Biomedical subjects

P A Torzilli

Publications and source records attributed to P A Torzilli.

At least 19 recordsLinked to original sources

PubMed2006

Effect of compressive strain on cell viability in statically loaded articular cartilage.

Physiological loading of articulating joints is necessary for normal cartilage function. However, conditions of excessive overloading or trauma can cause cartilage injury resulting in matrix damage and cell death. The objective of this study was to evaluate chondrocyte viability within mechanically compressed articular cartilage removed from immature and mature bovine knees. Twenty-three mature and 68 immature cartilage specimens were subjected to static uniaxial confined-creep compressions of 0-70% and the extent of cell death was measured using fluorescent microscopic imaging. In both age groups, cell death was always initiated at the articular surface and increased linearly in depth with increasing strain magnitude. However, most of the cell death was localized within the superficial zone (SZ) of the cartilage matrix with the depth never greater than approximately 500 microm or 25% of the thickness of the test specimen. The immature cartilage was found to have a significantly greater (> 2 times) amount (depth) of cell death compared to the mature cartilage, especially at the higher strains. This finding was attributed to the lower compressive modulus of the immature cartilage in the SZ compared to that of the mature cartilage, resulting in a greater local matrix strain and concomitant cell surface membrane strain in this zone when the matrix was compressed. These results provide further insight into the capacity of articular cartilage in different age groups to resist the severity of traumatic injury from compressive loads.

Aging↗

PubMed2004

Fourier transform infrared spectral analysis of degenerative cartilage: an infrared fiber optic probe and imaging study.

A preliminary investigation into the diagnostic potential of an infrared fiber optic probe (IFOP) for evaluating degenerative human articular cartilage is described. Twelve arthritic human tibial plateaus obtained during arthroplasty were analyzed using the IFOP. Infrared spectra were obtained from IFOP contact with articular surface sites visually graded normal or degraded (Collins Scale grade 1 and grade 3, respectively). Comparisons of infrared spectral parameters (peak heights and areas) were made to elucidate spectral indicators of surface degeneration. IFOP spectral analysis revealed subtle but consistent changes between grades 1 and 3 sites. Infrared absorbance bands arising from type II collagen were observed to change with degradation. More degraded tissues exhibited increased amide II (1590-1480 cm(-1))/1338 cm(-1) area ratio (p=0.034) and decreased 1238/1227 cm(-1) peak ratio (p = 0.017); similar changes were seen with Fourier transform infrared imaging spectroscopy (FT-IRIS) analysis. Grades 1 and 3 cartilage showed consistent spectral differences in the amide II, III, and 1338 cm(-1) regions that are likely related to type II collagen degradation that accompanies cartilage degeneration. These results suggest that it may be possible to monitor subtle changes related to early cartilage degeneration, allowing for IFOP use during arthroscopy for in situ determination of cartilage integrity.

Aged↗

PubMed2002

Cartilage viability after repetitive loading: a preliminary report.

OBJECTIVE: To assess matrix changes and chondrocyte viability during static and continuous repetitive mechanical loading in mature bovine articular cartilage explants. METHODS: Cartilage explants were continuously loaded either statically or cyclically (0.5 Hz) for 1-72 h (max. stress 1 megapascal). Cell death was assessed using fluorescent probes and detection of DNA strand breakage characteristic of apoptosis. Cell morphology and matrix integrity were evaluated using histology and transmission electron microscopy. RESULTS: Repetitive loading of articular cartilage at physiological levels of stress (1 megapascal) was found to be harmful to only the chondrocytes in the superficial tangential zone (STZ) and depended on the characteristics (static vs cyclic) and duration (1-72 h) of the applied load. The chondrocytes in the middle and deep zone remained viable at all times. Static loads caused cell death at an early time (3 h) as compared with cyclic loads (sinusoidal, 0.5 cycles per s for 6 h). The amount and extent of cell death peaked at 6 h of cyclic loading, and did not change in subsequent experiments run for longer periods of time (up to 72 h). There was no indication of fragmented nuclear DNA but there was evidence of injurious cell death (necrosis) by electron microscopy. Morphological analysis of cartilage repetitively loaded for 24 h showed matrix damage only in the uppermost superficial layer at the articular surface, reminiscent of the early stages of osteoarthritis. CONCLUSIONS: Cell death in mature cartilage explants occurred after 6 hours of continuous repetitive load or 3 h of static load. Cell death was directly related to the mechanical load, as control (free-swelling) explants remained viable at all times. The excessive, repetitive loading conditions imposed are not physiological, and demonstrate the deleterious effects of mechanical overload resulting in morphological and cellular damage similar to that seen in degenerative joint disease.

Animals↗

PubMed2001

FTIR microscopic imaging of collagen and proteoglycan in bovine cartilage.

Articular cartilage, a connective tissue that provides resistance to compressive forces during joint movements, has not been examined in detail by conventional Fourier transform infrared (FTIR) spectroscopy, microspectroscopy (FTIRM), or imaging (FTIRI). The current study reports FTIRM and FTIRI analyses of normal bovine cartilage and identifies the specific molecular components of cartilage that contribute to its IR spectrum. FTIRM data acquired through the superficial, middle, and deep zones of thin sections of bovine articular cartilage showed a variation in intensities of the absorbance bands that arise from the primary nonaqueous components of cartilage, collagen, and proteoglycan (primarily aggrecan) and thus reflected the differences in quantity of these specific components. The spectra of mixtures of model compounds, which had varying proportions of type II collagen and aggrecan, were analyzed to identify spectral markers that could be used to quantitatively analyze these components in cartilage. Collagen and aggrecan were then imaged by FTIRI based on markers found in the model compounds. Polarization experiments were also performed to determine the spatial distribution of the collagen orientation in the different zones of cartilage. This study provides a framework in which complex pathological changes in this heterogeneous tissue can be assessed by IR microscopic imaging.

Animals↗

PubMed2000

Proton spin-spin relaxation study of molecular dynamics and proteoglycan hydration in articular cartilage.

Spin-spin relaxation of proton magnetization in natural and deuterated articular cartilage is reported over a range of hydration. Information about macromolecular dynamics is deduced and a hydration stabilized macromolecular regime identified. There is good correspondence between NMR results and cartilage stoichiometry. A new measure for hydration of proteoglycans is found.

Animals↗

PubMed1999

Effect of serum and platelet-derived growth factor on chondrocytes grown in collagen gels.

In this in vitro study, cell proliferation, viability, and morphology; proteoglycan (PG) synthesis; and gel contraction were assessed over a 15-day period (on days 3, 6, 9, 12, and 15) for mature bovine chondrocytes cultured in collagen gels. The environment within the gel was varied by changing the concentration of fetal bovine serum (1% and 10%) and platelet-derived growth factor-BB (PDGF; 0, 10, 50, 100 ng/ml) within the gel and incubation media. Our results showed that the amount of serum or PDGF added to the gels had no effect on cell viability, with >95% of cells remaining alive throughout the experiment. There was a significant increase in cell number over time in all groups, with a higher rate of cell proliferation in gels containing 10% serum and higher concentrations of PDGF. In addition, the amount of serum significantly affected gel contraction with or without PDGF. Gels containing 10% serum contracted on day 10-12, while none of the gels containing 1% serum contracted over the course of the experiment. The PG content within each gel increased with incubation time only for the gels containing 1% serum, and 10 or 100 ng/ml of PDGF. However, on a per cell basis, there was no change in the PG content with time when only serum was used and a significant decrease in the rate of PG production with the addition of PDGF (9.1-27.8 pgPG/cell/day). Cell morphology was also affected by PDGF, with the cells becoming more spindle shaped. Cell alignment within the gels appeared to be most affected by gel contraction. Collagen gels can act as cell carriers for the purpose of tissue engineering. These gels provide a three-dimensional environment in which chondrocytes can proliferate and produce matrix. We have shown how this environment can be controlled to affect gel contraction, rates of cell growth and PG production, and cellular morphology while maintaining cell viability. This information will be useful in determining the conditions in which chondrocytes can be grown within collagen gels and combined with cytokines to create an ideal tissue construct.

Animals↗

PubMed1999

Effect of impact load on articular cartilage: cell metabolism and viability, and matrix water content.

Significant evidence exists that trauma to a joint produced by a single impact load below that which causes subchondral bone fracture can result in permanent damage to the cartilage matrix, including surface fissures, loss of proteoglycan, and cell death. Limited information exists, however, on the effect of a varying impact stress on chondrocyte biophysiology and matrix integrity. Based on our previous work, we hypothesized that a stress-dependent response exists for both the chondrocyte's metabolic activity and viability and the matrix's hydration. This hypothesis was tested by impacting bovine cartilage explants with nominal stresses ranging from 0.5 to 65 MPa and measuring proteoglycan biosynthesis, cell viability, and water content immediately after impaction and 24 hours later. We found that proteoglycan biosynthesis decreased and water content increased with increasing impact stress. However, there appeared to be a critical threshold stress (15-20 MPa) that caused cell death and apparent rupture of the collagen fiber matrix at the time of impaction. We concluded that the cell death and collagen rupture are responsible for the observed alterations in the tissue's metabolism and water content, respectively, although the exact mechanism causing this damage could not be determined.

Animals↗

PubMed1998

Diffusive properties of immature articular cartilage.

The diffusive properties of immature bovine articular cartilage were determined using two different-sized, uncharged solutes (glucose 180 Da, and dextran 10k Da). Radioactively tagged glucose and dextran were diffused into the cartilage for transport times of 5, 15, and 60 min, and the diffusion and partition coefficients were calculated by fitting the experimental data to a one-dimensional diffusion model. The diffusion and partition coefficients for the two solutes averaged 6.08 +/- 2.19 and 5.09 +/- 2.51 (x 10(-6) cm2/s) and 0.712 +/- 0.149 and 0.615 +/- 0.120, respectively. Both coefficients were significantly greater for glucose compared to the larger dextran. While no statistical differences could be found in the diffusive properties of these solutes in immature cartilage compared to their diffusive properties in mature cartilage, there was some evidence that the larger dextran solute might diffuse faster in the earlier time periods. Finally, the bulk fluid contents between the two types of cartilage were not different even though the immature tissue was significantly thicker (1.6 times) than the mature tissue. Our results indicate that the solute diffusion properties of articular cartilage, at least with respect to uncharged solutes, do not change during skeletal maturation.

Animals↗

PubMed1998

Continuous cyclic load reduces proteoglycan release from articular cartilage.

OBJECTIVE: To study the effect of a continuous cyclic mechanical load on the release of newly synthesized proteoglycans (PGs) from mature bovine articular cartilage. METHODS: Viable cartilage explants were continuously loaded with 1 MPa cyclic stress at 1 Hz frequency for 24 h, and the release of labeled (35SO4) PGs measured before, during and after application of the compressive load. To separate the effect of active chondrocyte catabolism from that of passive PG release, PG release in live explants, with and without protease inhibitors to inhibit PG breakdown, was compared to PG release in explants whose chondrocytes were killed prior to loading. RESULTS: In live explants, a continuous cyclic load significantly reduced PG release by as much as 50% compared to unloaded explants. In killed explants which were unloaded, the PG release increased five to 10 times, while a cyclic load reduced PG release to that found in viable, loaded explants. Twenty-four hours after load removal PG release in all loaded explants returned (increased) to that of the unloaded explants. CONCLUSIONS: These results indicate that PG release from the cartilage matrix is inhibited by continuous cyclic mechanical loading, independent of cellular metabolism, and suggest that a primary mechanism for reducing PG release is by decreasing the interstitial porosity through which the PGs can escape.

Animals↗

PubMed1998

Effects of scalpel, electrocautery, and CO2 and KTP lasers on wound healing in rat tongues.

OBJECTIVE: Evaluate wound healing of incisions created by the scalpel, electrocautery, CO2 laser, and potassium titanyl phosphate (KTP) laser in the upper aerodigestive tract in an animal model. STUDY DESIGN: Prospective randomized study in an animal model. METHODS: Postoperative oral intake, histologic depth of injury, and tensile mechanical strength were measured in rat tongues after creating incisions using a scalpel, electrocautery, CO2 laser, and KTP laser. An unpaired, two-tailed Student's t-test was used to compare results between the experimental groups. RESULTS: Oral intake, indirectly assessed by postoperative weight loss, by the third postoperative day was significantly decreased in the electrocautery (P = 0.004), CO2 laser (P = 0.001), and KTP laser (P = 0.0001) groups as compared with the scalpel group. The depth of the wound healing, as assessed by histologic examination, was successively greater for the scalpel (75 +/- 13 microm), electrocautery (110 +/- 10 microm), CO2 laser (145 +/- 10 microm), and KTP laser (195 +/- 23 microm) groups. However, this difference was only statistically significant for the CO2 laser (P = 0.006) and KTP laser (P = 0.01) groups relative to the scalpel group. Wounds created by the KTP laser had the lowest strength (76.5 +/- 6.9 kPa) as compared with the CO2 laser (156 +/- 28.4 kPa), electrocautery (153 +/- 15.7 kPa), and scalpel groups (249 +/- 61.8 kPa). This difference was only statistically significant for the KTP laser group (P = 0.02) when compared with the scalpel group. CONCLUSIONS: Wounds created in the upper aerodigestive tract of rats by scalpels result in the least postoperative weight loss, tissue destruction, and decrease in tensile strength, whereas wounds created by the KTP laser demonstrated a significantly greater postoperative weight loss, depth of wounding, and decrease in tensile strength.

Animals↗

PubMed1997

Characterization of cartilage metabolic response to static and dynamic stress using a mechanical explant test system.

A new mechanical explant test system was used to study the metabolic response (via proteoglycan biosynthesis) of mature, weight-bearing canine articular cartilage subjected to static and dynamic compressive stresses. Stresses ranging from 0.5 to 24 MPa were applied sinusoidally at 1 Hz for intervals of 2-24 h. The explants were loaded in unconfined compression and compared to age-matched unloaded explants. Both static and dynamic compressive stress significantly decreased proteoglycan biosynthesis (range 25-85%) for all loading time intervals. The inhibition was proportional to the applied stress but was independent of loading time. After rehydration upon load removal, the measured water content of the loaded explants was not different from the unloaded explants for all test variables. Autoradiographic and electron microscopic analysis of loaded explants showed viable chondrocytes throughout the matrix. Our results suggest that the decreased metabolic response of cyclically loaded explants may be dominated by the static component (RMS) of the dynamic load. Furthermore, the observed decreased metabolism may be more representative of the in situ tissue response than that of unloaded explants, in which we found an increasing rate of metabolism for up to 6 days after explant removal.

Animals↗

PubMed1997

Effect of proteoglycan removal on solute mobility in articular cartilage.

Transport of nutrients, cytokines, pharmacologic agents, and matrix components through articular cartilage is critical for the viability and structural integrity of the tissue. To understand the role of the extracellular matrix in regulating this process, we measured the diffusivity of three uncharged solutes of different molecular size (glucose, MW 180; inulin, MW 5000; dextran, MW 70,000) into intact cartilage and cartilage that had its proteoglycan (PG) component removed. Solute diffusivity was measured by performing transient (nonsteady state) one-dimensional diffusion tests using radiolabelled solutes. Compared to intact cartilage, the diffusivity of glucose was unchanged after PG removal, inulin was unchanged but dextran increased by 1.7 times after 71% PG removal, and both inulin and dextran increased by 1.6 and 2.0 times, respectively, after 93% PG removal. The diffusivities of inulin and dextran were inversely proportional to the PG content. While no change was found in the tissue's bulk fluid content, PG depletion resulted in an increase in fluid content in the upper regions of the tissue and a decrease in the lower regions. These results indicate that in intact tissue small uncharged solutes have free mobility through the inter-molecular and intra-molecular PG volumes, larger molecules have limited intra-molecular mobility, and very large molecules are excluded from the intra-molecular space.

Animals↗

PubMed1997

Intracellular calcium response of ACL and MCL ligament fibroblasts to fluid-induced shear stress.

This study examines the real-time intracellular calcium concentration, [Ca2+]i, response of canine medial collateral ligament (MCL) and anterior cruciate ligament (ACL) fibroblasts subjected to a fluid-induced shear stress of 25 dynes/cm2. In experiments using a modified Hanks' Balanced Salt Solution (HBSS) perfusate, both cell types demonstrated a significant increase in peak [Ca2+]i compared to respective no-flow controls, the response of MCL fibroblasts being nearly 2-fold greater than that of ACL fibroblasts. In studies where the cells were bathed in a medium of HBSS supplemented with 2% newborn bovine serum (NBS) and then introduced to flow with the same medium, ACL fibroblasts responded nearly 3-fold greater than MCL fibroblasts. Neomycin (10 mM), thapsigarigin (1 microM) and Ca(2+)-free media supplemented with EGTA (1 mM) were able to inhibit significantly the [Ca2+]i response to flow with HBSS in both fibroblasts. Thapsigargin also blocked the NBS flow response in both cell types, while neomycin and Ca(2+)-free media significantly inhibited the ACL response. Our findings demonstrate that ACL and MCL cells are not the same. These differences may be related to the disparate healing capacity of the ACL and MCL observed clinically.

Animals↗

PubMed1997

Effect of impact load on articular cartilage: development of an intra-articular fracture model.

OBJECTIVES: To investigate the biological and mechanical effects of a single-impact load on articular cartilage. DESIGN: An in vitro laboratory study was performed using mature bovine cartilage and bone, and isolated cartilage explants. Each specimen was impacted with a single load applied with a specially designed impactor and materials test machine. Chondrocyte metabolic activity and cartilage structural integrity was investigated using force displacement curves, radionuclide labeling, histology, and changes in water content. SETTING: Laboratory for Soft Tissue Research, New York, New York, U.S.A. SPECIMENS: Viable mature bovine cartilage and cartilage and bone explants. MAIN OUTCOME MEASUREMENTS: Mechanical failure, proteoglycan synthesis, water content, histology, radiography, and scanning electron microscopy changes occurring during the twenty-four-hour period immediately following impact. RESULTS: Force/displacement curves for the cartilage and bone explants demonstrated two failure-stress peaks, the first at fifty megapascals, representing cartilage failure, and a second peak at seventy-five megapascals, representing bone failure. Fine grain radiographs, histology, and scanning electron microscopy all confirmed the destruction of the cartilage in the area of direct impact (zone I) and subchondral bone failure and the detachment of the cartilage within the lesser impacted area (zone II). Proteoglycan synthesis was reduced significantly (p < 0.05) in the areas of direct impact (zone I) compared with areas with less or no impact (zones II and III, respectively). Significantly greater water content (p < 0.05) was found within the cartilage of zone I compared with zones II and III. CONCLUSIONS: Significant and possibly irreversible articular cartilage damage occurs after a single high-energy impact load.

Animals↗

PubMed1997

Biomechanical evaluation of the medial collateral ligament of the elbow.

UNLABELLED: Anatomical dissection and biomechanical testing were used to study twenty-eight cadaveric elbows in order to determine the role of the medial collateral ligament under valgus loading. The medial collateral ligament was composed of anterior, posterior, and occasionally transverse bundles. The anterior bundle was, in turn, composed of anterior and posterior bands that tightened in reciprocal fashion as the elbow was flexed and extended. Sequential cutting of the ligament was performed while rotation caused by valgus torque was measured. The anterior band of the anterior bundle was the primary restraint to valgus rotation at 30, 60, and 90 degrees of flexion and was a co-primary restraint at 120 degrees of flexion. The posterior band of the anterior bundle was a co-primary restraint at 120 degrees of flexion and a secondary restraint at 30 and 90 degrees of flexion. The posterior bundle was a secondary restraint at 30 degrees only. The reciprocal anterior and posterior bands have distinct biomechanical roles and theoretically may be injured separately. The anterior band was more vulnerable to valgus overload when the elbow was extended, whereas the posterior band was more vulnerable when the elbow was flexed. The posterior bundle was not vulnerable to valgus overload unless the anterior bundle was completely disrupted. The intact elbows rotated a mean of 3.6 degrees between the neutral position and the two-newton-meter valgus torque position. Cutting of the entire anterior bundle caused an additional 3.2 degrees of rotation at 90 degrees of flexion, where the effect was greatest. CLINICAL RELEVANCE: Physical findings in a patient who has an injury of the anterior bundle may be subtle, and an examination should be performed with the elbow in 90 degrees of flexion for greatest sensitivity. As the anterior bundle is the major restraint to valgus rotation, reconstructive procedures should focus on anatomical reproduction of that structure. Parallel limbs of tendon graft placed from the inferior aspect of the medial epicondyle to the area of the sublimis tubercle will simulate the reciprocal bands of the anterior bundle. Temporary immobilization with the elbow in flexion may relax the critically important anterior band of the reconstruction during healing.

Biomechanical Phenomena↗

PubMed1996

Effects of misoprostol and prostaglandin E2 on proteoglycan biosynthesis and loss in unloaded and loaded articular cartilage explants.

The effects of misoprostol, a prostaglandin E1 analog, and prostaglandin E2 on proteoglycan biosynthesis and loss were studied in unloaded and mechanically loaded mature bovine articular cartilage explants. The prostaglandins were administered daily at dosages of 0, 10, 100 and 1000 eta g/ml for up to seven days, and proteoglycan biosynthesis determined by measurement of radiolabelled sulfate incorporation. The presence of misoprostol lead to a significant (p < 0.001) dose-dependent inhibition (30%-50%) in proteoglycan biosynthesis which was also dependent on exposure time (p < 0.05). A significant decrease in biosynthesis (34%) was also found for prostaglandin E2, but only at the highest dose (1000 eta g/ml). Proteoglycan catabolism rates were not affected by either substance as assessed by loss of newly synthesized proteoglycan. The application of a continuous cyclic mechanical compressive load (stress of 1.0 MPa at 1 hertz for 24 hours) resulted in a significant inhibition of proteoglycan biosynthesis (up to 50%) as compared to unloaded explants. However, there was no additive effect when mechanical load and misoprostol or prostaglandin E2 were combined. These results suggest that prostaglandins may have a role in the degenerative and repair process in various forms of arthritis where elevated intra-articular levels of prostaglandin E2 are present.

Animals↗

PubMed1996

Gross, histological, and microvascular anatomy and biomechanical testing of the spring ligament complex.

In recent years there has been an increased interest in the treatment of acquired pes planus. The breakdown of the medial longitudinal arch is most often seen at the talonaviculocalcaneal articulation. This suggests a relationship between the ligamentous complex at this articulation and acquired pes planus. This study was undertaken to gain a better understanding of the gross, histologic, and microvascular anatomy, as well as the biomechanics of the ligamentous structures surrounding the talonaviculocalcaneal articulation. Cadaver dissections of 38 fresh-frozen feet were performed. Detailed descriptions of the gross anatomy of the superomedial calcaneonavicular ligament, inferior calcaneonavicular ligament, and the superficial deltoid ligament were recorded. Their relationships to the posterior tibialis tendon and to the bones of the talonaviculocalcaneal articulation are described. The histology and microvascularity of these structures were also studied. Preliminary biomechanical testing was performed. It was found there are two definitive anatomic structures that are commonly called the spring ligament: the superomedial calcaneonavicular ligament (SMCN) and the inferior calcaneonavicular ligament (ICN). The SMCN ligament was found to have histologic properties that suggest significant load bearing. The histology of the ICN ligament suggests a pure tensile load function. The deltoid ligament and the posterior tibialis tendon had direct attachments to the SMCN ligament in all specimens. An articular facet composed of fibrocartilage was found in each SMCN ligament specimen. The microvascular structures showed an avascular articular facet present in the ligament. The biomechanical testing showed that the SMCN ligament and ICN ligament had strength similar to ankle ligaments. This study suggests this "spring ligament complex" has more of a "sling" function for the talar head. It is hoped that the better understanding of this region will add to our understanding of the etiology of pes planus and possible treatment alternatives.

Biomechanical Phenomena↗

PubMed1995

Diminished material properties and altered bone structure in rat femora during pregnancy.

Pregnancy and lactation are known to cause structural and mechanical changes in bone, but the effects of pregnancy alone have not been evaluated thoroughly. This study used radiographic measurements, torsion testing, mineral analyses, and histological evaluation to determine whether there are changes in bone material and geometric properties during pregnancy in the growing rat, as implied by earlier biochemical and histological studies. The bones of pregnant 9 to 12-week-old rats and controls that were not pregnant and were matched by age (but not weight) were evaluated at times corresponding to 5, 10, 15, and 20 days of the 23-day gestation period to address the following questions: (a) How is the growth of whole bone affected by pregnancy in the growing rat (as determined by radiographic analyses)? (b) How are the mechanical properties (structural and material) of whole bone affected by pregnancy (as assessed by torsion testing)? (c) Are there changes in the characteristics of bone mineral during pregnancy (as determined by measurement of mineral content and x-ray diffraction analyses)? and (d) Are there detectable morphological or ultrastructural differences between the bones of pregnant and control rats (as assessed by analyses based on histology and back-scattered electron imaging)? The presence of statistically significant differences in this study was determined initially on the basis of a two-factor analysis of variance. In general, significant differences were noted only at late gestation (day 20), when the bones were longer and had a greater outer radius and cortical thickness; this indicates that more growth occurred during pregnancy.(ABSTRACT TRUNCATED AT 250 WORDS)

Animals↗