Louis S. Kidder

Osteogenic protein-1 induced bone formation in an infected segmental defect in the rat femur.

The goal of this study was to use a segmental defect model in the rat femur to determine if osteogenic protein‐1 (OP‐1) is capable of inducing bone formation in the presence of bacterial contamination. A 6 mm segmental defect was surgically created and stabilized with a polyacetyl plate and Kirschner wires in one femur in each of 126 Sprague–Dawley rats. The animals were divided into eight groups in which the defect was either left untreated, or subjected to various combinations of OP‐1 (11 or 50 μg), lyophilized bovine type I collagen (carrier for the OP‐1), and 105 colony‐forming units of Staphylococcus aureus. The animals were euthanized at either 2, 4, or 9 weeks. Quantitative radiographic and histologic analyses were performed on the harvested tissue. The initial contamination progressed to infection in all animals receiving bacteria, as determined by qualitative bacteriology. There was very little, if any, bone formation in the untreated defects, and in the contaminated defects with or without collagen carrier. Bone formation was significantly greater in contaminated defects with either dose of OP‐1, compared with contaminated defects without OP‐1. The 50 μg dose of OP‐1 induced significantly more bone formation than the 11 μg dose, both with and without bacteria. This investigation has demonstrated that OP‐1 maintains its osteoinductive capability in a contaminated segmental defect. OP‐1 may potentially be used in the clinical management of contaminated fractures.

Moderate Alcohol Consumption Suppresses Bone Turnover in Adult Female Rats

Chronic alcohol abuse is a major risk factor for osteoporosis but the effects of moderate drinking on bone metabolism are largely uninvestigated. Here, we studied the long‐term dose‐response (0, 3, 6, 13, and 35% caloric intake) effects of alcohol on cancellous bone in the proximal tibia of 8‐month‐old female rats. After 4 months of treatment, all alcohol‐consuming groups of rats had decreased bone turnover. The inhibitory effects of alcohol on bone formation were dose dependent. A reduction in osteoclast number occurred at the lowest level of consumption but there were no further reductions with higher levels of consumption. An imbalance between bone formation and bone resorption at higher levels of consumption of alcohol resulted in trabecular thinning. Our observations in rats raise the concern that moderate consumption of alcoholic beverages in humans may reduce bone turnover and potentially have detrimental effects on the skeleton.

Partial characterization of rat marrow stromal cells

SummaryFibroblast-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 grownin vitro in serum-free media, though higher levels (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 osteoblastsin 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.

Effects of orbital spaceflight on human osteoblastic cell physiology and gene expression

During long-term spaceflight, astronauts lose bone, in part due to a reduction in bone formation. It is not clear, however, whether the force imparted by gravity has direct effects on bone cells. To examine the response of bone forming cells to weightlessness, human fetal osteoblastic (hFOB) cells were cultured during the 17 day STS-80 space shuttle mission. Fractions of conditioned media were collected during flight and shortly after landing for analyses of glucose utilization and accumulation of type I collagen and prostaglandin E(2) (PGE(2)). Total cellular RNA was isolated from flight and ground control cultures after landing. Measurement of glucose levels in conditioned media indicated that glucose utilization occurred at a similar rate in flight and ground control cultures. Furthermore, the levels of type I collagen and PGE(2) accumulation in the flight and control conditioned media were indistinguishable. The steady-state levels of osteonectin, alkaline phosphatase, and osteocalcin messenger RNA (mRNA) were not significantly changed following spaceflight. Gene-specific reductions in mRNA levels for cytokines and skeletal growth factors were detected in the flight cultures using RNase protection assays. Steady-state mRNA levels for interleukin (IL)-1alpha and IL-6 were decreased 8 h following the flight and returned to control levels at 24 h postflight. Also, transforming growth factor (TGF)-beta(2) and TGF-beta(1) message levels were modestly reduced at 8 h and 24 h postflight, although the change was not statistically significant at 8 h. These data suggest that spaceflight did not significantly affect hFOB cell proliferation, expression of type I collagen, or PGE(2) production, further suggesting that the removal of osteoblastic cells from the context of the bone tissue results in a reduced ability to respond to weightlessness. However, spaceflight followed by return to earth significantly impacted the expression of cytokines and skeletal growth factors, which have been implicated as mediators of the bone remodeling cycle. It is not yet clear whether these latter changes were due to weightlessness or to the transient increase in loading resulting from reentry.

Pancreas Oxygen Persufflation Increases ATP Levels as Shown by Nuclear Magnetic Resonance

BACKGROUND Islet transplantation is a promising treatment for type 1 diabetes. Due to a shortage of suitable human pancreata, high cost, and the large dose of islets presently required for long-term diabetes reversal; it is important to maximize viable islet yield. Traditional methods of pancreas preservation have been identified as suboptimal due to insufficient oxygenation. Enhanced oxygen delivery is a key area of improvement. In this paper, we explored improved oxygen delivery by persufflation (PSF), ie, vascular gas perfusion. METHODS Human pancreata were obtained from brain-dead donors. Porcine pancreata were procured by en bloc viscerectomy from heparinized donation after cardiac death donors and were either preserved by either two-layer method (TLM) or PSF. Following procurement, organs were transported to a 1.5-T magnetic resonance (MR) system for (31)P nuclear magnetic resonance spectroscopy to investigate their bioenergetic status by measuring the ratio of adenosine triphosphate to inorganic phosphate (ATP:P(i)) and for assessing PSF homogeneity by MRI. RESULTS Human and porcine pancreata can be effectively preserved by PSF. MRI showed that pancreatic tissue was homogeneously filled with gas. TLM can effectively raise ATP:P(i) levels in rat pancreata but not in larger porcine pancreata. ATP:P(i) levels were almost undetectable in porcine organs preserved with TLM. When human or porcine organs were preserved by PSF, ATP:P(i) was elevated to levels similar to those observed in rat pancreata. CONCLUSION The methods developed for human and porcine pancreas PSF homogeneously deliver oxygen throughout the organ. This elevates ATP levels during preservation and may improve islet isolation outcomes while enabling the use of marginal donors, thus expanding the usable donor pool.

A controlled experimental model of revision implants: Part II. Implementation with loaded titanium implants and bone graft.

We used an experimental model producing an aggressive tissue response associated with implant loosening in humans: a 6 mm polymethylmethacrylate (PMMA) cylinder was pistoning 500 w m concentrically in a 7.5 mm hole, with polyethylene (PE) particles, for 8 weeks. At 8 weeks, the PMMA implant was revised with a titanium alloy (Ti) implant, and an identical primary Ti implant was inserted contralaterally for 4 weeks. With this protocol, we evaluated primary and revision plasma-sprayed Ti implants which were loaded under stable conditions with or without allograft, or under unstable conditions without allograft (bilateral primary and revision implants, n 8 per group, 48 implants in 24 dogs). Revision implants had lower interfacial shear strength, less bone in contact with and adjacent to the implant, and resulted in higher levels of IL-6 g and TNF f and lower levels of TGF g . In both the revision and primary settings, allograft increased shear strength, stiffness and energy, bone-implant contact, and bone area adjacent to the implant. Unstable implants could not generate a mechanically sound interface, and further exacerbated the difference between primary and revision. We conclude that factors important for improving the fixation of revision implants were bone graft and a stable interface.

Persufflation Improves Pancreas Preservation When Compared With the Two-Layer Method

Islet transplantation is emerging as a promising treatment for patients with type 1 diabetes. It is important to maximize viable islet yield for each organ due to scarcity of suitable human donor pancreata, high cost, and the large dose of islets required for insulin independence. However, organ transport for 8 hours using the two-layer method (TLM) frequently results in low islet yields. Since efficient oxygenation of the core of larger organs (eg, pig, human) in TLM has recently come under question, we investigated oxygen persufflation as an alternative way to supply the pancreas with oxygen during preservation. Porcine pancreata were procured from donors after cardiac death and preserved by either TLM or persufflation for 24 hours and subsequently fixed. Biopsies collected from several regions of the pancreas were sectioned, stained with hematoxylin and eosin, and evaluated by a histologist. Persufflated tissues exhibited distended capillaries and significantly less autolysis/cell death relative to regions not exposed to persufflation or to tissues preserved with TLM. The histology presented here suggests that after 24 hours of preservation, persufflation dramatically improves tissue health when compared with TLM. These results indicate the potential for persufflation to improve viable islet yields and extend the duration of preservation, allowing more donor organs to be utilized.

Effects of Growth Hormone and Low Dose Estrogen on Bone Growth and Turnover in Long Bones of Hypophysectomized Rats

Abstract. Pituitary hormones are recognized as critical to longitudinal growth, but their role in the radial growth of bone and in maintaining cancellous bone balance are less clear. This investigation examines the histomorphometric effects of hypophysectomy (Hx) and ovariectomy (OVX) and the subsequent replacement of growth hormone (GH) and estrogen (E), in order to determine the effects and possible interactions between these two hormones on cortical and cancellous bone growth and turnover. The replacement of estrogen is of interest since Hx results in both pituitary and gonadal hormone insufficiencies, with the latter being caused by the Hx-associated reduction in follicle stimulating hormone (FSH). All hypophysectomized animals received daily supplements of hydrocortisone (500 μg/kg) and l-thyroxine (10 μg/kg), whereas intact animals received daily saline injections. One week following surgery, hypophysectomized animals received either daily injections of low-dose 17 β-estradiol (4.8 μg/kg s.c.), 3 X/d recombinant human GH (2 U/kg s.c.), both, or saline for a period of two weeks. Flurochromes were administered at weekly intervals to label bone matrix undergoing mineralization. Whereas Hx resulted in reductions in body weight, uterine weight, and tibial length, OVX significantly increased body weight and tibial length, while reducing uterine weight. The combination of OVX and Hx resulted in values similar to Hx alone. Treatment with GH normalized body weight and bone length, while not affecting uterine weight in hypophysectomized animals. Estrogen increased uterine weight, while not impacting longitudinal bone growth and reduced body weight. Hypophysectomy diminished tibial cortical bone area through reductions in both mineral appositional rate (MAR) and bone formation rate (BFR). While E had no effect, GH increased both MAR and BFR, though not to sham-operated (control) levels. Hypophysectomy reduced proximal tibial trabecular number and cancellous bone area, and increased trabecular separation. Both GH and E reduced cancellous osteopenia, although employing different mechanisms. GH reduced the decrease in trabecular thickness, whereas E reduced the decrease in trabecular number and the increase in trabecular separation. Hypophysectomy reduced both Tb.MAR and Tb.BFR while treatment with GH enhanced them. This investigation has shown that Hx and GH have a dramatic impact on selected static and dynamic indices of rat cortical and cancellous histomorphometry. Furthermore, the mechanisms of action of GH and E differ, and suggest that some of the skeletal changes associated with Hx are caused by deficiencies in estrogen as well as deficiencies in growth hormone.

Calcium metabolism and bone mineralization in female rats fed diets marginally sufficient in calcium: effects of increased dietary calcium intake

Experiments were carried out to determine the ability of female rats with poorly mineralized skeletons to increase bone mineralization in response to increased dietary Ca consumption. We specifically addressed this question with regard to two different periods of the life cycle: the period of sexual maturation (6-9 weeks of age), and in animals that had attained adult rates of skeletal mineralization (100 days of age). We found that at both stages, increased dietary Ca consumption resulted in increased trabecular bone volume and total bone Ca. In the younger animals, it was found that dietary history influenced the disposition of bone mineral. Animals that were initially Ca-deprived exhibited increased trabecular bone and decreased cortical thickness compared to animals continuously fed 0.5% Ca. Ovariectomy of mature animals reduced but did not eliminate the response to increased Ca intake.

Continuous Real-time Viability Assessment of Kidneys Based on Oxygen Consumption

BACKGROUND Current ex vivo quality assessment of donor kidneys is limited to vascular resistance measurements and histological analysis. New techniques for the assessment of organ quality before transplantation may further improve clinical outcomes while expanding the depleted deceased-donor pool. We propose the measurement of whole organ oxygen consumption rate (WOOCR) as a method to assess the quality of kidneys in real time before transplantation. METHODS Five porcine kidneys were procured using a donation after cardiac death (DCD) model. The renal artery and renal vein were cannulated and the kidney connected to a custom-made hypothermic machine perfusion (HMP) system equipped with an inline oxygenator and fiber-optic oxygen sensors. Kidneys were perfused at 8 degrees C, and the perfusion parameters and partial oxygen pressures (pO(2)) were measured to calculate WOOCR. RESULTS Without an inline oxygenator, the pO(2) of the perfusion solution at the arterial inlet and venous outlet diminished to near 0 within minutes. However, once adequate oxygenation was provided, a significant pO(2) difference was observed and used to calculate the WOOCR. The WOOCR was consistently measured from presumably healthy kidneys, and results suggest that it can be used to differentiate between healthy and purposely damaged organs. CONCLUSIONS Custom-made HMP systems equipped with an oxygenator and inline oxygen sensors can be applied for WOOCR measurements. We suggest that WOOCR is a promising approach for the real-time quality assessment of kidneys and other organs during preservation before transplantation.