Class I and class II major histocompatibility molecules play a role in bone marrow-derived macrophage development.

Class I and class II major histocompatibility complex (MHC) molecules play significant roles in T cell development and immune function. We show that MHCI- and MHCII-deficient mice have low numbers of macrophage precursors and circulating monocytes, as well as abnormal bone marrow cell colony-stimulating factor type 1 secretion and bone composition. We suggest that MHCI and MHCII molecules play a significant role in macrophage development.

Bone changes in mucopolysaccharidosis VI in cats and the effects of bone marrow transplantation: mechanical testing of long bones.

Mucopolysaccharidosis VI (MPS VI) is a genetic lysosomal storage disease in which a defect in aryl sulfatase B leads to accumulation of the glycosaminoglycan dermatan sulfate and abnormalities in the development of cartilage and bone. A feline model of this disease was used to evaluate the efficacy of bone marrow transplant (BMT) therapy. Long bones from MPS VI cats (N = 6) and MPS VI + BMT cats (N = 7) were compared with control cats (N = 11) and control + BMT cats (N = 5) in mechanical tests. Dissected femurs and tibias were subjected to three-point bending and a subgroup of tibias were tested with the mechanical response tissue analyzer (MRTA) in which vibration is used to measure tissue impedance. Cats with MPS VI had markedly decreased stiffness and strength in both bone (p < 0.01). There was no significant difference in the MPS VI + BMT group. In the tibias, there was also decreased stiffness and strength in the control + BMT group as compared to controls (p < 0.05). However, when cross-sectional area was used to normalize for bone size there was good correlation with strength in both femurs (r = 0.907, p < 0.01) and tibias (r = 0.915, p < 0.1), and there were no significant differences between groups in the modulus of elasticity. In the tibias, in which stiffness was measured by MRTA, there was significant correlation with three-point bending stiffness. These results indicate that, in cats with MPS VI, the decreases in stiffness and strength of long bones can be largely accounted for by the decrease in bone size (osteopenia) that is present.

Histomorphometric, physical, and mechanical effects of spaceflight and insulin-like growth factor-I on rat long bones.

Previous experiments have shown that skeletal unloading resulting from exposure to microgravity induces osteopenia in rats. In maturing rats, this is primarily a function of reduced formation, rather than increased resorption. Insulin-like growth factor-I (IGF-I) stimulates bone formation by increasing collagen synthesis by osteoblasts. The ability of IGF-I to prevent osteopenia otherwise caused by spaceflight was investigated in 12 rats flown for 10 days aboard the Space Shuttle, STS-77. The effect IGF-I had on cortical bone metabolism was generally anabolic. For example, humerus periosteal bone formation increased a significant 37.6% for the spaceflight animals treated with IGF-I, whereas the ground controls increased 24.7%. This increase in humeral bone formation at the periosteum is a result of an increased percent mineralizing perimeter (%Min.Pm), rather than mineral apposition rate (MAR), for both spaceflight and ground control rats. However, IGF-I did inhibit humerus endocortical bone formation in both the spaceflight and ground control rats (38.1% and 39.2%, respectively) by limiting MAR. This effect was verified in a separate ground-based study. Similar histomorphometric results for spaceflight and ground control rats suggest that IGF-I effects occur during normal weight bearing and during spaceflight. Microhardness measurements of the newly formed bone indicate that the quality of the bone formed during IGF-I treatment or spaceflight was not adversely altered. Spaceflight did not consistently change the structural (force-deflection) properties of the femur or humerus when tested in three-point bending. IGF-I significantly increased femoral maximum and fracture strength.

Osteoprotegerin mitigates tail suspension-induced osteopenia.

Osteoprotegerin (OPG) is a recently discovered protein related to the tumor necrosis factor receptor family. It has been shown to inhibit ovariectomy (ovx)-induced resorption in rats and increase bone mineral density in young mice. Tail suspension is a procedure that inhibits bone formation in maturing rodents. This study was designed to quantify OPG's effect on cortical bone formation. Fifty-four mice were assigned to one of five groups (n = 10-11/group). A baseline control group was killed on day 0 of the 10 day study. The remaining groups were: vivarium housed (nonsuspended) control mice receiving 0.3 mg/kg per day OPG; vivarium control mice receiving daily placebo injections; tail-suspended mice receiving 0. 3 mg/kg per day OPG; and tail-suspended mice receiving placebo injections. Tetracycline was administered on days 0 and 8. OPG treatment of tail-suspended mice produced mechanical properties similar to those of placebo-treated, vivarium-housed mice: structural stiffness (8.5%, 20.7%) and elastic (13.9%, 10.1%) and maximum (4.7%, 8.1%) force were increased compared with placebo controls (vivarium, suspended groups). Percent mineral composition was highly significantly greater (p < 0.001 for all comparisons) for OPG-treated mice in the femur, tibia, and humerus, relative to placebo treatment. Matrix mass was also significantly increased in the femur, although not to the same degree as mineral mass. OPG decreased the amount of femoral endocortical resorption compared with the placebo-treated groups for both vivarium (27%) and suspended (24%) mice. Administration of OPG significantly decreased endocortical formation of the tibia. Periosteal bone formation rates were not altered by OPG. OPG-mitigated tail suspension induced osteopenia not by returning bone formation to normal levels, but by inhibiting resorption and increasing percent mineral composition.

Effect of MPC-11 myeloma and MPC-11 + IL-1 receptor antagonist treatment on mouse bone properties.

This study examines the effects of an IL-6-producing murine multiple myeloma cell line on trabecular and cortical mouse bone, and evaluates the efficacy of interleukin-1 receptor antagonist (IL-1ra) in mitigating bone destruction. Six-week-old BALB/c mice were assigned to two groups: normal controls and myeloma animals (5 x 10(7) MPC-11 cells on day 0). Myeloma animals were further assigned to three unique groups: MPC-11 only; MPC-11 treated with hyaluronic acid (HA); and MPC-11 + IL-1ra/HA (100 mg/kg). Disease development was assessed at 14 and 21 days via spleen, liver, and proximal tibia histology; histomorphometry at the femoral middiaphysis; and long bone composition and mechanical testing. Histologic analysis revealed marked myeloma infiltration into organs and bone marrow and gross bone resorption of the proximal tibia. IL-1ra tended to decrease bone resorption at the proximal tibia; however, it had no effect on quantitatively measured bone parameters. Whole femur and tibia, and tibial epiphysis, percent mineralization was decreased (3.0%, 2.9%, and 6.3%, respectively) in all MPC-11 groups. The presence of myeloma did not affect long bone stiffness, strength, or length over the 3 week study. The percent of the femoral endosteal perimeter showing excessive resorption ( approximately 60%) in the MPC-11 groups increased significantly after 21 days. MPC-11 cell presence caused no change in bone formation or morphology. Normal growth mechanisms were not impacted, as the bones lengthened and increased in size and mass despite the presence of myeloma. IL-1 does not appear to be a primary factor in in vivo bone destruction caused by the MPC-11 cell line. These findings reveal the stochastic nature of bone lesions in multiple myeloma and suggest that IL-1 is not a cytokine critical to this disease pathology.

Effects of spaceflight and PEG-IL-2 on rat physiological and immunological responses.

Sprague-Dawley rats were subjected to two 8-day spaceflights on the space shuttle. Rats housed in the National Aeronautics and Space Administration's animal enclosure were injected (iv or sc) with pegylated interleukin-2 (PEG-IL-2) or a placebo. We tested the hypothesis that PEG-IL-2 would ameliorate some of the effects of spaceflight. We measured body and organ weights; blood cell differentials; plasma corticosterone; colony-forming units (macrophage and granulocyte macrophage); lymphocyte mitogenic, superantigenic, and interferon-gamma responses; bone marrow cell and peritoneal macrophage cytokine secretion; and bone strength and mass. Few immunological parameters were affected by spaceflight. However, some spaceflight effects were observed in each flight. Specifically, peritoneal macrophage spontaneous secretion of tumor necrosis factor-alpha occurred in the first but not in the second flight. A significant monocytopenia and lymphocytopenia were detected in the second but not in the first flight. The second mission produced bone changes more consistent with past spaceflight investigations. PEG-IL-2 did not appear to be beneficial; however, this was mostly due to the lack of spaceflight effects. These studies reflect the difficulty in reproducing experimental models by using current space shuttle conditions.

Skeletal unloading causes organ-specific changes in immune cell responses.

The effects of skeletal unloading using antiorthostatic tail suspension on the mouse immune system are tissue specific. This phenomenon was demonstrated by analyzing cells from the lymph nodes, spleen, and bone marrow. Phytohemagglutinin-induced T-cell proliferation was depressed in lymph nodes after 11 days of antiorthostatic suspension. In contrast, splenic T-cell proliferation in response to phytohemagglutinin was enhanced. Splenic natural killer cell cytotoxicity was unchanged after suspension, which demonstrated the organ- and cell-specific effects of skeletal unloading. Whereas antiorthostatic suspension induced minimal changes in bone, there was a significant depression in the number of macrophage precursors in the bone marrow. Overall, skeletally unloaded animals had slightly higher blood corticosterone levels than did control animals; however, it did not appear to be responsible for the observed changes. In conclusion, skeletal unloading produces organ- and cell-specific changes in the murine immune system rather than a generalized immunosuppression.

Osteoprotegerin ameliorates sciatic nerve crush induced bone loss.

This study examines the ability of osteoprotegerin (OPG) to prevent the local bone resorption caused by sciatic nerve damage. Sixty-five 18-week-old male mice were assigned to one of six groups (n = 10-11/group). A baseline control group was sacrificed on day zero of the 10-day study. The remaining groups were placebo sham operated, placebo nerve crush (Plac NC) operated, 0.1 mg/kg/day OPG + nerve crush (LOW), 0.3 mg/kg/day OPG + nerve crush (MED), and 1.0 mg/kg/day OPG + nerve crush (HI). Nerve crush or sham operations were performed on the right leg. The left leg served as a contralateral control to the nerve crushed (ipsilateral) leg. The difference in mass between the right and left femur and tibia was examined. Additionally, quantitative histomorphometry was performed on the right and left femur and tibia diaphyses. Nerve crush resulted in a significant loss of bone mass in the ipsilateral side compared to the contralateral side. Bone mass for the ipsilateral bones of the Plac NC group were significantly reduced by 3.8% in the femur and 3.5% in the tibia compared to the contralateral limb. The percent diminution was reduced for OPG treated mice compared to the Plac NC group for both the femur and tibia. In the femur, the percent reduction of ipsilateral bone mass was reduced to 1.0% (LOW), 1.3% (MED) and 1.6% (HI) compared to the contralateral limb. In the tibia, loss of bone mass in the ipsilateral limb was reduced to 1.4% (LOW), 1.4% (MED), and 2.4% (HI) compared to the contralateral. OPG also decreased the amount of tibial endocortical resorption compared to the Plac NC group. In summary, OPG mitigated bone loss caused by damage to the sciatic nerve.

Material and compositional properties of selectively demineralized cortical bone.

Timed immersion in buffered ethylenediamine-tetraacetic acid (EDTA) was used to selectively alter the mineral content at each level in the cortical bone structural hierarchy. The effects on the mechanical behavior were investigated using a combination of experimental techniques which provide collectively a wide range of resolution (5 microns to 3 mm). Optical microscopy and histological analysis demonstrated a heterogeneous structure consisting of a mineralized tissue core surrounded by a layer of demineralized tissue (collagen) whose thickness varied depending on the immersion time. The mechanical behaviors of treated samples with (intact) and without (core) the surrounding demineralized layer were evaluated using three-point flexure. Overall, the intact specimens became significantly less brittle with increased immersion time in buffered-EDTA. For the core specimens, there was a systematic decrease in the elastic flexural properties (E, sigma e, epsilon e). The site-specific properties of the specimens were determined using microhardness testing, scanning acoustic microscopy, and wavelength dispersive analysis. The mineralization and site-specific properties of the mineralized cores were not significantly affected by buffered-EDTA immersion; however, histomorphometric analysis showed a decrease in the mineralized volume fraction via widening of the pre-existing vascular channels. The experimental hierarchy was effective in discerning site-specific property changes and the localized heterogeneities resulting from the buffered-EDTA treatment. Based on the results of this study, buffered-EDTA treatment can be used to facilitate the determination of material and physical properties of intact and demineralized tissues within a single cortical bone sample.

The physical and mechanical effects of suspension-induced osteopenia on mouse long bones.

The present investigation addresses the extent of tail-suspension effects on the long bones of mice. The effects are explored in both sexes, in both forelimb and hindlimb bones, and in both diaphyseal and metaphyseal/epiphyseal bones. Two weeks of suspension provided unloading of the femora and tibiae and an altered loading of the humeri. Whole-bone effects included lower mass (approximately 10%) and length (approximately 4%) in the bones of suspended mice compared to controls. The geometric and material properties of the femora were considered along the entire length of the diaphysis and in the metaphysis/epiphysis portions as a unit. Geometric effects included lower cross-sectional cortical area (16%), cortical thickness (25%) and moment of inertia (21%) in the femora of suspended mice; these differences were observed in both distal and proximal portions of the femur diaphysis. The relative amount of bone comprising the middle 8 mm of the diaphysis was greater (3%) in the control mice than in the suspended mice. Significant mass differences between the group in the metaphysis/epiphysis were not observed. Material effects included lower %ash (approximately 2%) in the femora and tibiae as well as in the humeri of suspended mice compared to controls. With respect to the measured physical and material properties, suspension produced similar bone responses in male and female mice. The effects of suspension are manifested largely through geometric rather than through material changes.

Errors due to measuring voltage on current-carrying electrodes in electric current computed tomography.

Electric current computed tomography is a process for determining the distribution of electrical conductivity inside a body based upon measurements of voltage or current made at the body's surface. Most such systems use different electrodes for the application of current and the measurement of voltage. This paper shows that when a multiplicity of electrodes are attached to a body's surface, the voltage data are most sensitive to changes in resistivity in the body's interior when voltages are measured from all electrodes, including those carrying current. This assertion is true despite the presence of significant levels of skin impedance at the electrodes. This conclusion is supported both theoretically and by experiment. Data were first taken using all electrodes for current and voltage. Then current was applied only at a pair of electrodes, with voltages measured on all other electrodes. We then constructed the second data set by calculation from the first. Targets could be detected with better signal-to-noise ratio by using the reconstructed data than by using the directly measured voltages on noncurrent-carrying electrodes. Images made from voltage data using only noncurrent-carrying electrodes had higher noise levels and were less able to accurately locate targets. We conclude that in multiple electrode systems for electric current computed tomography, current should be applied and voltage should be measured from all available electrodes.

Effects of space flight and IGF-1 on immune function.

We tested the hypothesis that insulin-like growth factor-1 (IGF-1) would ameliorate space flight-induced effects on the immune system. Twelve male, Sprague-Dawley rats, surgically implanted with mini osmotic pumps, were subjected to space flight for 10 days on STS-77. Six rats received 10 mg/kg/day of IGF-1 and 6 rats received saline. Flight animals had a lymphocytopenia and granulocytosis which were reversed by IGF-1. Flight animals had significantly higher corticosterone levels than ground controls but IGF-1 did not impact this stress hormone. Therefore, the reversed granulocytosis did not correlate with serum corticosterone. Space flight and IGF-1 also combined to induce a monocytopenia that was not evident in ground control animals treated with IGF-1 or in animals subjected to space flight but given physiological saline. There was a significant increase in spleen weights in vivarium animals treated with IGF-1, however, this change did not occur in flight animals. We observed reduced agonist-induced lymph node cell proliferation by cells from flight animals compared to ground controls. The reduced proliferation was not augmented by IGF-1 treatment. There was enhanced secretion of TNF, IL-6 and NO by flight-animal peritoneal macrophages compared to vivarium controls, however, O2(-) secretion was not affected. These data suggest that IGF-1 can ameliorate some of the effects of space flight but that space flight can also impact the normal response to IGF-1. Grant Numbers: NAGW-1197, NAGW-2328.

The role of sex and genotype on antiorthostatic suspension effects on the mouse peripheral skeleton.

Previous antiorthostatic suspension studies have used a single sex and strain of rat or mouse. Nonetheless, broadly similar effects of suspension on the two species indicates a generalized effect of suspension not attributed to specific genetic, behavioral, or sex-linked etiology. In order to directly test genetic and sex-linked factors, the effects of suspension on the appendicular bone of male and female BALB-CJ, C57BL-6J, and DBA-2J mice were compared. These genotypes were selected based on their widely different developmental and behavioral characteristics as well as on past research involving a heterogeneous strain derived from these strains. The effects of suspension on the geometric, mechanical, and material properties of the femora, humeri, and tibiae were determined. Among the bone types, the femora were most significantly affected by suspension. The effects of suspension were similar in nature in male and female mice aged 1.7 months. Strain-dependent suspension effects may be indicative of bone developmental differences in the strains at the age chosen.

Effect of microgravity on collagenase deproteinization and EDTA decalcification of bone fragments.

Undecalcified (n = 140) and decalcified (n = 11) bone fragments were treated with either collagenase (to remove collagen portion; undecalcified n = 64, decalcified n = 11) or EDTA (to remove mineral portion; n = 76) under the reduced gravity environment on US Space Shuttle mission STS-57. The fragments were initially stored in Dulbecco's phosphate buffer solution. After orbit had been established, fragments were exposed to either a neutral buffered collagenase or EDTA solution. Reactions were terminated (neutral buffered formalin for collagenase, 21% CuSO4 5H2O for EDTA) before reentry to earth's atmosphere. Differences in bone samples mass from before flight to after flight were measured. EDTA-treated sample mass was corrected for CuSO4 content. Flight and matched ground (gravitational control) sample showed similar EDTA-induced loss of mineral mass. Collagenase treatments, however, appeared to be more effective in flight samples compared to ground control samples. The flight-exposed, collagenase-treated samples showed significantly more loss of mass than did ground samples. The microgravity environment appeared to promote proteolytic reactions in bone more than the EDTA decalcification reaction.

Effect of microgravity, temperature, and concentration on fibrin and collagen assembly.

In purified form collagen and fibrin can be processed into gel-like matrices of interconnecting fibers. The microscopic structure of materials produced from these macromolecules is critical to their utility as biomaterials. Varying the conditions of the assembly environment allows for the production of a wide range of morphologies. In this study, changes in gravity, temperature, and concentration were examined. Contrary to protein crystal growth studies which indicate substantial increases in organization and size in microgravity, the gravitational environment had no repeatable effect on collagen and fibrin fiber diameters and matrix porosity. However, fibrin gels formed in microgravity appeared more homogeneous than ground samples. Changes in temperature and concentration of both protein and buffer had substantial effects on fiber diameters and material porosity for both collagen and fibrin. Temperature experiments were performed over the range 23.8 to 39 degrees C for fibrin and 22 to 33 degrees C for collagen. Thrombin concentration was varied from 0.02 to 0.10 units/ml for fibrin experiments and buffer concentration was varied by means of a dialysis membrane for collagen experiments. Consequently, the temperature and concentration controls developed for flight experiments are being considered for their potential in developing fibrin and collagen based materials with well-defined microscopic structures. The increased homogeneity of fibrin gels produced in microgravity suggests the possibility of using this environment for the production of optimal biomaterials.

An autonomous module for supporting mice during spaceflight.

The Animal Module for Autonomous space Support (A-MASS) was developed to enable 30-day spaceflight for mice on the first Commercial Experiment Transporter mission. Because space hardware did not previously exist to support mice without astronaut intervention, the A-MASS presented considerable technical and animal care challenges. The technical challenges included maintaining a 42.5l payload volume and 20-g structural conformance while providing 30 days of autonomous mouse support. Sensors, video, a pressurized oxygen supply system and an internal data logging system were incorporated. The A-MASS met NIH guidelines for temperature, humidity, food and water access, oxygen supply, air quality and odor control. These technical and animal care challenges, along with power and mass constraints, were addressed using a novel design which ensures a fresh food and water supply, a clean view path into the cage for the camera system, and removal of the wastes from the air supply. The payload was successfully tested in an enclosed chamber and passed animal health, vibrational, mechanical, and electrical tests. The physiological, tactical and animal support information gathered will be applicable to the development of mouse support modules for the Shuttle Middeck and Space Station Freedom Express Rack environments.

A novel combination of methods to assess sarcopenia and muscle performance in mice.

A novel combination of assays was developed to assess sarcopenia and muscle performance. Three techniques were tested to assess muscle function both during and upon termination of treatments designed to induce sarcopenia. In unsuspended (US) and hindlimb suspended (HS) mice, a Hindlimb Exertion Force Test (HEFT), cage wheel running, and in vitro muscle electrophysiology were performed. Twelve-week old, mature male C57BL/6J mice were HS (n = 24) for two weeks, or served as US controls (n = 26). Both groups were subjected to a HEFT on day 13; that is, the maximum force exerted against a beam force transducer (2 lb. linear range, Transducer Techniques, Temecula CA) following applied tail shock stimulus (0.15 mA, 300 msec). This test primarily evaluated the hindlimb muscles used for an escape response (i.e., hamstrings, quadriceps and calf muscles). Mice (n = 10-11/group) were given voluntary access to running wheels for 7 days post treatment to evaluate muscle endurance. On day 13, HS mice showed a mean 18.9% (p = 0.002) decrease in the maximum force exerted compared to US mice. After 7 days of wheel running, HS running distance tended to decrease (13.2%, p = 0.084). HS mice ran an average of 2.0 km/day less than US control mice, with similar running patterns: distance declined on day 2 following completion of HS but increased steadily thereafter. With in vitro testing, the maximum soleus tetanus response decreased by 31.8% (p = 0.01) with HS, in agreement with the changes observed by the other assays. These three assays, combined, appear to provide effective and complementary ways to measure muscle performance and functional differences.

Comparison of tail-suspension and sciatic nerve crush on the musculoskeletal system in young-adult mice.

Musculoskeletal unloading and disuse result in significant muscle and bone loss. These phenomena can be modeled using sciatic nerve crush or tail-suspension. Mature animals eliminate the complication of growth superimposed on bone and muscle loss. In the current study, young-adult (12-week old male) C57BL/6J mice were subjected to sciatic nerve crush (NC; n = 9) or tail-suspension (TS; n = 9) for 14 days, with a normal ambulatory control (n = 10). The soleus, gastrocnemius, and EDL muscles were collected and weighed at sacrifice. Femurs were analyzed in three-point bending for stiffness, elastic force and maximum force. Muscle masses in tail suspended mice were reduced by 41.9% (p < 0.001), 17.5% (p < 0.001), and 9.1% (N.S.) for the soleus, gastrocnemius, and EDL, respectively. In NC mice, muscle masses were reduced by 18.6% (p = 0.004), 37.2% (p < 0.001), and 22.5% (p = 0.003). Femur stiffness, elastic and maximum forces were reduced by 20.9% (p = 0.014), 14.7% (N.S.), and 11.6% (N.S.) in TS, compared to NC where masses were reduced by 15.5% (p = 0.022), 0.2% (N.S.) and 11.2% (N.S.) in the crushed leg compared to the contralateral control. NC resulted in a greater reduction of muscle mass in the gastrocnemius and EDL muscle; whereas tail-suspension had a greater effect on the soleus. Tail-suspension had the greatest effect on bone mechanical properties. When comparing these results to actual spaceflight data, it appears as though TS most closely models muscle loss, and NC most closely models changes in bone mechanical properties. These unloading models have tissue-specific effects that impact their applications for musculoskeletal research.

Determining the efficacy of edge detection algorithms.

Edge detection is an important process in the interpretation of image data. Four types of edge detection algorithms (the Local Variance, Correlation, Laplacian and Frequency Peak algorithms) are compared for their ability to discriminate the edges present in an image with precisely defined edges. The variety of techniques are discussed in terms of their sensitivity to the presence of edges and their sensitivity to the applied noise. Both computational and empirical analyses are presented, and the relative merits of each discussed in terms of signal-to-noise ratio, sensitivity and computational difficulty. It is shown that local variance edge detection is an acceptable method for edge detection which simplifies the computation needed for image convolution. The means through which to devise more sophisticated edge detection algorithms is outlined, and the use of difference images to discriminate edges in an image is discussed. The results are also discussed in terms of the implementation of edge detection algorithms as part of a computer visual system which has an architecture modeled after the mammalian visual system.

Cranial bone apposition and ingrowth in a porous nickel-titanium implant.

A 5 x 5 x 1-mm uncoated porous nickel-titanium (nitinol) implant was placed 4 mm to either side of the midsection of the frontal bone and 4 mm anterior to the coronal suture of the cranial bone of New Zealand White rabbits. In the other frontal location, a 5 x 5 x 1-mm coralline hydroxyapatite (HA) (Interpore 200, a well-known craniofacial implant material) implant was fitted. Rabbits were killed at each of three postsurgical intervals (2, 6, and 12 weeks), and the implants were evaluated for gross biocompatibility, bony contact, and ingrowth. No adjacent macrophage cells were observed for either implant type, and overlaying soft tissues and connective tissues readily adhered to the implants even after 2 weeks. Both materials made bone contact with the surrounding cranial hard tissue, and percent ingrowth increased with surgical recovery time. Measurements of microhardness and bone histologic parameters indicated that bone in contact with and grown into the implants was similar in properties to the surrounding cranial bone. Porous nitinol implants therefore appear to allow for significant cranial bone ingrowth after as few as 12 weeks, and thus nitinol appears to be suitable for craniofacial applications. Compared to HA, the nitinol implants demonstrated a trend for less total apposition and more total ingrowth after 6 and 12 weeks of implantation.