Arthur C. Vailas

The Effect of Nonablative Laser Energy on Joint Capsular Properties An In Vitro Histologic and Biochemical Study Using a Rabbit Model

The purpose of this study was to evaluate the effect of laser energy at nonablative levels on joint capsular histologic and biochemical properties in an in vitro rabbit model. The medial and lateral portions of the femoropatellar joint capsule from both stifles of 12 mature New Zealand White rabbits were used. Speci mens were divided into three treatment groups (5 watts, 10 watts, and 15 watts) and one control group using a randomized block design. Specimens were placed in a 37° bath of lactated Ringers solution and laser energy was applied using a holmium:yttrium-alu minum-garnet laser in four transverse passes across the tissue at a velocity of 2 mm/sec with the handpiece set 1.5 mm from the synovial surface. Histologic anal ysis revealed thermal alteration of collagen (fusion) and fibroblasts (pyknosis) at all energy densities, with higher laser energy causing significantly greater mor phologic changes over a larger area (P < 0.05). Ap plication of laser energy did not significantly alter the biochemical parameters evaluated, including type I col lagen content and nonreducible crosslinks (P > 0.05). This study demonstrated that nonablative laser energy caused significant thermal damage to the joint capsular tissue in an energy-dependent fashion, but type I col lagen content and nonreducible crosslinks were not significantly altered.

Ultrasonic Wave Velocity Measurement in Small Polymeric and Cortical Bone Specimens

A system was refined for the determination of the bulk ultrasonic wave propagation velocity in small cortical bone specimens. Longitudinal and shear wave propagations were measured using ceramic, piezoelectric 20 and 5 MHz transducers, respectively. Results of the pulse transmission technique were refined via the measurement of the system delay time. The precision and accuracy of the system were quantified using small specimens of polyoxymethylene, polystyrene-butadiene, and high-density polyethylene. These polymeric materials had known acoustic properties, similarity of propagation velocities to cortical bone, and minimal sample inhomogeneity. Dependence of longitudinal and transverse specimen dimensions upon propagation times was quantified. To confirm the consistency of longitudinal wave propagation in small cortical bone specimens (< 1.0 mm), cut-down specimens were prepared from a normal rat femur. Finally, cortical samples were prepared from each of ten normal rat femora, and Youngs moduli (Eii), shear moduli (Gij), and Poisson ratios (Vij) were measured. For all specimens (bone, polyoxymethylene, polystyrene-butadiene, and high-density polyethylene), strong linear correlations (R2 > 0.997) were maintained between propagation time and distance throughout the size ranges down to less than 0.4 mm. Results for polyoxymethylene, polystyrene-butadiene, and high-density polyethylene were accurate to within 5 percent of reported literature values. Measurement repeatability (precision) improved with an increase in the wave transmission distance (propagating dimension). No statistically significant effect due to the transverse dimension was detected.

Mechanical and biochemical analyses of tibial compartment fascia in chronic compartment syndrome

Increases in compartment pressure associated with chronic compartment syndrome (CCS) may be due to changes in the mechanical properties and/or thickness of fascia (4,22). To explore this possibility, we compared the mechanical and biochemical characteristics (stiffness, thickness, time-dependent response, collagen content, and collagen crosslinking) of fascia from patients with symptomatic anterior compartment syndrome to fascia from adjacent collateral compartments. We tested 43 specimens harvested from 20 individuals during surgical fasciectomy. Properties of normal (lateral)-compartment (NC) and pathological (anterior)-compartment (PC) fascia were mechanically tested in the axial and transverse directions forming four groups. An external control group (EX) of six specimens of anterior and lateral-compartment fascia harvested from amputated legs was also included in the study. PC fascia was found to be thicker and structurally stiffer (elastic modulus times thickness) in the axial direction than was NC fascia (p≤0.05). No significant differences were found between NC and PC time-dependent response, although significant differences between percent relaxation in the pooled axial and transverse direction specimens were observed. No differences were found in the collagen content, as measured by hydroxyproline (Hyp) concentration, between NC and PC fascia. PC fascia was found to have less collagen crosslinking by hydroxylyslpyridinoline (HP) concentration. In conclusion, although this study does not elucidate etiological factors in CCS, the changes found in PC fascia suggest that fascial mechanical properties contribute to the pathology.

Systemic administration of IGF-I enhances healing in collagenous extracellular matrices: evaluation of loaded and unloaded ligaments

BackgroundInsulin-like growth factor-I (IGF-I) plays a crucial role in wound healing and tissue repair. We tested the hypotheses that systemic administration of IGF-I, or growth hormone (GH), or both (GH+IGF-I) would improve healing in collagenous connective tissue, such as ligament. These hypotheses were examined in rats that were allowed unrestricted activity after injury and in animals that were subjected to hindlimb disuse. Male rats were assigned to three groups: ambulatory sham-control, ambulatory-healing, and hindlimb unloaded-healing. Ambulatory and hindlimb unloaded animals underwent surgical disruption of their knee medial collateral ligaments (MCLs), while sham surgeries were performed on control animals. Healing animals subcutaneously received systemic doses of either saline, GH, IGF-I, or GH+IGF-I. After 3 weeks, mechanical properties, cell and matrix morphology, and biochemical composition were examined in control and healing ligaments.ResultsTissues from ambulatory animals receiving only saline had significantly greater strength than tissue from saline receiving hindlimb unloaded animals. Addition of IGF-I significantly improved maximum force and ultimate stress in tissues from both ambulatory and hindlimb unloaded animals with significant increases in matrix organization and type-I collagen expression. Addition of GH alone did not have a significant effect on either group, while addition of GH+IGF-I significantly improved force, stress, and modulus values in MCLs from hindlimb unloaded animals. Force, stress, and modulus values in tissues from hindlimb unloaded animals receiving IGF-I or GH+IGF-I exceeded (or were equivalent to) values in tissues from ambulatory animals receiving only saline with greatly improved structural organization and significantly increased type-I collagen expression. Furthermore, levels of IGF-receptor were significantly increased in tissues from hindlimb unloaded animals treated with IGF-I.ConclusionThese results support two of our hypotheses that systemic administration of IGF-I or GH+IGF-I improve healing in collagenous tissue. Systemic administration of IGF-I improves healing in collagenous extracellular matrices from loaded and unloaded tissues. Growth hormone alone did not result in any significant improvement contrary to our hypothesis, while GH + IGF-I produced remarkable improvement in hindlimb unloaded animals.

Extracellular matrix maturation in the left ventricle of normal and diabetic swine

The main objective of this study is to determine the transmural distribution of extracellular matrix (ECM) collagen and maturation in non-diabetic and diabetic hearts. The Yucatan miniature swine heart ECM was analyzed in eight streptozotocin (STZ) induced diabetic pigs (Diabetic-Swine) and age matched normal control pigs (Nondiabetic-Swine). After 12 weeks of STZ induced diabetes, transmural biopsies were obtained from the left ventricular free wall divided into subendocardial, mid- and subepicardial layers. Collagen concentration and maturation were measured by RP-HPLC determination of hydroxyproline (Hyp) and content of hydroxylysylpyridinoline (HP) cross-links, respectively. Results showed a significant elevation in arterial glucose (P<0.05) and reduction in arterial plasma insulin levels in the Diabetic-Swine. Hyp concentration was significantly greater (P<0.05) in the subendocardial layers in both the Diabetic and Nondiabetic animals. The HP cross-link content was significantly greater (17%) in the Diabetic-swine subendocardial layer compared to Nondiabetic-Swine (P<0.05), but not in other layers. In summary, the accumulation and/or increase in HP cross-link content in the Diabetic-Swine subendocardial layer suggests that myocardial fibrosis may be greater in this specific region.

The effect of in situ freezing on rabbit patellar tendon A histologic, biochemical, and biomechanical analysis

Cell necrosis has been well documented as one of the many changes that occur in autogenous tendon when it is used to reconstruct the anterior cruciate ligament. The purpose of this experiment was to isolate cell necrosis as a variable and study its effect on the patellar tendon. To accomplish this, both knees of 25 New Zealand White rabbits were operated on. In one knee, a 5-mm wide band of patellar tendon was subjected to two rapid freeze-thaw cycles, while the other knee underwent sham surgery. Histologic evaluation showed a zone of necrosis at 2 and 4 weeks with cellular repopulation complete at 8 weeks. Patellar tendon cross-sectional area was 0.118 cm2 at 8 weeks for the frozen specimens compared to 0.102 cm2 for the sham- operated controls. This difference was significant at the P = 0.025 level. Mechanical testing at 4 and 8 weeks revealed no significant changes in tendon length, max imum load, or stiffness. The collagen content was also unchanged at both 4 and 8 weeks.

Effect of a growth hormone treatment on bone orthotropic elasticity in dwarf rats.

A refinement of the current ultrasonic elasticity technique was used to measure the orthotropic elastic properties of rat cortical bone as well as to quantify changes in elastic properties, density, and porosity of the dwarf rat cortex after a treatment with recombinant human growth hormone (rhGH). The ultrasonic elasticity technique was refined via optimized signal management of high-frequency wave propagation through cubic cortical specimens. Twenty dwarf rats (37 days old) were randomly assigned to two groups (10 rats each). The dwarf rat model (5–10% of normal GH) was given subcutaneous injections of either rhGH or saline over a 14-day treatment period. Density was measured using Archimedes’ technique. Porosity and other microstructural characteristics were also explored via scanning electron microscopy and image analysis. Statistical tests verified significant decreases in corticla orthotropic Young’s (−26.7%) and shear (−16.7%) moduli and density (−2.42%) concomitant with an increase in porosity (+125%) after rhGH treatments to the dwarf model (p<0.05). A change in material symmetry from orthotropy toward planar isotropy within the radial-circumferential plane after GH treatments was also noted. These results demonstrate some alteration in bone properties at this time interval. Structural implications of these changes throughout physiological loading regimens should be explored.

Adaptations of immature trabecular bone to moderate exercise : geometrical, biochemical, and biomechanical correlates

Strenuous endurance exercise can adversely affect the mechanical integrity of immature bone, but it is unclear whether a more moderate exercise regimen would have a positive effect. Thus, to investigate the response of immature trabecular bone to moderate exercise, we randomly assigned female Sprague-Dawley rats (8 weeks old) to either a basal-control, exercise, or age-matched control group. The basal-control rats were killed at 8 weeks of age, while the other two groups were killed at 18 weeks of age. Between 8 and 18 weeks, one group remained sedentary, while another group was trained progressively on a motor-driven treadmill at a moderate level of intensity. Rat femoral necks (FN) were tested in cantilever bending to failure, and the sixth lumbar vertebral bodies (L6) were compressed to 50% of their initial height. Both tissues were analyzed for calcium, hydroxyproline, and collagen-crosslinking concentrations, and for changes in geometry. The adrenal mass per unit body mass was significantly greater in the exercised group, compared to the age-matched controls. L6 calcium content, compressional stress, and elastic modulus were significantly less in the exercise group as compared to the age-matched control group. Nonreducible collagen crosslinks (hydroxylysylpyridinoline [HP] and lysylpyridinoline [LP]) were significantly greater in the older exercise and age-matched control L6 and FN. In the weightbearing FN--but not L6--the LP concentration of the exercise group was significantly greater than the age-matched controls.(ABSTRACT TRUNCATED AT 250 WORDS)

Acute modification of biomechanical properties of the bone-ligament insertion to rat limb unweighting.

We investigated the acute adaptation of the rat femur‐medial collateral ligament‐tibia (FMT) complex to 7 days of limb unweighting by means of a hind‐limb suspension protocol. Male, young adult, Harlan Sprague‐Dawley rats were randomly assigned to either control or suspended groups. Rats deprived of hind limb‐to‐ground contact forces had a 42% decrease in soleus muscle mass compared with the control group. Medial collateral ligament (MCL) length and cross‐sectional area were measured, and each FMT complex was tension tested to failure. All failed at their tibia‐MCL insertion. The ultimate load in the FMT and the peak Kirchhoff stress in the MCL (occurring immediately before insertion site failure) were significantly reduced in the suspended group. The suspended MCLs were 9.7% larger in area and 5.7% shorter in length than the controls under the same preload (0.25 N). We found no significant differences between the control and suspended MCLs in Green strain, stretch, or deformation immediately before insertion site failure, nor did we find a significant difference in the MCL tangent modulus. This study indicates that even acute periods of limb unweighting can structurally compromise bone‐ligament insertions. Further, this study implies that the adaptations responsible for this structural compromise must involve acute changes in the intrinsic zone (or zones) of the bone‐ligament insertion.— Vanderby, R., Jr.; Vailas, A. C.; Graf, B. K.; Thielke, R. J.; Ulm, M. J.; Kohles, S. S.; Kunz, D. N. Acute modification of biomechanical properties of the bone‐ligament insertion to rat limb unweighting. FASEB J. 4: 2499‐2505; 1990.

Growth and maturational changes in dense fibrous connective tissue following 14 days of rhGH supplementation in the dwarf rat

The purpose of this study was to investigate the impact of recombinant human growth hormone (rhGH) on patella tendon (PT), medial collateral ligament (MCL), and lateral collateral ligament (LCL) on collagen growth and maturational changes in dwarf GH-deficient rats. Twenty male Lewis mutant dwarf rats, 37 days of age, were randomly assigned to Dwarf + rhGH (n = 10) and Dwarf + vehicle (n = 10) groups. The GH group received 1.25 mg rhGH/kg body wt twice daily for 14 days. rhGH administration stimulated dense fibrous connective tissue growth, as demonstrated by significant increases in hydroxyproline specific activity and significant decreases in the non-reducible hydroxylysylpyridinoline (HP) collagen cross-link contents. The increase in the accumulation of newly accreted collagen was 114, 67, and 117% for PT, MCL, and LCL, respectively, in 72 h. These findings suggest that a short course rhGH treatment can affect the rate of new collagen production. However, the maturation of the tendon and ligament tissues decreased 18-25% during the rapid accumulation of de novo collagen. We conclude that acute rhGH administration in a dwarf rat can up-regulate new collagen accretion in dense fibrous connective tissues, while causing a reduction in collagen maturation.