Twenty-four hour hormone profiles of TSH, Free T3 and free T4 in hypothyroid patients on combined T3/T4 therapy.

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.

Plaster of Paris as bone substitute in spinal surgery.

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.

Comparative morphology of the marrow sac.

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.

Endogenous parathyroid hormone-related peptide enhances proliferation and inhibits differentiation in the osteoblast-like cell line ROS 17/2.8.

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 complicating metastatic malignant melanoma.

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.

Plaster of Paris as an osteoconductive material for interbody vertebral fusion in mature sheep.

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.

Functional oxytocin receptors discovered in human osteoblasts.

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.

Transforming growth factor-beta2 mRNA level in unloaded bone analyzed by quantitative in situ hybridization.

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.

Long-term remodeling of vascularized and nonvascularized onlay bone grafts: a macroscopic and microscopic analysis.

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.

Expression of BMP-2 by rat bone marrow stromal cells in culture.

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 injury resulting in disc degeneration: I. An in vivo rabbit model.

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.

Torsional injury resulting in disc degeneration in the rabbit: II. Associative changes in dorsal root ganglion and spinal cord neurotransmitter production.

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.

Winner of the AlbertTrillat Young Investigator Award. The effects of aggressive notchplasty on the normal knee in dogs.

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.

Prevention of corticosteroid-induced bone loss with alendronate.

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.

Positively charged dextran resin inhibits trabecular bone repair in the rabbit tibial physis.

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 origin of bone formed by heterotopic periosteal autografts.

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.

Prevention of corticosteroid-induced bone loss with nitric oxide donor nitroglycerin in male rats.

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.

Antitumor effect of positively charged resin in the hamster cheek pouch model.

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.

Burn-associated bone disease in sheep: roles of immobilization and endogenous corticosteroids.

To determine the role of immobilization in the pathogenesis of burn-associated bone disease, we selected the sheep as a model to study the effects of burn injury compared with a sham-burned control group. Seven of the sheep were subjected to controlled 40% flame burn, and seven underwent anesthesia with arterial and venous cannulation but without burn. After labeling newly formed bone with tetracycline and calcein, the sheep were killed 2 weeks after burn or sham burn, and the iliac crest and lumbar vertebrae were analyzed for histomorphometry. Analysis failed to demonstrate a significant reduction of bone formation rate in the burned sheep. Osteoid area and surface and osteoblast surface, which correlated significantly with bone formation rate (r = 0.49, p < 0.025), were reduced in the burned sheep. Results suggest that immobilization may play a primary role in the pathogenesis of burn-associated bone disease, but the presence of differences in other histomorphometric features indicates the bone disease is multifactorial.