Reed A. Ayers

Bone development and age-related bone loss in male C57BL/6J mice

The objective of this study was to examine changes in the long bones of male C57BL/6J mice with growth and aging, and to consider the applicability of this animal for use in studying Type II osteoporosis. Male C57BL/6J mice were aged in our colony between 4 and 104 weeks (n=9-15/group). The right femur and humeri were measured for length and subjected to mechanical testing (3-point flexure) and compositional analysis. The left femurs were embedded and thick slices at the mid-diaphysis were assessed for morphology, formation indices, and bone structure. In young mice, rapid growth was marked by substantial increases in bone size, mineral mass, and mechanical properties. Maturity occurred between 12 and 42 weeks of age with the maintenance of bone mass and mechanical properties. From peak levels, mice aged for 104 weeks experienced decreased whole femur mass (12.1 and 18.6% for dry and ash mass, respectively), percentage mineralization (7.4%), diminished whole bone stiffness (29.2%), energy to fracture (51.8%), and decreased cortical thickness (20.1%). Indices of surface-based formation decreased rapidly from the onset of the study. However, the periosteal perimeter and, consequently, the cross-sectional moments of inertia continued to increase through 104 weeks, thus maintaining structural properties. This compensated for cortical thinning and increased brittleness due to decreased mineralization and stiffness. The shape of the mid-diaphysis became increasingly less elliptical in aged mice, and endocortical resorption and evidence of subsequent formation were present in 20-50% of femurs aged > or =78 weeks. This, combined with the appearance of excessive endocortical resorption after 52 weeks, indicated a shift in normal mechanisms regulating bone shape and location, and was suggestive of remodeling. The pattern of bone loss at the femoral mid-diaphysis in this study is markedly similar to that seen in cortical bone in the human femoral neck in Type II osteoporosis. This study has thus demonstrated that the male C57BL/6J mouse is a novel and appropriate model for use in studying endogenous, aging-related osteopenia and may be a useful model for the study of Type II osteoporosis.

Porous Materials for Bone Engineering

Maintaining bone geometric and structural integrity is a necessity for normal mobility. After fracture, bone disease or other conditions resulting in skeletal loss or compromise, porous materials offer the possibility for near faultless replacement of the normal bone material. Ceramics, and to a lesser extent, metals, are the predominant porous materials currently used in bone engineering. This latter term is used as a blanket term for orthodontics, orthopedics and related fields in which the replacement of bone is either required or selectively chosen. Because bone is a porous material, there is a physiological rationale for the use of porous materials in its replacement. Moreover, porous bone implant material is advantageous for the early incorporation of the implant into or apposed to the bony tissue surrounding it. There is, however, a difference in the size and extent of the bone and implant porosities for optimal bone incorporation of the material. This review intends to clarify both the nature of and reason for this difference. Accordingly, a review of the principal types of porous materials (organics, ceramics, metals, metallorganics and organoapatites and composites) used in bone engineering will be provided. This will springboard a consideration of the important engineering considerations of material property matching, machining and forming, corrosion and biocompatibility, fatigue and lifecycle, coating, and interfacial properties. The importance of matching the porous material to the particular bone engineering application will then be discussed. In providing this review, the authors hope to bring an appreciation of the complexity of the field to the fore, while also demonstrating how much has already been accomplished due to the efforts of many research groups. The ultimate porous bone implant, perhaps, is yet to be designed; however, there is reason to believe that such a material is not long in coming. We hope to demonstrate some possible pathways to this material. Outline The paper is constructed as follows.

Effect of nitinol implant porosity on cranial bone ingrowth and apposition after 6 weeks

The present study addresses two aspects of the use of nitinol in cranial bone defect repair. The first is to verify that there is substantial bone ingrowth into the implant after 6 weeks; the second is to determine the effect of pore size on the ability of bone to grow into the implant during the early (6-week) postoperative period. Porous equiatomic (equal atomic masses of titanium and nickel) nickel-titanium (nitinol) implants with three different morphologies (differing in pore size and percent porosity) were implanted for 6 weeks in the parietal bones of New Zealand White rabbits. Ingrowth of bone into the implants and apposition of bone along the exterior and interior implant surfaces were calculated. The mean pore size (MPS) of implant type #1 (353 +/- 74 microm) differed considerably from implant types #2 (218 +/- 28 microm) and #3 (178 +/- 31 microm). There was no significant difference among implant types in the percentages of bone and void/soft tissue composition of the aggregate implants. The amount of bone ingrowth also was not significantly different among the implant types. Implant #1 was significantly higher in pore volume and thus had a significantly higher volume of ingrown bone (2.59 +/- 0.60 mm3) than implant #3 (1. 52 +/- 0.66 mm3) and a greater amount, but not significantly greater, than implant #2 (1.76 +/- 0.47 mm3). Pore size does not appear to affect bone ingrowth during the cartilaginous period of bone growth in the implant. This implies that within the commonly accepted range of implant porosities (150-400 microm), at 6 weeks bone ingrowth near the interface of nitinol implants is similar.

Long-term bone ingrowth and residual microhardness of porous block hydroxyapatite implants in humans.

PURPOSE This study examined the ingrowth of bone into coralline, porous hydroxyapatite (HA) block (Interpore 200) over long periods after orthognathic surgery and analyzed their microhardness as a measure of the structural integrity of the ingrown bone as well as of the HA. MATERIALS AND METHODS Twenty-five maxillary HA implants (4 to 138 months of implantation; mean, 32 months) were removed from 17 patients. These implants had been placed into the lateral maxillary wall, juxtapositioned to the maxillary sinus during orthognathic surgery, and were harvested for analysis after voluntary consent. RESULTS Microscopic examination showed normal bone morphology in all implants; no inflammatory response was observed. Histomorphometric measurements indicated that there was significant bone ingrowth in all implants, with an overall mean of 23+/-7% bone (range, 7% to 31%), 51%+/-7% HA matrix (range, 39% to 65%), and the remainder being soft tissue or void at 26%+/-9% (range, 10% to 40%). No significant difference in microhardness values between the bone in the implant and the bone surrounding the implant was noted, indicating that the structural integrity of the porous block HA/bone aggregate had been maintained. Bone ingrowth appeared to plateau around 20 months, reaching an equilibrium in which the relative amount of osseous tissue remained constant. CONCLUSION Based on the findings in this study, porous block HA is a viable material for long-term implantation in the maxilla during orthognathic surgery.

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 OPGs 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.

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.

Investigation of inductively coupled Ar and CH4/Ar plasmas and the effect of ion energy on DLC film properties

Gas-phase and surface analysis techniques were utilized to investigate the effects of gas-phase species on plasma deposited diamond-like carbon (DLC) thin films. A vacuum system was built to perform Langmuir probe and energy analysis-based mass spectrometry measurements to characterize the gas-phase of low pressure, 13.56?MHz inductively coupled plasma molecular beams. Low-energy peaks contributed significantly to the total area of the ion energy distributions (IEDs) measured for Ar+ in Ar and CH4/Ar plasmas. In contrast, for all other ions in these systems, the low-energy peaks had a lower contribution to the IEDs as a result of the low probability of energy exchange via ion?neutral collisions. Hydrogenated DLC films were deposited on silicon wafers at different substrate potentials to determine the effect of ion bombardment on film properties. Films were characterized via Fourier transform infrared spectroscopy, scanning electron microscopy, atomic force microscopy and nanoindentation measurements. The hydrogen content, surface roughness and deposition rate decreased, whereas the hardness of the films increased when a negative bias voltage was applied. These results demonstrate that ion energy has a significant effect on the composition and morphology of plasma deposited DLC films.

Quantification of bone ingrowth into porous block hydroxyapatite in humans

This study sought to quantify bone ingrowth from a single bone-implant surface into porous block hydroxyapatite used in maxillofacial applications. Seventeen maxillary hydroxyapatite implants (implant time of 4-138 months, 39-month mean) were harvested for analysis from 14 patients. The implants had been placed into the lateral maxillary wall during orthognathic surgery, juxtapositioned to the maxillary sinus. Ingrowth was measured in 100-microm increments from a bone-implant interface to a depth of 1500 microm. Bone ingrowth averaged over the 14 patients (0-1100 microm depth) is described by the equation % ingrowth - 20% * (depth in millimeters) + 41.25% (R2 = 0.98, n = 10 incremental depths). Beyond 1100 microm, the average ingrowth remained constant at 15.0 +/- 0.7%. The duration of implantation also showed as affect on the percent ingrowth into the implants at the incremental depths, and the percent ingrowth asymptotically approached a maximum. Overall, the composite average data from all depths is best described by the logarithmic function % ingrowth = 15% * ln(implantation time in months) - 24.0% (R2 = 0.71, n = 14 patients). Several factors may come into play in determining bone ingrowth including the mechanical environment, the osteoconductivity of the implant material, and the osteogenic capability of the tissues in the pore spaces. Measurements of bone ingrowth are most influenced by the depth into the implant and the time the implant was in the body; the age of the patient had little affect on bone ingrowth.

Effects of gravity on combustion synthesis of functionally graded biomaterials

Combustion synthesis, or self-propagating, high temperature synthesis is currently being used at the Colorado School of Mines to produce advanced materials for biomedical applications. These biomaterials include ceramic, intermetallic, and metal-matrix composites for applications ranging from structural to oxidation- and wear-resistant materials, e.g., TiC-Ti, TiC-Cr3C2, MOSi2-SiC, NiAl-TiB2, to engineered porous composites, e.g., B4C-Al2O3, Ti-TiBx, Ni-Ti, Ca3(P04)2 and glass-ceramic composites, e.g., CaO-SiO2-BaO-Al2O3-TiB2. The goal of the functionally graded biomaterials project is to develop new materials, graded in porosity and composition, which will combine the desirable mechanical properties of implant, e.g., NiTi, with the bone-growth enhancement properties of porous biodegradable ceramics, e.g., Ca3(PO4)2. Recent experiments on the NASA parabolic flight (KC-135) aircraft have shown that gravity plays an important role in controlling the structure and properties of materials produced by combustion synthesis. The results of these studies, which will be presented at the conference, will provide valuable input to the design of experiments to be done in Space-DRUMSTM, a containerless materials processing facility scheduled to be placed on the International Space Station in 2003.

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.