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We assessed the possible association between an aggressive intercondylar notchplasty and histopathologic, radiographic, and gait changes to the knee. Three groups of six adult greyhounds were observed for 6 months. Group I dogs had a sham operation. Group II dogs had a 4-mm notchplasty of the lateral femoral condyle where it articulates with the lateral tibial spine. Group III dogs had a 7- to 8-mm notchplasty of the lateral femoral condyle to simulate the long-term effects of an overly aggressive notchplasty. Force plate gait analyses were not significantly different for any dogs at 3 and 6 months. Histopathologic studies (hematoxylin and eosin and safranin O stains) revealed notchplasty area remodeling with a thin layer of lamellar bone covered by fibrous connective tissue. Both Group II and III dogs had significant loss of lateral femoral condyle and trochlear groove articular surface proteoglycans. The radiographic notch width index remained unchanged throughout the study for Group I; the indexes increased immediately after surgery in Groups II and III because of the notchplasty, but after 6 months these values returned to near-preoperative measurements. An aggressive intercondylar notchplasty caused articular cartilage histopathologic changes at 6 months consistent with those found in knees with early degenerative arthritis. Significant refilling of a non-impinged notchplasty occurred by 6 months after surgery. Our results raise concern about the effects of aggressive intercondylar notch widening in humans. [ABSTRACT FROM AUTHOR]

Objective: To investigate the association of timing of commencing glucose management with glycemia, glycemic variability, and pregnancy outcomes among women with early gestational diabetes mellitus (GDM)., Research Design and Methods: In this substudy among participants of a trial of immediate vs. delayed treatment of early GDM diagnosed by 2013 World Health Organization criteria, all women treated immediately and those with delayed diagnosis at 24-28 weeks' gestation (treated as if late GDM) were instructed to monitor capillary blood glucose (BG) four times a day (fasting and 2-h postprandial) until delivery. Optimal glycemia was defined as ≥95% of BG measurements between 70 and 140 mg/dL (3.9-7.8 mmol/L)., Results: Overall, 107,716 BG values were obtained from 329 of 549 (59.9%) women (mean age 32.3 ± 4.9 years, BMI 32.0 ± 8.0 kg/m2, 35% European, gestation at GDM diagnosis 15.2 ± 2.4 weeks). Women treated early (n = 213) showed lower mean glucose (MG) and mean fasting glucose (MFG) compared with those treated late (n = 116) (MG: 5.7 ± 0.4 vs. 5.9 ± 0.5, P < 0.001; MFG: 5.2 ± 0.3 vs. 5.3 ± 0.4, P = 0.004), with greater optimal glycemia (74.6% vs. 59.5%, P = 0.006) and similar glycemic variability. MG was similar from 30 weeks' gestation. Overall, optimal glycemia was achieved in 69% of women and associated with lower birth weight, fewer large-for-gestational-age infants (14.4% vs. 26.7%, P = 0.01), more small-for-gestational-age infants (15.3% vs. 5.9%, P = 0.02), and lower gestational weight gain (4.9 ± 6.4 vs. 7.6 ± 6.2 kg, P = 0.001). Suboptimal glycemia was associated with non-European ethnicity, prior GDM, 1-h glucose at booking oral glucose tolerance test, and insulin use., Conclusions: Both early and delayed treatment of early GDM resulted in similar glycemia toward the end of pregnancy. Early treatment was associated with improved glycemia overall., (© 2024 by the American Diabetes Association.)

Introduction: Systemic therapies have been associated with clinically significant events (CSEs) in patients with unresectable hepatocellular carcinoma (uHCC). We evaluated the incidence of CSEs (bleeding, clotting, encephalopathy, and portal hypertension), and their impact on healthcare resource utilization (HCRU) and costs, in patients with uHCC treated with first-line (1L) atezolizumab plus bevacizumab (A + B), lenvatinib (LEN), or sorafenib (SOR) in the USA., Methods: A retrospective cohort study was performed using medical/pharmacy claims from Optum ® Clinformatics ® Data Mart. Patients diagnosed with HCC who initiated 1L A + B between June 01, 2020 and December 31, 2020 or LEN/SOR between January 01, 2016 and May 31, 2020 were included. Outcomes included incidence rates of CSEs, HCRU, and costs. Subgroup analysis was performed in patients with no CSEs or ≥ 1 CSE., Results: In total, 1379 patients were selected (A + B, n = 271; LEN, n = 217; SOR, n = 891). Clotting (incidence rate per 100 patient-years [PY] 94.9) and bleeding (88.1 per 100 PY) were the most common CSEs in the A + B cohort. The most common CSEs in the LEN cohort were clotting (78.6 per 100 PY) and encephalopathy (66.3 per 100 PY). Encephalopathy (73.0 per 100 PY) and portal hypertension (72.3 per 100 PY) were the most common CSEs in the SOR cohort. Mean total all-cause healthcare costs per patient per month (PPPM) were $32,742, $35,623, and $29,173 in the A + B, LEN, and SOR cohorts, respectively. Mean total all-cause healthcare costs PPPM were higher in patients who had ≥ 1 CSE versus those who did not (A + B $34,304 versus $30,889; LEN $39,591 versus $30,621; SOR $31,022 versus $27,003)., Conclusion: Despite improved efficacy of 1L systemic therapies, CSEs remain a concern for patients with uHCC, as well as an economic burden to the healthcare system. Newer treatments that reduce the risk of CSEs, while improving long-term survival in patients with uHCC, are warranted., (© 2024. The Author(s).)

Previous studies show Republicans have lower risk perceptions of COVID-19 than Democrats. Has this gap in risk perception been consistent throughout the course of the pandemic? Using longitudinal data from the Understanding America Study's Understanding Coronavirus in America longitudinal panel survey we examine how time and vaccination status have changed Republican and Democratic risk perceptions of engaging in various activities (such as grocery shopping, visiting friends and family etc.) as well as risk perceptions of infection, hospitalization, and death due to COVID-19. We find that while Republicans have lowered their perceived risk of activity more than Democrats over time, vaccinated Democrats have lowered their perceived risk of infection, hospitalization, and death more than vaccinated Republicans. These results are robust to inclusion of media consumption and social media use. This divergence on the two measures of risk from COVID-19 may complicate leaders' efforts to move on from the politics of the pandemic toward a return to "normal.", Competing Interests: Disclosure Statement No potential conflict of interest is reported by the authors.

Context: As COVID-19 vaccines were rolled out in early 2021, governments at all levels in the United States experienced significant difficulty in consistently and efficiently administering injections in the face of vaccination resistance among a public increasingly politically polarized on vaccination preferences before the beginning of mass vaccinations., Methods: Using an original conjoint experiment fielded to a nationally representative sample before the mass proliferation of COVID-19 vaccines, the authors examined how different incentives (e.g., employer mandates, state-organized or health care provider-organized vaccination clinics, and financial incentives) affect the public's preference to get vaccinated. They also tested how financial incentive preferences correlated with self-reported vaccination intention using observational data from the June 2021 Kaiser Family Foundation Health Tracking Poll., Findings: The authors found financial incentives positively influenced vaccine preferences among the mass public and all partisan groups, including Republicans who were initially "unlikely" to be vaccinated. The authors used the observational data to replicate their experimental findings, showing positive financial incentive attitudes positively correlated with self-reported vaccination disclosures., Conclusions: These results provide support for direct financial incentives, rather than other incentives, as being a valuable tool for policy makers tasked with alleviating vaccination resistance among a US mass public increasingly polarized along partisan lines., (Copyright © 2023 by Duke University Press.)

Background: Durvalumab is approved for the treatment of adults with unresectable stage III non-small cell lung cancer (NSCLC) post-chemoradiotherapy (CRT). This real-world study describes patient characteristics and durvalumab treatment patterns (number of doses and therapy duration; treatment initiation delays, interruptions, discontinuations, and associated reasons) among VHA-treated patients., Methods: This was a retrospective cohort study of adults with unresectable stage III NSCLC receiving durvalumab at the VHA between 1 January 2017 and 30 June 2020. Patient characteristics and treatment patterns were presented descriptively., Results: A total of 935 patients were included (median age: 69 years; 95% males; 21% Blacks; 46% current smokers; 16% ECOG performance scores ≥ 2; 50% squamous histology). Durvalumab initiation was delayed in 39% of patients ( n = 367). Among the 200 patients with recorded reasons, delays were mainly due to physician preference (20%) and CRT toxicity (11%). Overall, patients received a median (interquartile range) of 16 (7-24) doses of durvalumab over 9.0 (2.9-11.8) months. Treatment interruptions were experienced by 19% of patients ( n = 180), with toxicity (7.8%) and social reasons (2.6%) being the most cited reasons. Early discontinuation occurred in 59% of patients ( n = 551), largely due to disease progression (24.2%) and toxicity (18.2%)., Conclusions: These real-world analyses corroborate PACIFIC study results in terms of the main reasons for treatment discontinuation in a VHA population with worse prognostic factors, including older age, predominantly male sex, and poorer performance score. One of the main reasons for durvalumab initiation delays, treatment interruptions, or discontinuations was due to toxicities. Patients could benefit from improved strategies to prevent, identify, and manage CRT and durvalumab toxicities timely and effectively.

Vaccines are the most effective means at combating sickness and death caused by COVID-19. Yet, there are significant populations within the United States who are vaccine-hesitant, some due to ideological or pseudo-scientific motivations, others due to significant perceived and real costs from vaccination. Given this vaccine hesitancy, twenty state governors from May 12th to July 21st 2021 implemented some form of vaccination lottery aiming to increase low vaccination rates. In the aftermath of these programs, however, the critical question of whether these lotteries had a direct effect on vaccination remains. Previous literature on financial incentives for public health behaviors is consistent: Financial incentives significantly increase incentivized behaviors. Yet, work done specifically on state vaccine lotteries is both limited in scope and mixed in its conclusions. To help fill this gap in the literature, we use synthetic control methods to analyze all 20 states and causally identify, for eighteen states, the effects of their lotteries on both first-dose and complete vaccination rates. Within those eighteen states, we find strong evidence that all but three states' lotteries had positive effects on first-dose vaccination. We find for complete vaccinations, however, over half the states analyzed had negative or null effects. We explore possibilities related to these mixed results including the states' overall partisanship, vaccine hesitancy, and the size of their lotteries finding null effects for each of these explanations. Therefore, we conclude that the design of these programs is likely to blame: Every state lottery only incentivized first-doses with no additional or contingent incentive based on a second dose. Our findings suggest that the design of financial incentives is critical to their success, or failure, but generally, these programs can induce an uptake in vaccination across diverse demographic, ideological, and geographic contexts in the United States., Competing Interests: The authors have declared that no competing interests exist.

Background: Snail, a transcriptional factor and repressor of E-cadherin is well known for its role in cellular invasion. It can regulate epithelial to mesenchymal transition (EMT) during embryonic development and in epithelial cells. Snail also mediates tumor progression and metastases. Silencing of Snail and its associate member Slug in human A2780 ovarian epithelial carcinoma cell line was investigated to identify its role in tumor neovascularization., Methods: Live cell sialidase, WST-1 cell viability and immunohistochemistry assays were used to evaluate sialidase activity, cell survival and the expression levels of tumor E-cadherin, N-cadherin, VE-cadherin, and host endothelial CD31+(PECAM-1) cells in archived paraffin-embedded ovarian A2780, A2780 Snail shRNA GIPZ lentiviral knockdown (KD) and A2780 Slug shRNA GIPZ lentiviral KD tumors grown in RAGxCγ double mutant mice., Results: Oseltamivir phosphate (OP), anti-Neu1 antibodies and MMP-9 specific inhibitor blocked Neu1 activity associated with epidermal growth factor (EGF) stimulated A2780 ovarian epithelial carcinoma cells. Silencing Snail in A2780 cells abrogated the Neu1 activity following EGF stimulation of the cells compared to A2780 and A2780 Slug KD cells. OP treatment of A2780 and cisplatin-resistant A2780cis cells reproducibly and dose-dependently abated the cell viability with a LD50 of 7 and 4 μm, respectively, after 48 h of incubation. Heterotopic xenografts of A2780 and A2780 Slug KD tumors developed robust and bloody tumor vascularization in RAG2xCγ double mutant mice. OP treatment at 50 mg/kg daily intraperitoneally did not significantly impede A2780 tumor growth rate but did cause a significant reduction of lung metastases compared with the untreated and OP 30mg/kg cohorts. Silencing Snail in A2780 tumor cells completely abrogated tumor vascularization, tumor growth and spread to the lungs in RAGxCγ double mutant mice. A2780 and A2780 Slug KD tumors expressed high levels of human N- and VE-cadherins, and host CD31+ endothelial cells, while A2780 Snail KD tumors expressed E-cadherin and reduced host CD31+ cells. OP 50mg/kg cohort tumors had reduced numbers of host CD31+ cells compared to a higher expression levels of CD31+ cells in tumors from the untreated control and OP 30mg/kg cohorts., Conclusion: Snail transcriptional factor is an important intermediate player in human ovarian tumor neovascularization.

The benefits of using thyroxine (T4) plus triiodothyronine (T3) in combination in thyroid hormone replacement are unproven but many individuals continue to be treated with this regime. When T3 is used alone for hypothyroidism, it results in wide fluctuations of thyroid hormone levels. However, only limited data exists on combined T3/T4 therapy. In this study, we have compared 24-hour profiles of thyroid stimulating hormone (TSH), free T4 (fT4) and free T3 (fT3) and cardiovascular parameters in 10 hypothyroid patients who had been on once daily combined T3/T4 therapy for more than 3 months with 10 patients on T4 alone. Twenty patients, who were part of a larger study, investigating the long-term benefits of combined T3/T4 therapy, were recruited into this sub-study. Over 24-hours, 12 samples were taken for thyroid hormones. Their 24-hour pulse and BP is also monitored on a separate occasion. On T4 alone, a modest 16% rise in fT4 with no change in fT3 was seen in the first 4-hours post-dose. In contrast, on combined treatment, fT3 levels showed a marked rise of 42% within the first 4-hours post-dose (T3/T4:T4=6.24: 4.63 mU/L, p<0.001). Mean exposure to fT3 calculated by area under the curve (AUC) was higher (T3/T4:T4=1148:1062, p<0.0001) on T3. Circadian rhythm of TSH was maintained on both treatments. No difference in pulse or blood pressure over the 24-hours was seen between the groups. Our data suggests that despite chronic combined T3/T4 therapy, wide peak-to-trough variation in fT3 levels persists. Although no immediate cardiovascular effects were seen, the long-term consequences for patients on combined therapy are unknown.

Conflicting results have recently been published about the benefits of combined T(4) and T(3) in treating hypothyroid patients. However, these studies may have been underpowered to detect differences in psychological well-being specifically related to T(4) replacement. We conducted a large, double-blind, randomized controlled trial of partial substitution of 50 microg T(4) by 10 microg T(3) vs. the original dose of T(4) in 697 hypothyroid patients. Thyroid function showed a rise in TSH (132%), a fall in free T(4) (35%, P < 0.001), and unchanged basal free T(3) levels (P = 0.92). At 3 months, there was a large (39%) placebo effect improvement in psychiatric caseness defined by the General Health Questionnaire (GHQ) 12 score in the control group compared with baseline, and this was sustained at 12 months. Differences vs. the intervention (T(3)) group were more modest with improvements in GHQ caseness (odds ratio, 0.61; 95% confidence interval, 0.42, 0.90; P = 0.01) and Hospital Anxiety and Depression questionnaire-anxiety scores at 3 months (P < 0.03) but not GHQ Likert scores, Hospital Anxiety and Depression questionnaire-depression, thyroid symptoms, or visual analog scales of mood and the initial differences were lost at 12 months. These results may be consistent with a subgroup of patients showing transient improvement after partial substitution with T(3) but do not provide conclusive evidence of specific benefit from partial substitution of T(4) by T(3) in patients on T(4) replacement. They also emphasize the large and sustained placebo effect that can follow changes in thyroid hormone administration.

In order to assess the effectiveness of calcium sulphate (plaster of Paris; POP) as a substitute for autologous bone graft, we performed lumbar intervertebral fusion in mature sheep using POP and a variety of other graft materials, and reviewed the literature. The osteoconductivity of the POP grafts was compared to that of grafts carried out with autogenous iliac crest, frozen allogeneic bone, and ProOsteon 500 coralline bone. We also compared the osteogenicity of POP to admixtures of autogenous iliac crest bone with POP and coralline bone, and to an osteoinductive demineralized sheep bone preparation (DBM). The substrates were loaded into tubular titanium mesh, implanted into excavated disc spaces and recovered after a period of 4 months. Fusion mass segments tested in flexion and tension showed that POP was equal to autogenous bone and most other substrates. The POP fusions were significantly tougher than the DBM fusions, even though histomorphometry failed to reveal differences in the amount of trabecular bone. We conclude that POP can be used to achieve a biomechanically stable interbody lumbar vertebral fusion. In addition, our literature review indicated that POP can be used as a vehicle for local delivery of antibiotics in bone infections.

Electron microscopic techniques have been used to profile the morphologies of marrow sacs in different laboratory species. These structures all comprise a condensed layer of overlapping fibroblast-like stromal cells and apparently confine the medullary and endosteal osteoblast/lining cells to separate histiotypic compartments. There were some variations in the morphology of the sac cells in the different species. In rats, cats, and sheep, scanning electron microscopy (SEM) showed a seamless arrangement of marrow sac cells which resembled a thin, flat simple squamous epithelium; they displayed few intercellular cytoplasmic processes. In the rabbit and pigeon, the sac comprised a more woven, multilayered fabric of broadly elongate flat fibroblast-like cells which displayed numerous intercellular processes. Transmission electron microscopy (TEM) showed that all marrow sac cells were attenuated with elongated nuclei, a few small round mitochondria, and a sparse rough endoplasmic reticulum. In the majority of animals, the sac was one to two cell layers thick. The rabbit and pigeon sacs were multilayered, and never less than three to four cells deep. The cell layers were not closely apposed. Tight or gap junctions were absent at the points of intercellular contact. These morphological results suggest that marrow sacs are common elements of the vertebrate skeleton with species specific morphologies., (Copyright 2000 Wiley-Liss, Inc.)

To investigate potential effects of endogenous parathyroid hormone-related peptide (PTHrP) on osteoblast function, ROS 17/2.8 cells were transfected with full-length PTHrP cDNA in a sense or antisense orientation to alter PTHrP production. Compared with vector-transfected control cells, PTHrP-overproducing (sense-transfected) cells showed increased DNA synthesis ([(3)H]-thymidine incorporation) and increased growth (cell number). The extent of apoptosis was compared for the different clones using the terminal deoxynucleotide-mediated dUTP nick-end-labeling assay (TUNEL) and Hoechst staining. No differences in percentages of apoptotic cells were found under basal culture conditions or after 3 days of serum deprivation, which, itself, markedly increased numbers of apoptotic cells. The effect of PTHrP on osteoblast differentiation was assessed by examining two protein markers of differentiation, alkaline phosphatase, and bone morphogenetic protein (BMP)-2. Alkaline phosphatase activity was decreased in sense-transfected cells and increased in antisense-transfected cells, compared with cells transfected with empty vector. PTHrP-overproducing cells also showed decreased numbers of BMP-2-positive cells, whereas antisense-transfected cells showed no difference compared with vector control. The results indicate that: (a) endogenously produced PTHrP can increase growth of these osteoblastic cells by stimulating proliferation while not affecting apoptosis; and (b) the increased cell proliferation produced by PTHrP was accompanied by a reduction in activity or amount of two proteins normally expressed by differentiated osteoblasts.

Compartment syndrome is well documented in the literature. Neoplasia as a cause is a rare. We report a patient with known metastatic malignant melanoma presenting with a compartment syndrome of the arm caused by a relatively slow growing, non-invasive metastatic deposit. This was excised and the patient made an uneventful recovery., (Copyright 2000 The British Association of Plastic Surgeons.)

Study Design: In adult female sheep, histologic and biomechanical criteria were used to determine whether the osteoconductive performance of plaster of paris would promote the incorporation of the tubular titanium mesh implants used for interbody vertebral fusions., Objectives: To compare the osteogenicity of plaster of paris with that of autogenous iliac crest bone and bone marrow 6 months after they were loaded into tubular titanium mesh cages and implanted as L3-L5 bridges after L4 corpectomies., Summary of Background Data: One of the aims of surgery for vertebral pathology is to stabilize the spine by interbody fusions. The morbidity associated with the use of iliac crest autograft bone for fusion grafts prompted trials using plaster of paris as an osteoconductive substrate., Methods: The total volume of bone that invested the L3-L5 mesh cages after 6 months was quantitated by computed tomography scans. All specimens subsequently were cut into fusion mass segments for biomechanical testing in flexion, extension, compression, and torsion, and then embedded in plastic for sectioning and histomorphometry to determine the trabecular bone volume within the titanium mesh., Results: In each experimental model, implants of plaster of paris were the osteoconductive equal of autogenous iliac crest bone/marrow preparations. The volumes of bone formed around and within the titanium mesh were identical, and the tissues were biomechanically indistinguishable. A partial mechanism was determined by modifying the system for midshaft femoral defects., Conclusions: In the sheep, a tubular titanium mesh packed with plaster of paris forms an osteoconductive conduit to achieve a biomechanically stable interbody lumbar vertebral fusion.

Undifferentiated or differentiated human trabecular bone cells with osteogenic capacity in primary culture express oxytocin receptors (OTRs). OTR expression then persists upon differentiation to an osteoblast phenotype. A human epithelial osteosarcoma cell line, Saos-2, also expresses OTRs. Expression was determined both at mRNA and protein levels. Functional OTRs are evidenced by an increase in intracellular calcium concentration, [Ca2+]i, in response to 10 nM oxytocin (OT). An oxytocin antagonist (OTA) blocked this effect, demonstrating specificity for OT. OT also stimulated prostaglandin E2 (PGE2) synthesis in both confluent undifferentiated and differentiated human trabecular bone cells. This is the first report of OTR mRNA and protein expression and of prescribed OT signal pathways in osteoblastic cells. Since PGE2 has been shown to increase bone turnover in favor of bone formation, OT may be a new class of a bone anabolic agent.

The effects of tail suspension hypokinesia on the gene expression for TGF-beta2 at different sites within bone were evaluated. TGF-beta2 mRNA signal levels were determined quantitatively by an image analysis system. The osteopenia induced by tail suspension was verified by histomorphometry. In the periosteum of nonsuspended control rats, TGF-beta2 mRNA was highly expressed in the preosteoblasts and osteoblast-rich cambial layers; very little signal was present within the middle and outer fibroblastic layers. Gene expression was significantly reduced in suspended rats, and this was evident both in terms of the number of silver grains in unit area or length of tissue and in each osteoblast and preosteoblast. Hypokinesia also reduced the expression of TGF-beta2 mRNA level in cortical and trabecular bone osteocytes, but did not adversely affect the mRNA level in chondrocytes in growth plate. The results affirm the site-specific response of TGF-beta2 gene expression in rats, and suggest that the cortical and trabecular bone osteopenia associated with hypokinesia in rats may be associated with a deficit in osteoblastic and osteocytic TGF-beta2 level.

The present study was performed to compare vascularized and nonvascularized onlay bone grafts to investigate the potential effect of graft-to-recipient bed orientation on long-term bone remodeling and changes in thickness and microarchitectural patterns of remodeling within the bone grafts. In two groups of 10 rabbits each, bone grafts were raised bilaterally from the supraorbital processes and placed subperiosteally on the zygomatic arch. The bone grafts were oriented parallel to the zygomatic arch on one side and perpendicular to the arch on the contralateral side. In the first group, vascularized bone grafts were transferred based on the auricularis anterior muscle, and in the second group nonvascularized bone grafts were transferred. Fluorochrome markers were injected during the last 3 months of animal survival, and animals were killed either 6 or 12 months postoperatively. The nonvascularized augmented zygoma showed no significant change in thickness 6 months after bone graft placement and a significant decrease in thickness 1 year after graft placement (p < 0.01). The vascularized augmented zygoma showed a slight but statistically significant decrease in thickness 6 months after graft placement (p < 0.003), with no significant difference relative to its initial thickness 1 year after graft placement. In animals killed 6 months after bone graft placement, both the rate of remodeling and the bone deposition rate measured during the last 3 months of survival were significantly higher in the vascularized bone grafts compared with their nonvascularized counterparts (p < 0.02). By 1 year postoperatively, there were no significant differences in thickness, mineral apposition rate, or osteon density between bone grafts oriented perpendicular and parallel to the zygomatic arch. These findings indicate that the vascularity of a bone graft has a significant effect on long-term thickness and histomorphometric parameters of bone remodeling, whereas the direction of placement of a subperiosteal graft relative to the recipient bed has minimal effect on these parameters. In vascularized bone grafts, both bone remodeling and deposition are accelerated during the initial period following graft placement. Continued bone deposition renders vascularized grafts better suited for the long-term maintenance of thickness and contour relative to nonvascularized grafts.

To investigate the role of bone morphogenetic protein (BMP-2) in ossifying rat bone marrow stromal cell cultures, we determined the population of fibroblast-like stromal cells that expressed BMP-2 immunocytochemically (anti-rhBMP-2 monoclonal antibody), and compared that to alkaline phosphatase (AP) and collagen synthesis formed in culture over a 4-week period in control and dexamethasone-supplemented mineralizing media. In control media, the percentage of BMP-2-positive stromal cells (BMP-2(+)) increased from 12 to 25% within the first 4 days of culture. In mineralizing media, the level of BMP-2(+) cells was significantly increased (43-44%). The intensity of immunostaining gradually increased with time. The levels of AP were undetectable at 1 week in both control and mineralizing media, but increased gradually over the next 2 weeks and peaked at 3 weeks. ALP levels were significantly greater in cultures grown in mineralizing medium (P < 0.05 at 3 weeks, P < 0.01 at 4 weeks). Collagen synthesis peaked and was significantly greater at 3 weeks (P < 0.05) in cultures grown in mineralizing medium. The levels of AP and collagen synthesis most closely reflected the changes in the percentage of BMP-2(+) cells from 7 to 28 days. Though these changes may reflect a primary action of BMP-2 on marrow osteoprogenitor-like stromal cells, they do not exclude a mechanism that involves the induction of other members of the BMP family known to stimulate AP and collagen synthesis. We conclude that BMP-2 expression in cultures of fibroblast-like marrow stromal cells is enhanced when those cells are induced to become osteoblasts by exposure to dexamethasone.

Torsional injuries may be a precursor to intervertebral disc degeneration, but published rabbit models indicate a latent time of 6 months. We describe a rabbit model in which instability and disc degeneration appear within 3 months. Sixty-five male New Zealand rabbits underwent presurgical irradiation to inhibit heterotopic bone formation. Control animals then underwent either a soft-tissue release or facetectomy and capsulotomy, whereas experimental animals received surgery and an acute 30 degrees torsional lumbar injury. Capsulotomy, as well as facetectomy without torsion, failed to effect disc degeneration. However, the rabbits that received torsion exhibited clear indications of degenerative disc changes (thinning, increased PLA2 levels, and decreased nucleus pulposus volume) within 60-90 days. The observations associate disc degeneration with a destabilizing acute torsional injury.

The mechanism mediating the chronic pain associated with lumbar disc degeneration may involve neurotransmitters elaborated by dorsal root ganglion (DRG). This hypothesis has been tested in an applicable rabbit model of disc degeneration. Twenty control male rabbits underwent a soft-tissue release; 20 experimental rabbits sustained a facetectomy and capsulotomy and received an acute torsional lumbar injury. The levels of calcitonin gene-related peptide, vasoactive intestinal peptide, and substance P were measured in the DRG, spinal cord, and disc at 10, 30, 60, and 90 days postoperatively. Torsional injury was associated with a statistically significant increase in most DRG and spinal cord neurotransmitter values after 60-90 days. These points in time marked the periods of maximum biomechanical instability and disc narrowing. Such data support concepts about the association between chronic lumbar spinal instability, disc degeneration, and pain.

Because exposure to positively charged dextran resin (PCDR) inhibits the growth of cultured rat and human bone cells, we tested the hypothesis that PCDR might inhibit bone repair in vivo. Central physeal defects were created by drilling 3.0-mm holes from the proximal tibial plateau into the metaphysis. The defects in left tibiae were packed with neutral resin (control); those in right tibiae were filled with PCDR. At the end of the 1st, 2nd, 3rd, and 10th postoperative weeks, the outcomes were quantitated by documenting the percent trabecular bone volume within the defect. The PCDR-filled defects showed a significant decrease in trabecular bone formation as early as the 2nd week. By the 10th postoperative week, formation of trabeculae had been reduced by nearly 40%. The inhibition conferred by PCDR suggests that the resin could be used as a suppressive interpositional material.

The putitive bone-sparing effect of alendronate was tested in two animal models of osteopenia: estrogen-deficient female rats and glucocorticoid-treated male rats. In the first study, 18 female Sprague-Dawley rats, 4 months of age, were ovariectomized (OVX), and an additional 6 rats were sham-operated. The OVX rats were treated with either vehicle, 17beta-estradiol (E2) (100 microg/rat/week, s.c.), or alendronate (1 mg/kg/day, on alternate days, orally). In the second study, 24 8-month-old male Wistar rats were treated with either vehicle, methyl prednisolone (7 mg/kg once a week, s.c.), prednisolone plus testosterone (16 mg/kg once every 3 weeks, i.m.), or prednisolone plus alendronate (20 microg/kg twice a week, s.c.). Prior to treatment and at the end of the 6-week treatment period, bone mineral density (BMD) of the lumbar spine was measured by dual energy x-ray absorptiometry, and mean femur weights were calculated. The OVX rats had subnormal BMD (-3.91 +/- 1.0% vs control +5.19 +/- 3.92%, P < 0.05) and femur weights (720 +/- 6 mg vs %; 746 +/- 11 mg, P < 0.05). OVX-induced bone loss was completely abolished by the administration of E2 (7.01 +/- 2.32%, P < 0.005; 748 +/- 6 mg, P < 0.01), or alendronate (24.2 +/- 2.73%, P < 0.0001; 779 +/- 11 mg, P < 0.001). In the second study in older male rats, glucocorticoids significantly decreased BMD (-9.70 +/- 3.44% vs -1.10 +/- 1.75%, P < 0.05), and femur weight (1070 +/- 14 mg vs 1180 +/- 24 mg, P < 0.01). Concomitant administration of testosterone (BMD 4.23 +/- 1.84%, P < 0.005; femur weight 1260 +/- 56 mg, P < 0.02), or alendronate (BMD 8.18 +/- 1.36%, P < 0.001; femur weight 1360 +/- 50 mg) with prednisolone, abolished the corticosteroid-induced bone loss. Bone histomorphometry showed a 34% loss of trabecular bone volume in glucocorticoid-treated rats (P < 0.05), which was prevented with both testosterone and alendronate therapies. However, at the doses used in both models, alendronate was more efficacious than either E2 or testosterone in increasing BMD and femur weight. In summary, this study demonstrated that alendronate therapy is highly effective in counteracting the osteopenia of OVX and glucocorticoid therapy.

Purpose: This study tested the hypothesis that a significant amount of the new bone produced by heterotopic periosteal autografts is derived osteoinductively because proliferating periosteal cells express the bone morphogenetic protein (BMP)., Materials and Methods: Rabbit ulnar and radial periosteum were autografted as free grafts (FGs) to the forelimb musculature, and as millipore diffusion chambers grafts (MDCGs) to the rectus abdominus muscle. The grafts were recovered at 3, 5, 7, 14, and 28 days postoperation, fixed in 4% paraformaldehyde, demineralized in 0.6N HCL, and 4.0 microns paraffin-embedded sections were immunostained with monoclonal antibody against recombinant human (rh) BMP-2., Results: Sections from FGs recovered 5 to 28 days postoperatively exhibited cartilage and bone; fibrous tissue, cartilage, bone, and osteochondroid differentiated within MDCGs. Although BMP-2 was expressed by mesenchymal cells, osteoblasts, osteocytes, and osteoclasts, none of the MDCGs produced the osteoinductive signature of transmembrane bone formation., Conclusions: These observations indicated that the larger fraction of the new bone produced by heterotopic periosteal autografts is derived from the graft cells.

Nitric oxide (NO) has been reported to inhibit osteoclastic bone resorption. We examined the bone sparing effect of NO on prevention of corticosteroid-induced bone loss in older male rats. Recently, we demonstrated that NO donor nitroglycerin (NG) can alleviate ovariectomy-induced bone loss, and the protective effects of estrogens on bone are mediated through NO [Bone 18(4):301-304; 1996]. Therefore, we chose to study a different model (i.e., steroid-induced osteoporosis in males) to evaluate whether NG can inhibit the bone loss associated with corticosteroid therapy. Twenty-five 32-week-old male Wistar rats were randomly assigned to five groups (n = 5/group). They received either vehicle, methylprednisolone (7 mg/kg per week), NO synthase inhibitor L-NAME (25 mg/kg per day), NO donor nitroglycerin (NG, 0.2 mg twice daily), a combination of prednisolone+NG, or prednisolone plus L-NAME, respectively. Prior to treatment and at the end of the 6 week treatment period, bone mineral density (BMD) of the lumbar spine was measured by dual energy X-ray absorptiometry scanning. Administration of prednisolone significantly decreased BMD (-9.50%, p < 0.05). The group receiving NG with prednisolone (-2.34%) and the group treated with NG alone (-0.36%) were not statistically different from the control group (-0.11%). Similar to the changes in BMD, femur weights were also significantly lower in prednisolone-treated rats (1.09 +/- 0.01 g vs. 1.17 +/- 0.03 in controls; p < 0.05). However, the rats receiving prednisolone together with NG were able to maintain their femur weights (1.13 +/- 0.02). There was a reduction of 9.5% of BMD (p < 0.05) and 7.8% of femoral weight (p < 0.05) in rats treated with L-NAME. A 50%-70% reduction of the percentage trabecular bone volume in the proximal tibia and distal femur and a 50% reduction of the midshaft cortical area was seen after corticosteroid therapy, and these too were prevented by administration of NG. Here, we demonstrate, for the first time, that supplementation with a NO donor compound can counteract prednisolone-induced bone loss.

Following the signal observation that contact with positively charged dextran resin (PCDR) inhibited the growth of cultured mammary (Hs578T and MDA-MB-231), pancreatic (H2T), and myeloma (RR-658) tumor cell lines, studies were developed in the hamster cheek pouch model using hamster H2T pancreatic tumor cells to determine if the antiproliferative effect of PCDR could inhibit tumorigenesis. In these studies, the control population represented groups injected with H2T cells alone or in combination with either neutral or negatively charged resin. When cells (5 x 10(2) to 1 x 10(5)) and PCDR were administered simultaneously, the tumor incidence (percent engraftment) and growth of tumors that already had been established were significantly reduced. When PCDR was injected into already established 1-35-mm2 H2T tumors (engraftment for 21 days = 96%), the resin suppressed the growth of the smallest tumors (< 10 mm2). In none of these trials was the somatic growth of the host hamsters affected. PCDR contact with H2T cells in vitro for 4 days or used to treat growing solid tumors for 72 days significantly reduced cellular ornithine decarboxylase activity. While the mechanism of PCDR action has not been established, the observations have implications for in vivo tumor therapeutic models.

In 13 New Zealand White rabbits with a mean age of 6 months, vascularized bone transfers incorporated as paired auricular anterior myo-osseous flaps were harvested; they were placed in either an inlay or an onlay position relative to the zygomatic arch. The onlay bone transfers were placed either in full contact or in partial contact with the zygomatic arch. The animals were sacrificed 1 year after transfer. At 1 year, the inlay transfer simulated the adjacent zygoma in width and thickness. Onlay full contact transfers maintained significant aug mentation in thickness of the zygoma, while the onlay partial contact transfers did not; the thickness of the augmented zygoma in the onlay full contact subgroup was significantly greater than that in the onlay partial contact transfers. The onlay partial contact grafts had remodeled into the zygoma in bone contact, where the orientation of mismatched osteons within the bone transfers had transformed to match that of the native zygoma. In areas of bone contact between the onlay and the host bone, full-thickness conversion from a cortical to a trabecular architecture had occurred in both the transfer and host bones. These findings have numerous implications regarding mechanisms that could be exploited clinically to optimize the survival of a bone transfer; they also raise questions regarding alteration of the recipient bed after placement of an onlay bone transfer.

The fibroblast-like cells in the marrow stromal system were separated from endothelial cells and macrophages by negative selection of magnetic beads. Immunocytochemistry confirmed that these fibroblast-like cells expressed fibronectin and collagen Type III, but not Factor VIII and epithelial membrane antigen (endothelial cell markers) or Mac I (macrophage marker). The fibroblast-like stromal cells (FSC) synthesized the insulin-like growth factors (IGF)-I and -II in amounts equivalent to that produced by unfractionated marrow stromal cells (UMSC); in both, the concentration of IGF-II was 10 times higher than that of IGF-I. Northern analysis revealed that FSC and UMSC expressed identical patterns of mRNAs for IGF-I and transforming growth factor (TGF) -beta 2, for osteopontin, and for procollagen Types I and III (Type I > Type III). Type II procollagen mRNA was not expressed in both cell populations. The TGF-beta 2 gene mRNA was expressed at a lower level by the FSC than UMSC. The pattern of gene expression in these cells is consistent with an osteoprogenitor phenotype. Both FSCs and UMSCs express parathyroid hormone (PTH) and estrogen receptor genes (rtPCR technique). The study provides additional evidence that fibroblast-like marrow stromal cells have an osteoblast signature, and that they are largely responsible for the osteogenic performance of cells in unfractionated marrow.

We previously reported that bone marrow stromal cells produce insulin-like growth factors (IGF-I and -II), and that medium conditioned by marrow stromal cells stimulates osteoblast proliferation in vitro. The present study employed the rat tail-suspension model to unload the hindlimbs. It was designed to test the hypothesis that the development of osteopenia or osteoporosis could be due to a deficit in the osteogenic function of marrow stromal cells. Although tail suspension suppressed body weight during the first 3 days of an 11-day pair-fed study, the overall weight gain recorded by these animals was normal. Nevertheless, bone growth was inhibited by suspension. Similarly, the total adherent marrow stromal cell population harvested from the femurs and tibias was decreased by tail suspension, and only half the normal number of fibroblastic stromal cell colonies grew when they were cultured. The proliferation of alkaline-phosphatase-positive cells in the stroma was also inhibited. Northern hybridization revealed that the messenger RNA level for transforming growth factor-beta 2 and IGF-II in stromal cell was reduced by tail suspension. The production of IGF-II by marrow stromal cells was also decreased. The steady-state level of five different transcript sizes of IGF-I mRNA was altered differentially by tail suspension. Osteopontin mRNA was also reduced in marrow stromal cells from tail-suspended rats compared with the normal rats. These data suggest that skeletal unloading not only alters the mRNA level for growth factors and peptide production, but also affects the proliferation and osteogenic differentiation of marrow stromal cells. These changes may be responsible for the reduced bone formation in osteopenia and osteoporosis.

A C-terminal analog of parathyroid hormone-related peptide (PTHrP), PTHrP(107-139), was found to stimulate cAMP production in three osteoblast cell preparations. The effect was studied most extensively in ROS 17/2.8 cells. The effect was dose-related and comparable in magnitude to that produced by PTHrP(1-34), but potency was lower. The functional significance of the cAMP effect is unknown, but preliminary findings indicated that PTHrP(107-139) also inhibited osteopontin mRNA levels in ROS 17/2.8 cells treated with peptide for 48 h. The results suggest that the carboxy-terminal region of PTHrP may play a role in bone metabolism by influencing osteoblast activity.

The osteogenic response to subperiosteal injection of negatively charged ion exchange resins was compared in the tibiae of one-month and 16- to 22-month-old rats. The resins were administered either in the form of beads (CM Sephadex) or as particles (CM cellulose, carboxymethylcellulose), and the animals were killed at two weeks and at one month after injection. Histologically, the resins did not produce an inflammatory response. Periosteal bone formation was observed wherever resin was in contact with bone, and in the resin bed the connective tissues that invested the charged materials ossified within the first month. Marrow spaces commonly formed where periosteal growth was most rapid. The osteogenic effect was independent of resin conformation, and it was more pronounced in the younger rats.

The adjunctive role of demineralized bone matrix (DBM) in enhancing the incorporation of segmental freeze dried (FD)-allogeneic bone grafts has been studied in the rabbit ulna and fibula. The studies compared the healing patterns of fresh segmental autografts, FD-allografts, DBM-allografts, and FD-allografts supplemented at the graft-host junctions with FD- or DBM-allografts as particulates (ulna) or as segmental struts (fibula). The outcome was evaluated at five and ten weeks by a radiologic score, by biomechanical properties (breaking strength, energy to failure, stiffness), and histology. The ulnar autografts healed most rapidly (ten weeks = 100%), followed by DBM allografts (60%). By all criteria, FD-allografts were poorly incorporated (20-40%), and the process was not improved by supplements of FD- or DBM-particulate/strut bone. Histologically, the DBM component of composite fibular strut grafts was osteoinductive and united with host tissues within 35 days. The contiguous FD-allograft struts were not incorporated, showing fibrocartilaginous nonunions and resorptive foci. While the addition of DBM does not protect FD-allograft integrity in the rabbit, segmental mineralized FD-allografts could provide mechanical support for some intervals until, in such composite grafts, osteoinductive processes produced biomechanically competent new bone.

The anatomical relationships between the fiber tracts and bone lining cells within the rat calvarial periosteum have been studied by electron microscopy. Classical Sharpey's fibers were not observed in this location. Rather, thin unmineralized fibers originating from the periosteum traversed the cambial layer and passed to the bone surface between individual osteoblasts or groups of osteoblasts. The organizational relationship suggests that the osteogenic calvarial cell populations are compartmentalized into domains that might be particularly sensitive and responsive to the biomechanical forces of masticatory muscles.

Aluminum (Al) loading is associated with reduced bone formation and osteomalacia in human and certain animal models. However, uncertainty exists as to the cellular effect(s) of Al as both inhibition and stimulation of osteoblast proliferation have been reported. Furthermore, the extent to which Al affects osteoprogenitor cell populations is unknown. To determine the cellular effects of Al in the rat, an animal model in which Al bone disease has been produced, we compared the in vitro effect of 10-50 microns Al on the proliferation and hydroxyproline collagen formation of marrow osteoprogenitor stromal cell populations and perinatal rat calvarial osteoblasts. In subconfluent cultures, Al suppressed proliferation of both marrow fibroblast-like stromal cells and calvarial osteoblasts. In confluent cultures, however, Al selectively stimulated periosteal fibroblast and osteoblast DNA synthesis and collagen (hydroxyproline) production, both in the presence or absence of 1,25-dihydroxyvitamin D. Osteocalcin was not detected in osteoblast-conditioned media or extracellular matrix. These observations suggest that the bone formation defect associated with Al toxicity in growing rats may be a function of impaired patterns of osteoprogenitor/osteoblast proliferation. Furthermore, the Al-stimulated increase in collagen formation is consistent with the development of osteomalacia in Al-toxic humans and animals. The mechanism by which Al stimulated DNA synthesis and collagen production in more mature cultures awaits further study.

Decreased muscular activity results in weakness and muscular atrophy. Coincident with this protein catabolic state is glucose intolerance and hyperinsulinemia. Rats were tail suspended for 7 to 14 days to accomplish unloading of the hindlimbs. Insulin resistance was documented in these animals by a 14 day tail suspension-related 26% increase in serum glucose in spite of a 253% increase in serum insulin concentration. Microsomal membranes were prepared from hindlimb muscles and specific binding of insulin and insulin-like growth factor I (IGF-I) were determined in these membranes. Insulin binding was decreased by 27% at 7 days and by 21% at 14 days. In contrast, IGF-I binding was unchanged at 7 days and was increased by 24% at 14 days. Liver membrane insulin receptors also had declined by 14 days of suspension, suggesting that the change in insulin receptors was a generalized, humorally-mediated phenomenon. These data suggest that tail suspension in rats results in insulin resistance, hyperinsulinemia, a decline in insulin receptors in liver and muscle, and a relative increase in muscle membrane IGF-I receptors. These data are consistent with the hypothesis that resistance to insulin's effects on protein metabolism in skeletal muscle may contribute to the protein catabolism associated with decreased muscular activity.

The pathogenesis of nutritional rickets is not well-understood. While the etiologies include deficiencies of vitamin D, calcium (Ca) or phosphate (PO4), and perhaps aluminium toxicity, the role these nutrients play in the development of tissue level anomalies characteristic of rachitic cartilage and bone has yet to be defined. Reported alterations in the biochemistry of rachitic bone and cartilaginous matrix which could adversely affect mineralization and endochondral ossification are of questionable significance since the tissues mineralize rapidly when exposed to Ca and PO4 salts in vivo and in vitro. The low Ca and PO4 concentrations of the extracellular fluid (ECF) bathing rachitic cartilage and bone matrix suggest that local mechanisms operate to impair mineralization. In healing rickets, the Ca and PO4 content of these tissue fluids increases in the same time-frame it takes to experimentally remineralize the matrices. However, it is not certain what determines the Ca and PO4 content of the ECF. Cytokines which may play a role in the cellular regulation of Ca and PO4 and maintain processes which contribute to normal patterns of endochondral ossification could provide a mechanism common to the pathogenesis of rickets from a variety of causes.

A large animal model to assess fetal fracture repair and the ability to close excisional bony defects is presented. Incisional and excisional ulnar fractures were made in 14 midgestation fetal lambs, harvested at serial time points, and subjected to high-resolution low-kilovolt magnification radiographs, magnetic resonance imaging scans, and histologic analysis. Fetal fracture healing was characterized by early closure of excisional defects and rapid fracture healing with minimal or no soft-tissue inflammation or callus formation. Magnetic resonance imaging scans of the fractures revealed a characteristic pattern compatible with the histologic findings, namely, minimal inflammation in soft tissue adjacent to the fracture site. Histologic and magnification radiographic findings indicated that complete bony repair occurred within 21 days in incisional defects and within 40 days in excisional defects. In both cases, healed fetal bone resembled normal bone matrix. Excisional defects, including periosteum, of greater than three times the width of the bony cortex closed rapidly with virtually normal-appearing bony matrix and with minimal or no callus formation.

Fibroblast-like marrow stromal cells are believed to play a role in the maintenance of osteoblast populations at the marrow-bone interface. We now report that this interaction may be very specific. Stromal cell conditioned medium (SC-CM) stimulated DNA synthesis and proliferation in culture of neonatal rat calvarial osteoblasts at low concentrations (1.25-5%), but was inhibitory at 10%. At growth promoting effective concentrations, the activity of osteoblast alkaline phosphatase was decreased. This action was selective since SC-CM failed to promote the growth of rat calvarial fibroblasts. Characterization of the SC-CM indicated the cells produced IGF-I and -II and a wide range of molecular weight fractions with putative stimulatory action (FPLC analysis using Superose 12 and 6 gel permeation columns). HPAE-PAD analysis showed that some elements were glycosylated, and the composition suggested the presence of N- and O-linked oligosaccharide chains. Because rat marrow stromal fibroblast-like cells produce a number of osteotropic factors which affect calvarial osteoblast growth, these interactions may be important to considerations about the etiology of the osteoporoses.

Fibroblast-like rat marrow stromal cell (CFU-F) cultures have been characterized in terms of their responsiveness to calciotropic hormones, metal ions, the nonsteroidal antiinflammatory drug, and by their putative paracrine role in the maintenance of active populations of osteoblasts at the marrow-bone interface. These studies indicate that CFU-Fs lack a complete osteoblast signature. Subconfluent CFU-Fs grown in the presence or absence of 10(-7) M dexamethasone lack receptors for PTH and calcitonin, and fail to show enhanced cAMP or cGMP responses to 10(-7) M 1-34 PTH (rat), or any evidence of osteocalcin production [+/- 10(-9) M 1,25-(OH)2D3]. Low concentrations of fluoride [10(-12) and 10(-9) M] stimulated CFU-F grown in vitro in serum-free media, though higher levels (10(-7) and 10(-6) M), inhibited growth in vivo and in vitro. Aluminum (10(-12)-10(-7) M) and ibuprofen (10(-7) M) did not alter normal growth patterns, indicating an action on bone cells more differentiated than CFU-Fs. Serum-free conditioned medium (CM) from control and ovariectomized (OVX)/OVX+ dihydrotachysterol-Rx rat CFU-F cultures was mitogenic for neonatal rat calvarial osteoblasts in vitro, but not for ROS 17/2.8 cells. The studies affirm the mesenchymal-like character of CFU-Fs and project their significant role in sustaining functional endosteal osteogenic cell populations.