Dynamic pressure transmission through agarose gels.

In biomedical research, agarose gel is widely used in tissue culture systems because it permits growing cells and tissues in a three-dimensional suspension. This is especially important in the application of tissue engineering concepts to cartilage repair because it supports the cartilage phenotype. Mechanical loading, especially compression, plays a fundamental role in the development and repair of cartilage. It would be advantageous to develop a system where cells and tissues could be subjected to compression so that their responses can be studied. There is currently no information on the pressure response of agarose gel when pressure is applied to the gas phase of a culture system. To understand the transmission of pressure through the gel, we set up an apparatus that would mimic an agarose suspension tissue culture system. This consisted of a sealed metal cylinder containing air as well as a layer of agarose submerged in culture medium. Pressure responses were recorded in the air, fluid, gel center, and gel periphery using various frequencies, pressures, gel volumes, and viscosities. Regression analyses show an almost perfect linear relation between gas and gel pressures (r(2) = 0.99987, p < 0.0001, f(x) = 0.9982 x - 0.0286). The pressure transmission was complete and immediate, throughout the range of the applied pressures, frequencies, volumes, and viscosities tested. Applying dynamic pressure to the gas phase results in reproducible pressure in the agarose and, therefore, validates the use of agarose tissue culture systems in studies employing dynamic pressurization in cartilage tissue engineering.

Fetal lung maturatio. III. Amniotic fluid phosphatidic acid phosphohydrolase (PAPase) and its relation to the lecithin/sphingomyelin ratio.

The purpose of this study was to establish 1) the sequential changes in specific activity of phosphatidic acid phosphohydrolase (PAPase) in amniotic fluid and its relation to the lecithin/sphingomyelin (L/S) ratio; and 2) the origin of amniotic fluid PAPase. the increase in the amniotic fluid L/S ratio is preceded by an increase in PAPase activity, rising from 15 nmoles of phosphate released per milligram of protein per hour at 30 weeks to 100 nmoles at 37 weeks. The mean PAPase activity in the nasopharyngeal fluid of the infant is 456 nmoles of phosphate released per ml per hour, the amniotic fluid mean PAPase activity at delivery being 129 nmoles (P less than 0.01). These findings are consistent with the view that amniotic fluid PAPase originates, in part, from the fetal lung and likely participates in the regulation of the synthesis of lecithin.

Initiation of human parturition. III. Fetal membrane content of prostaglandin E2 and F2alpha precursor.

Unesterified arachidonic acid is the obligatory precursor of the prostaglandins (PG), PGF2alpha and PGE2. In order to ascertain whether or not the human fetal membranes could represent a storage site for prostaglandin(s) precursor, the fatty acid content of human fetal membranes was measured. Approximately 20% of the fatty acids found in fetal membranes obtained from near-term, non-laboring women was arachidonic acid, whereas only 0.4% of the fatty acids of the parietal peritoneum of the mother is arachidonic acid. A small but significant decrease in the arachidonic acid concentration was found in the fetal membranes obtained from laboring women compared to that found prior to labor. On the other hand, the concentration of palmitic acid was increased in membranes obtained during labor while no significant changes in concentration in the remaining fatty acids were observed in membranes from laboring compared to non-laboring near-term gravidas. The significance of these observations in relation to the availability of prostaglandin precursor and the initiation of human parturition is considered.

Tendons attached to prostheses by tendon-bone block fixation: an experimental study in dogs.

To develop a method of tendon attachment to a metallic endoprosthesis, we evaluated fixation strength, clinical function of the tendon, and morphological changes in an experimental model. The canine supraspinatus tendon was removed from the greater tubercle of the humerus and attached to a titanium prosthesis. In 12 animals, the bone block underlying the tendon insertion was preserved and attached in one limb; the soft part of the tendon was attached directly to the prosthesis in the contralateral limb. Fixation strength was evaluated after 16 weeks of in vivo implantation (12 specimens) and compared with the in vitro fixation strength (12 specimens) and with intact normal controls (six specimens from cadavera). Function of the tendon in vivo was evaluated by force-plate analysis (at 3-week intervals). All specimens were evaluated histologically. Sixteen weeks after surgery, the tendon-bone block attachment was significantly stronger (mean, 16%) than the direct tendon attachment and not significantly different from the normal control, and the direct tendon attachment was significantly weaker (mean, 68%) than the normal control. There was significantly more weight-bearing on the limbs with a tendon-bone block attachment than on the limbs with a direct tendon attachment at both 3 and 6 weeks postoperatively. Both front legs showed increased weight-bearing with time, but the differences were not statistically significant. Anchorage by tissue ingrowth to the titanium prosthesis was found consistently--there was bone ingrowth in the tendon-bone block attachments and fibrous tissue ingrowth in the direct tendon attachments. When a bone block was preserved, the strength and stiffness were comparable with those of a normal tendon insertion.(ABSTRACT TRUNCATED AT 250 WORDS)

Tensile properties of the supraspinatus tendon.

The tensile properties of the supraspinatus tendon were investigated in 11 shoulders from fresh cadavers. The tendon was divided into three longitudinal strips: anterior, middle, and posterior. Each specimen was mounted on a materials testing machine, with four fluorescent markers placed on both surfaces of the tendon strip. The positions of these markers were recorded during the test by two synchronized video cameras. Load-deformation and strain curves were determined, and the stress-strain curve, strength, and modulus of elasticity were calculated. The posterior strip was thinner in cross section than the others (p = 0.0355). The ultimate load and ultimate stress were significantly greater in the anterior strip (16.5 +/- 7.1 MPa) than in the middle (6.0 +/- 2.6 MPa) and posterior (4.1 +/- 1.3 MPa) strips (p < 0.0001). The modulus of elasticity also was significantly greater in the anterior strip (p < 0.0001), but there was no significant difference between the superficial and deep surfaces. It is concluded that the anterior portion of the supraspinatus tendon is mechanically stronger than the other portions, and it seems to perform the main functional role of the tendon.

The enhancement of periosteal chondrogenesis in organ culture by dynamic fluid pressure.

Cartilage repair by autologous periosteal arthroplasty is enhanced by continuous passive motion (CPM) of the joint after transplantation of the periosteal graft. However, the mechanisms by which CPM stimulate chondrogenesis are unknown. Based on the observation that an oscillating intra-synovial pressure fluctuation has been reported to occur during CPM (0.6-10 kPa), it was hypothesized that the oscillating pressure experienced by the periosteal graft as a result of CPM has a beneficial effect on the chondrogenic response of the graft. We have developed an in vitro model with which dynamic fluid pressures (DFP) that mimic those during CPM can be applied to periosteal explants while they are cultured in agarose gel suspension. In this study periosteal explants were treated with or without DFP during suspension culture in agarose, which is conducive to chondrogenesis. Different DFP application times (30 min, 4 h, 24 h/day) and pressure magnitudes (13, 103 kPa or stepwise 13 to 54 to 103 kPa) were compared for their effects on periosteal chondrogenesis. Low levels of DFP (13 kPa at 0.3 Hz) significantly enhanced chondrogenesis over controls (34 +/- 7% vs 14 +/- 5%; P < 0.05), while higher pressures (103 kPa at 0.3 Hz) completely inhibited chondrogenesis, as determined from the percentage of tissue that was determined to be cartilage by histomorphometry. Application of low levels of DFP to periosteal explants also resulted in significantly increased concentrations of Collagen Type II protein (43 +/- 8% vs 10 +/- 5%; P < 0.05). New proteoglycan synthesis, as measured by 35S-sulphate uptake was increased by 30% in periosteal explants stimulated with DFP (350 +/- 50 DPM vs 250 +/- 75 DPM of 35S-sulphate uptake/microg total protein), when compared to controls though this difference was not statistically significant. The DFP effect at low levels was dose-dependant for time of application as well, with 4 h/day stimulation causing significantly higher chondrogenesis than just 30 min/day (34 +/- 7 vs 12 +/- 4% cartilage; P < 0.05) and not significantly less than that obtained with 24 h/day of DFP (48 +/- 9% cartilage, P > 0.05). These observations may partially explain the beneficial effect on cartilage repair by CPM. They also validate an in vitro model permitting studies aimed at elucidating the mechanisms of action of mechanical factors regulating chondrogenesis. The fact that these tissues were successfully cultured in a mechanical environment for six weeks makes it possible to study the actions of mechanical factors on the entire chondrogenic pathway, from induction to maturation. Finally, these data support the theoretical predictions regarding the role of hydrostatic compression in fracture healing.

Calibration of measured center of pressure of a new stairway design for kinetic analysis of stair climbing.

A stairway that allows the collection of kinetic data is essential for biomechanical studies on stair climbing. There is a need to validate the measured center of pressure (COP) on the surface of a stair in order to verify the accuracy of the calculation of joint kinetics. The purpose of this study was to validate a new stairway design for kinetic analysis of stair climbing through a calibration and error analysis of the COP obtained from this system. The new stairway design allows the collection of kinetic data for multiple steps without any constraint to foot placement. Known vertical forces were applied to known locations on the surface of each stair and each force plate. Multiple regression analyses were conducted to determine the distribution pattern of the error in the measured COP. It was found that the error in the COP was a function of location on the stair or force plate. The magnitude of the vertical force had no significant effect on the error in the measured COP. The distribution pattern of the error in the measured COP on the force plates used in this study matched the results in the literature. A healthy female subject was used as a subject in a stair climbing test. The error in the measured COP had a significant effect on the calculated joint resultant moments, especially the abduction-adduction and internal-external rotation moment. The correction of these errors should make the kinetic calculation in stair climbing more accurate.

The synthesis of higher glycerides via the monoglyceride pathway in hamster adipose tissue.

The monoglyceride pathway for the synthesis of triglycerides has been investigated employing subcellular fractions and whole cell preparations of white and brown adipose tissue. Conclusive evidence has been obtained for the monoglyceride pathway in these tissues by employing the 2-monoether analogue of 2-monoolein as the substrate. The monoglyceride and alpha-glycerophosphate pathways were primarily found in the microsomal fraction. In these in vitro systems the activity of the monoglyceride pathway compared with the alpha-glycerophosphate pathway was of the same order of magnitude in whole cell preparations and was approximately one-half the activity of the alpha-glycerophosphate pathway when the microsomal fraction was employed.

Initiation of human parturition. IV. Demonstration of phospholipase A2 in the lysosomes of human fetal membranes.

In this study we sought to define the subcellular localization of phospholipase A2 in human fetal membranes. Others have postulated a role for decidual lysosomes in the initiation of human parturition. We hypothesized that if phospholipase A2 were localized within lysosomes of fetal membranes the accelerated expression of the activity of this enzyme could be prevented until such time as the metabolic events of parturition begin. At parturition a perturbation of the lysosomal membrane within the chorio-amnion could result in an increased release of free arachidonic acid through an accelerated activity of phospholipase A2. The results of this study suggest that at least a portion of phospholipase A2 in the chorion laeve and amnion is localized in the lysosomal fraction.

Initiation of human parturition. II. Identification of phospholipase A2 in fetal chorioamnion and uterine decidua.

In this study, the presence of a phospholipase has been demonstrated in the human chorioamnion and uterine decidua. That the chorioamnionic enzyme is of the phospholipase A2 type was established by product identification following the incubation of the enzyme with either radioactive phosphatidylcholine or phosphatidylethanolamine. The potential relationship between the expression of the activity of this enzyme and the regulation of arachidonic acid release, prostaglandin formation, and the initiation of labor is considered.

Regulation of triglyceride biosynthesis in adipose and intestinal tissue.

The synthesis of phosphatidic acid and di- and triglycerides via the glycerol-3-phosphate pathway is markedly inhibited by 2-monooleyl ether in microsomal and whole cell preparations obtained from adipose and intestinal tissue. Monoglycerides are also inhibitors under conditions in which their hydrolysis is minimized. A correlation between inhibition by, and the hydrolysis of, monoglycerides has been demonstrated. 2-Monooleyl ether is the most effective inhibitor of the several mono- and di- ethers and esters studied. The specificity of the inhibition of glycerol-3-phosphate acylation by 2-monoethers or 2-monoesters has been demonstrated because microsomal NADH- and NADPH-cytochrome c reductase activities were not significantly inhibited. The reported control mechanism for triglyceride biosynthesis is discussed in relation to the regulation of fatty acid uptake and release in adipose tissue and the absorption and metabolism of triglycerides by the intestinal mucosa.

Fixation of canine tendons to metal.

For the purpose of developing a method to attach tendons directly to the prosthesis, canine supraspinatus tendons were attached in vitro to a metallic surface, using 3 different fixation devices: a spiked polyacetal washer (Synthes), a spiked soft tissue fixation plate (Synthes), and a newly designed Enhanced Tendon Anchor (ETA), which straddled the tendon with interlocking spikes oriented at a 20-degree angle. 2 methods were used: 1) the tendon was fixed directly to the metallic surface, or 2) a bone block containing the tendon insertion was fixed to the metallic surface. The specimens were tested for initial fixation strength in tension to failure; intact bone-muscle-tendon-bone units were used as controls. Bone block fixations were stronger than direct tendon fixations when the spiked washer or the ETA was used; this was not true of the fixation plate. The ETA was stronger than the other techniques in ultimate strength in both direct tendon fixation and bone block fixation. The soft tissue fixation plate was found to be weaker than the other techniques in bone block fixation.