensile responses or failure characteristics were noted for irradiated and nonirradiated grafts in the drip, in the bath environment the nonirradiated tissues had greater strength and modulus. This resulted in there being a significant difference between irradiated and nonirradiated tissue responses in a heated saline bath environment. These experimental results exemplify the need to control in vitro test environments in the evaluation of various sterilization and preservation protocols for soft tissue allografts.