The effects of sustained delivery of alendronate on the kidney in ovariectomized female rats.

Bisphosphonates are indicated for the treatment and prevention of osteoporosis in adults; the treatment and prevention of glucocorticoid-induced osteoporosis; the treatment of Paget’s disease; and the treatment of multiple myeloma in patients with documented bone metastases. Two long-term trials in cancer patients demonstrated an increase in serum creatinine (SCr) when 8 mg of bisphosphonate was administered, prompting a recommendation to reduce the dose to 4 mg. The risk for renal toxicity remains a possibility following chronic administration of bisphosphonate treatment for osteoporosis. The goal of the study was to evaluate the kidney of osteoporotic female rats following chronic administration of alendronate using a drug delivery device for 4 weeks and compare the finding with control non-ovariectomized animals, ovariectomized control animals (OVX), and ovariectomized animals with an empty drug delivery system. The results of the study showed significant increases in body weights in the ovariectomized animals compared with non-ovariectomized animals. The organ wet-weights were not statistically different between the control and treatment groups or the ovariectomized and non-ovariectomized animals. Histological and histomorphometric analysis of the kidney revealed significant changes in the glomerular area on alendronate treated animals at 4 weeks when compared with ovx, ovx-sham and control non-ovariectomized animals. The results indicate chronic use of alendronate for osteoporosis may impair renal function or increase renal related problems in patients with existing kidney disease.

Delivery of antifungal agents using bioactive and nonbioactive bone cements

BACKGROUND: Management of fungal osteomyelitis is prolonged and frequently unsuccessful. Antifungal-impregnated cement is sometimes used as adjunctive therapy. OBJECTIVE: To examine the release of antifungals from biodegradable and nonbiodegradable cement carriers. METHODS: In vitro methods were used to assess antifungal drug release and antifungal activity of impregnated cements commonly used as adjunctive treatment of osteomyelitis. Cements included thermoplastic, nonbioactive polymers (polymethylmethacrylate [PMMA]) or bioactive agents (hydroxyapatite [HAP], beta-tricalcium phosphate [beta-TCP]) and were formed into spheres (beads). RESULTS: Amphotericin B provided consistent supernatant concentrations (release), between 1.75 and 2.0 microg/mL, over 110 days from all bone cements. Flucytosine and fluconazole were observed for 33-42 days before becoming undetectable from a nonbioactive sphere and 18-22 days from a bioactive sphere. Serum concentrations for micafungin, terbinafine, and anidulafungin impregnated into PMMA rapidly became undetectable, regardless of the matrix used. Investigational beta-TCP spheres prolonged release for fluconazole and micafungin, but had no effect on amphotericin B. Serum calcium concentrations decreased 60-80% in all HAP-impregnated drug sphere supernatants. Only amphotericin B-impregnated PMMA impacted supernatant calcium, decreasing concentrations by 50-60%. The antifungal-impregnated beads did not appear to be toxic to osteoblasts during 72 hours of exposure in tissue culture medium. CONCLUSIONS: Elution characteristics of most antifungals from bone cement spheres are probably not optimal for treatment of deep-seated fungal infections if a similar phenomenon of antifungal release manifests in vivo. Ceramic nonabsorbable impregnated devices must be removed after their lifespan expires and may necessitate another surgical procedure that can increase surgical risk and cost. Bioactive osteoconductive materials may provide a surgical alternative to nonabsorbable matrices. However, there have been no controlled trials demonstrating improved therapeutic outcomes with local therapy and assessing whether biodegradable materials act as a new focus for infection.

Delivery of antifungal agents using bioactive and nonbioactive bone cements.

BACKGROUND: Management of fungal osteomyelitis is prolonged and frequently unsuccessful. Antifungal-impregnated cement is sometimes used as adjunctive therapy. OBJECTIVE: To examine the release of antifungals from biodegradable and nonbiodegradable cement carriers. METHODS: In vitro methods were used to assess antifungal drug release and antifungal activity of impregnated cements commonly used as adjunctive treatment of osteomyelitis. Cements included thermoplastic, nonbioactive polymers (polymethylmethacrylate [PMMA]) or bioactive agents (hydroxyapatite [HAP], beta-tricalcium phosphate [beta-TCP]) and were formed into spheres (beads). RESULTS: Amphotericin B provided consistent supernatant concentrations (release), between 1.75 and 2.0 microg/mL, over 110 days from all bone cements. Flucytosine and fluconazole were observed for 33-42 days before becoming undetectable from a nonbioactive sphere and 18-22 days from a bioactive sphere. Serum concentrations for micafungin, terbinafine, and anidulafungin impregnated into PMMA rapidly became undetectable, regardless of the matrix used. Investigational beta-TCP spheres prolonged release for fluconazole and micafungin, but had no effect on amphotericin B. Serum calcium concentrations decreased 60-80% in all HAP-impregnated drug sphere supernatants. Only amphotericin B-impregnated PMMA impacted supernatant calcium, decreasing concentrations by 50-60%. The antifungal-impregnated beads did not appear to be toxic to osteoblasts during 72 hours of exposure in tissue culture medium. CONCLUSIONS: Elution characteristics of most antifungals from bone cement spheres are probably not optimal for treatment of deep-seated fungal infections if a similar phenomenon of antifungal release manifests in vivo. Ceramic nonabsorbable impregnated devices must be removed after their lifespan expires and may necessitate another surgical procedure that can increase surgical risk and cost. Bioactive osteoconductive materials may provide a surgical alternative to nonabsorbable matrices. However, there have been no controlled trials demonstrating improved therapeutic outcomes with local therapy and assessing whether biodegradable materials act as a new focus for infection.

The use of androgen loaded tcpl delivery devices to assess the proinflammatory cytokines profiles.

The goal of this study is to investigate the pathophysiological changes, of both local and systemic control factors (cytokines and structural alterations), associated with continuous and sustained delivery of T and DHT using tricalcium-phosphate-lysine (TCPL) delivery devices. A total of 32 adult male Sprague Dawley rats were equally divided into four equal groups. Animals in group 1 were left untreated and served as intact control. Animals in group 2 were implanted with an empty ("sham") drug delivery device, and animals in groups 3 and 4 were implanted with a drug delivery carrier containing either DHT or T, respectively. Animal weights were recorded weekly and blood was collected bi-weekly over a six week period. At the end of 21 days, the animals were euthanized and blood and organs were collected, analyzed, and statistically evaluated. Data obtained from this study demonstrated that supraphysiological levels of androgens given in a sustained manner for long duration to adult male rats resulted in an increased level of IL-6, as well as, major alterations in lipoprotein profiles (total cholesterol, VLDL/LDL, and HDL). Histopathological evaluations of vital (heart, kidney and adrenal tissues) and major reproductive organs revealed structural damage. Histological evaluation of myocardial (ventricle and apex) tissues suggests evidence of hypertrophy and early inflammatory infiltrate. As androgens are considered for potential treatments of pathophysiological disorders, more research is needed to determine if the changes which occur as a result of sustained release of high levels of androgens can be reversed upon removal of the drug.

The evaluation of macrophages following bacterial or cytokine challenge.

Phosphatidic acid (PA) has been shown to be involved in several pathophysiological processes, including cellular signaling and inflammation. The present study was conducted to evaluate the viability of RAW264.7 macrophages following treatment with lipopolysaccharides (LPS) or TNFa. Our hypothesis is that activation of the phosphatidylcholine-phospholipase D (PC-PLD) pathway leading to phosphatidic acid production plays a role in both cytokine and LPS signaling in macrophages. Results from this study show that RAW264.7 macrophages treated with LPS or TNFa did not affect cellular viability or cause increased cellular damage. This was evident by similar cellular viability results in treated and untreated cells as compared to the control. Cells treated with 2 microg/ml of LPS or 5 ng/ml of TNFa displayed similar changes in membrane phospholipids. PA formation in cells treated with LPS increased four-fold, compared to a two-fold increase in the TNFa treatment group. These increases in PA are likely related to the increase in cytokine production of both groups. DAG production increased nearly three-fold for both the LPS and TNFa treated groups. The addition of an inhibitor, D609, did not affect PA levels or cytokine production. Regardless to the stimuli used, cellular signaling leading to cytokine production was mediated via a common event, an increase in PA. Results from this study suggest that the PAPLD signaling pathway may promote inflammatory cytokine production.

Evaluation of insulin secretion by pancreatic cells in response to increasing amounts of glucose.

Panc-1 is a human pleiomorphic epithelioid carcinoma of the exocrine pancreas. There is evidence these cells can be manipulated into insulin producing cells, by transfection with an insulin promoter or by altering the culture medium. In experiments where the cultures were altered, the insulin secreting cell types were aggregates of round cells not stellate shaped cells. The goal of this experiment was to select for survival of the round phenotype by altering glucose concentrations and to evaluate glucose dependent insulin secretion. Initially cells were grown in flasks with control medium, and then split into three separate groups containing control media, or media containing an extra 2.5% or 5% glucose. After selection for round phenotypes, the cells were maintained in control media for several passages. Cells from the three cultures were plated (1 x 105 cells/well) and treated with control, 1%, 2.5%, or 5% glucose for 24, 48, and 72 hours. Cells and supernatants were harvested and cell number, cellular glutathione, morphology, and insulin levels were determined. Control cells grown in 2.5 or 5% glucose showed significant decreases in cell number at 48-72 hours. Glutathione levels decreased as malondialdehyde (MDA) levels increased. Cells grown initially in 2.5 or 5% glucose were not affected significantly by increasing glucose concentrations. Evaluation of insulin production by enzyme linked immuno sorbent assay (ELISA) showed trends of dose and time dependency in all groups, with the highest levels in cells grown at 5% glucose and treated with 5% glucose. Overall, it appears Panc-1 cells can be converted into insulin producing cell types.

Evaluation of endotoxin binding to uhmwpe and inflammatory mediator production by macrophages.

The purpose of this investigation was 1) to determine if endotoxin can bind to the surface of high molecular weight polyethylene (UHMWP) implants in concentrations that elicit an inflammatory response, and 2) to determine if endotoxin differentially adheres to polyethylene surfaces of either oxidized ("aged") or unoxidized ("new"). Endotoxins are a major constituent of gram-negative bacteria and can stimulate the release of inflammatory mediators (e.g. IL-1) and nitric oxide (NO). Activation of IL-1 can stimulate osteolysis, therefore contributing to implant failure. Results suggest that a dose as low as 1microg/mL endotoxin was enough to elicit an inflammatory response in macrophage cell cultures without hindering cellular viability. Levels of NO were elevated in all groups treated with 1 microg/mL, 2 microg/mL, and 10 microg/mL of endotoxin, however, dose dependence was not observed. Similar findings were detected for cytokine levels of IL-1. Studies have shown wear particle debris can result in increased levels of IL-1 and NO, which lead to osteolysis. Our findings suggest that endotoxin can bind both "aged" and "new" UHMWP in a similar manner. Endotoxin also causes increases in IL-1 and NO, which lead to osteolysis. Additional studies are needed to determine if phagocytosis of wear particles can significantly increase NO and lead to osteolysis.

Comparison of two different fixation techniques for a segmental defect in a rat femur model.

Studies have attempted to identify the osteogenic effects of bone morphogenetic proteins using a rat femur model, which commonly involves the creation of a critical size defect followed by internal fixation of the femur. Among the most familiar fixation methods are either plating or intramedullary placement of a Kirschner wire (K-wire). There are advantages and disadvantages to each method; however, this study attempts to identify the best method by exploring the histological effects of each technique. The experiment involved two groups with no added treatment: Group P (plate fixation method) and Group K (K-wire fixation method). The animals were allowed a 4-week interval for the femurs to heal, and proximal, distal, and two midshaft cuts were examined under high-power microscopy after the fixation apparatus was removed. Group K exhibited a peculiar fibrotic healing pattern that followed the shaft of the then vacated K-wire and there was minimal new viable bone formation. Group P, however, exhibited a more natural ingrowth of newly formed bone that began at the proximal and distal cuts and proceeded centrally into the core of the defect. Due to the fibrotic tissue in Group K, this study shows that the model is insufficient due to the micromotion created and thus supports plating of critical defects as the fixation method of choice due to the creation of a stable healing environment.

Effects of sustained delivery of IGF-1 in a rat degenerative disc model.

Degenerative disc disease is a leading source of pain as well as increased health care costs in the United States, and research efforts into understanding the pathophysiology of this disease are necessary for the development of new management strategies. Addition of growth factors to stimulate chondrocyte development are on the horizon as new treatment modalities for degenerative disc disease, but increasing growth factor concentrations in the body may have adverse affects on vital organs. The objective of this study was to investigate the use of sustained delivery of insulin-like growth factor-1 (IGF-1) for treatment of degenerative discs using the adult male rat as a model. The results showed increased chondrocyte proliferation and decreased apoptosis at the traumatized disc after 28 days. Analysis of the vital organs revealed slight increases in kidney wet weights, and closer histomorphometric evaluation of the tissue revealed changes in the proximal tubules. Further investigation to evaluate the potential physiological or pathophysioloigcal effects of the growth factors at the organ levels is warranted before use as therapeutic agent to treat degenerative disc disease.

Evaluation of short-term healing following sustained delivery of osteoinductive agents in a rat femur drill defect model.

The bone morphogenetic proteins (BMPs) carry a crucial role in bone formation and bone healing. Previous studies have identified the osteoinductive capabilities of demineralized bone matrix (DBM) and the individual factor osteogenic protein-1 (OP-1), also known as BMP-7. The purpose of this study is to identify the short-term healing effects of targeted drug delivery of demineralized bone matrix and osteogenic protein-1 following bony trauma in terms of mechanical strength and histological changes. Animals in Group 1 acted as the control and Group 2 received a unicortical drill defect and placement of a calcined tricalcium phosphate lysine (TCPL) capsule containing antibiotic (sham). Group 3 and 4 animals had a created drill defect and received a TCPL carrier containing antibiotic along with DBM or OP-1, respectively. After 2 weeks post-implantation, animals in each group were sacrificed before the retrieval of the bone. The femurs were analyzed biomechanically for return of strength and histologically for bone growth. The average rupture strength for the femurs of the control group was shown to be significantly higher than all other groups (p<0.001 compared to sham, DBM, and OP-1). There was no significant difference between the two treatment groups (p=0.984) and there was no significant difference between the sham and DBM or OP-1 treated groups (p=0.908 and p=0.991, respectively). Analysis of the gross specimens showed minimal signs of bone regeneration at the defect site for both DBM and OP-l treated femurs when compared to the shams. Histological sections did show increased cortical thickness and new bone formation in the DBM and OP-1 groups, with the OP-1 group appearing slightly thicker. Despite the histological changes seen, the results indicate that there is no significant improvement in mechanical strength during the early stages of healing regardless of treatment.

The effects of demineralized bone matrix proteins and osteogenic protein-1 on bone cells isolated in culture.

With the growing number of bone-related traumas and the limitations of traditional bone repair, alternative methods of bone management must be investigated. Demineralized bone matrix protein (DBX) has been used to reconstruct bone. DBX, a type of demineralized bone matrix, is a combination of several different proteins including osteogenic protein-1 (OP-1). Osteogenic protein-1 or Bone Morphogenic Protein-7 (BMP-7) was the first BMP approved for clinical use in the United States. Previous studies have shown that proliferation of osteoblasts (bone forming cells) was stimulated by OP-1. However, the effects of DBM and OP-1 at the cellular level have not been clearly defined. MG-63 osteosarcoma cells were utilized as a model and subsequently plated onto 24 well tissue culture plates at a density of 1x 10(5) ml/well. Cells were exposed to different concentrations of DBX demineralized bone matrix and OP-1 for periods of 24, 48, and 72 hours and compared with untreated controls. After each incubation period, cell morphology, cell damage, cell number, and protein concentrations were determined. Results indicate a significant increase in cell number at 72 hours in cells treated with 30% (5.66 x 10(5)) and 100% (6.3 x 10(5)) DBX treated groups when compared with the control (1.4 x 10(5)). OP-1 results do not indicate a significant increase in cell number at the 24 and 48 hour treatment phases when compared with the control (p > 0.05), however, results do show a statistically significant difference (approximately twofold, p < 0.05) between the control cells (1.9 x 10(4)) and those cells treated with low (3.9 x 10(4)) and high (4.1 x 10(4)) concentrations of OP-1 at the 72 hour time phase. The increases in cell number indicate that both DBX and OP-1 are effective in stimulating cell growth. When comparing the results of the DBX treatments with those of the OP-1 treatments, the cells treated with DBX showed a more substantial increase in bone cell proliferation after treatment than those cells treated with OP-1. This does suggest that DBX provides the most effective treatment for bone cell proliferation. Closer evaluation of the morphology especially the changes occurring at the nuclear level need to be addressed in future studies.

Effects of polystyrene particle density on type II pneumocytes.

Studies into the effects of ultrafine particles on the lung have shown adverse outcomes and are do in part to the particle size. Inhalation of small particles is associated with exacerbation of respiratory diseases in epidemiological studies. Ultrafine particles have been hypothesized to play an important role, but it is unclear as to whether a dose response type of relationship exists. The aim of the present study was to investigate the role of ultrafine particle number on lung cells and to describe the effects due to phagocytosis of particles by the cells. A549 cells are a transformed cell line similar to type II lung pneumocytes. A549 cells seeded at a density of 5 x 10(4) cells were treated with 1000, 5000, and 10,000 polystyrene particles and incubated at 37 degrees C for periods of 24, 48, and 72 hours. Cell number, protein, and MDA levels were determined on the treated cells and compared with untreated controls. The lung cells were also observed microscopically to assess cell damage. Lung cells exposed for 24 hours with 1,000 and 5,000 particles showed the greatest increase in cell number. Cellular protein levels were similar for all groups (p > 0.05) for the duration of the study. MDA levels for treated groups at 24 and 72 hours were not statistically different from the control groups. Cells treated at 48 hours with 5,000 and 10,000 particles showed slight increases in the MDA levels above low particle number and control treated groups. Morphological evaluation of the cells revealed increased inclusions with increasing dose. The results from this study indicate the ability of A549 cells to respond to a challenge with ultrafine particles. The concentrations tested caused an initial stimulation cell proliferation at 24 hours followed by increased damage at 48 hours. Future studies will focus on the inflammatory products formed by ingestion of the ultrafine particles.

Comparison between mevinolin and PTH action on MG63 osteoblast-like cells.

Increasing osteoblast activity in an anabolic fashion may offer an ideal therapeutic treatment for various orthopedic complications including osteoporosis. The purpose of this study was to evaluate the effect of mevinolin, a clinical statin drug, on osteoblast function (MG 63 Cell Line) and compare its mode of action with the conventionally utilized parathyroid hormone (PTH). MG63 cells were treated with different concentrations (control, low (100 nM), medium (1 uM), and high (10 uM)) of mevinolin or Parathyroid hormone. The cells were incubated for 24, 48, and 72 hours at 37 degrees C in a 95% air and 5% CO2 environmental chamber. Data obtained in this study revealed that: (I) there were significant decreases in cell number after 24 hours upon the exposure of medium and high doses of mevinolin, (II) cell numbers rebounded back toward control after 48 hours and were similar in number at 72 hours, and (III) there were no significant changes in calcium or alkaline phosphatase activity were observed throughout the study. Morphologically, the cells treated with various doses of Mevinolin expressed similar structural changes to those observed using PTH. These changes included pleomorphic characteristics and an occasional hyperchromatic pattern during the entire duration of the study (72 Hours). Other structural features observed were spindle shapes, cluster arrangements and multinucleation. The majority of cells had multiple nucleoli in all treated groups compared to controls. The overall conclusion of this investigation demonstrated that the concentrations used (100 nM and 10 microM) did not appear to affect the mitotic activities of immature phenotypic MG-63 cells. In addition, the concentrations of mevinolin used did not trigger the differentiation process of the cells throughout the experimental phases. This observation led us to suggest that the reason for such an outcome could be attributed to the lack of a response in calcium production or alkaline phosphatase activity (stimulator to differentiation and mineralization process).

Mechanical strength repercussions of various fixative storage methods on bone.

This study compensates for the lack of literature on the actual effects that various fixative storage methods have on the mechanical strength characteristics of bone and attempts to identify the ideal fixative method for preservation of all tissues while maintaining in vivo bone strength. Researchers currently use a wide variety of storage methods that lessen the mechanical strength to varying degrees. Differences could introduce error into a great number of bone fracture studies if an inexact discrepancy in the mechanical properties of fixed bone does actually exist. Furthermore, such disparities could go on to pose clinical risks for patients. This study focuses on the mechanical strength testing of four different groups of rat femora that were retrieved at various times and subjected to differing storage procedures. The first, Group N, were fresh, new femora retrieved just days before testing. The second, Group F, were femora that have been fixed in a 10% formalin bath for just over a year prior to testing. The third, Group W, are femora that have also been fixed in 10% formalin for just over a year but were washed out just prior to testing. The fourth, Group P, were femora that were retrieved from rats that were perfused with formalin immediately following euthanasia. Mechanical strength tests on the four groups revealed that fixing bone in a 10% formalin bath significantly reduces the mechanical fracture strength properties of the bone regardless of whether the formalin is washed out prior to testing. Testing also revealed that bone from perfused animals behaves more similarly to fresh bones from non-perfused animals suggesting that the formalin did not entirely infiltrate the bone and permanently fix the material. These results could have profound implications on how studies equate the behavior of in vitro bone to in vivo bone which could manifest as clinical complications for patients.

Evaluation of bioflavonoids as potential chemotherapeutic agents.

Bioflavonoids are compounds that are natural plant antioxidants. Antioxidants have been shown to prevent damage caused by free radicals to DNA and other molecules. Bioflavonoids have demonstrated several cancer preventive properties. In addition to antioxidant activity, these compounds may reduce abnormal cell growth and inflammation; help the body get rid of cancer-causing agents; and restore communication between different cells in the body. The objective of this study is to compare nontoxic phenols as cancer chemotherapeutic, agents and to apply these agents in treating human cancers. In this study the effects of a single dose of 10 microM Green Tea Extract (EGCG), 5 microM Tannic Acid (TA), or 5 microM Thymoquinone (TQ) on Hep-2 cells were determined. Compounds were evaluated in their effectiveness to reduce cell number. The cells were also evaluated for membrane damage, and alterations in cellular morphology after 24, 48, and 72 hours in culture. The results showed a 50% reduction in Hep-2 cell numbers after 24 hours in TQ treated cells. After 48 hours, the cells treated with TQ and TA exhibited a four-fold decrease in total cell number compared to the control and EGCG treated cells. Cell numbers were similar in all treatment groups by 72 hours. At 48 hours the only significant increase in cell damage was seen in cells treated with EGCG. The results indicated that naturally occurring bioflavonoids given in a bolus dose could alter cellular viability. It appears the drug is either utilized by the cells or chemically degraded in the media leading to increased cell numbers back toward control values with time in culture. In order for the compounds to be effective chemotherapeutic agents, the data also suggests a need for continuous administration over bolus dosing.

Glomerular response to adrenocortical hormone alone or in combination with selenomethionine.

Increased levels of glucocorticoids (GC) can result in major complications such hypertension and vascular injury. Chronically, this condition may lead to impairment of renal function. Glucocorticoid excess was considered the etiologic agent that triggers over production of reactive oxygen species (ROS). The mode of action of ROS was implicated to disrupt nitric oxide availability in the vascular endothelium, leading to vascular complications. To circumvent this damage attempts were made to use antioxidants in order to counter-balance the oxidative process. The objectives of this study were: (1) to establish an animal model of increased glucocorticoid levels by sustained delivery and (2) to determine if sustained delivery of selenomethionine in combination with glucocorticoids could protect kidney tubular structures using adult rats. Sixteen female rats were divided into four equal groups (control and 3 experimental groups implanted with tricalcium phosphate lysine drug delivery systems (TCPL) charged with either 50mg selenomethionine (Se), 50 mg corticosterone (C), or 50 mg of both C and Se). At the end of 24 days, the rats were sacrificed and both kidneys were removed for histopathological analysis. Quantitative analysis was performed on serum calcium levels, body weights and kidney weights in animals from all groups. Kidney slides were screened for possible structural damage. Sustained release of Se and Se+C resulted in a significant reduction of glomerular area (p < 0.05). Data obtained indicated that C, Se and Se+C administration caused a reduction in serum calcium levels compared to control animals. The reduction may be in part to changes in calcium-filtered load, changes in glomerular filtration rates or interference of calcium absorption from the gut. In conclusion, data obtained from this investigation provided the literature with significant information regarding the role of sustained delivery of supraphysiological levels of corticosterone in modifying kidney structure and function (possibly alter blood pressure).

Sustained release of OP-1 and antibiotics in treatment of femoral defects in male rats.

Osteogenic proteins (OP-1) promote osteoinduction. Formation of new bone growth in patients receiving OP-1 is not consistent, and is possibly due to the short half-life of the drug. In order to test the capacity of OP-1 to consistently produce bone in a fracture model, a drug delivery system was developed to prolong the action of OP-1. Fifteen Sprague Dawley male rats were randomly divided into three equal groups; Animals in group 1 served as control. Animals in groups 2 and 3 had a 5mm defect created in the left femur using a number six dental burr and a drug delivery capsule (TCPL) containing either antibiotic alone (sham) or antibiotic +OP-1. Body weights, blood, and X-rays were taken weekly. Femurs and organs were harvested 30 days post-op, and processed for histomorphometry. Data was analyzed using ANOVA and significant difference between the groups was determined using Student Newman Kuels (p < 0.05). The results showed complete bone healing in the OP-1 group with an evident callus formation. The osteoid tissue exhibited a proliferation of osteoblasts, which differentiated from the vascularized mesenchymal tissue. The complete bone healing using OP-1 sharply contrasted sham treatment, where an obvious injury was still seen at 30 days. Histologically sham animals exhibited the early stage of repair with evidence of blood clotting and mesenchyme with early formation of osteoblasts. Overall, OP-1 delivered in a sustained manner for 30 days caused increased bone formation in a defect model.

Stimulation of fracture healing by continuous delivery of demineralized bone matrix proteins and tobramycin.

Demineralized bone matrix (DBM) is an allogenous, bioabsorbable material that has long been used for its osteoinductive and osteoconductive properties. A significant complication experienced by physicians who perform bone defect filling surgery is the risk of subsequent bacterial infections and the inefficiency of oral antibiotics to provide adequate prophylaxis against microorganisms, especially Staphylococcus aureus, Pseudomonas aeruginosa, and Staphylococcus epidermidis. In order to deliver both DBM and an efficient antiobiotic at high local concentrations without deleterious systemic effects, a ceramic sustained delivery system was implanted and monitored over the course of 30 days for bone regeneration, infection, and systemic effects. Twenty-five adult Sprague Dawley albino male rats were used in the experiment. They were randomly divided into five equal groups. Animals in group 1 were used as control, group 2 animals had a created 5mm defect, in group 3 defect induced and implanted with devices loaded with antibiotic (tobramycin) alone, group 4 had a created defect plus DBM, and group 5 had a created defect, plus tobramycin (which is effective against the aforementioned organisms) and DBM. At 30 days post-implantation, the experimental animals showed no significant difference in weight when compared to the control and sham animals. X-rays taken at this time showed the experimental femurs to be totally healed and virtually indistinguishable from control. Initial dissections revealed that the implants were accepted by the hosts as shown by the fibrous, vascularized sheath that surrounded the femurs and capsules. The implants were found to be in close contact with the cancellous bone and none of the sheaths showed signs of infection. Macroscopically, no defect could be detected in the experimental animals, while little regeneration was observed in the femurs of the sham animals.

The effect of agonists and antagonists on Hep-2 cells.

Androgens may play an important role in promoting the growth of laryngeal carcinomas. The aims of this investigation were to investigate the effects of (testosterone (T) and androstendione (AED)) in the presence of the anti-androgen, spironolactone (S), n Hep-2 cellular proliferation and damage after 24, 48 and 72 hours. Hep-2 cells were divided into six groups (n = 5) control, S, T, AED, S+T, and S+AED, respectively. The cells were harvested after each incubation period into two different fractions: suspended cells and adhered cells. Cell counts and cellular damage determinations were performed on each fraction. Analysis of variance was used to determine significance at p < 0.05. Data for cell counts revealed an interesting phenomenon between the two fractions. Adhered cells showed decreased cell numbers in the presence of S and T for 24 - 48 hours followed by a significant increase at 72 hours. Cells in the adhered fraction incubated in the presence of AED or AED + S were significantly lower for the duration of the experiment. However AED or AED + S treatment caused significant increase in cell number in suspended fraction for the duration of the experiment. All treatments after 72 hours showed a slight reduction in MDA levels indicating treatments did not cause cell damage. Overall, the data suggests the possibility of two populations of cells that respond differently to the AED. T had no significant effect on either cell fraction for the first 48 hours followed by a significant increase in cell number at 72 hours suggesting T may need to be converted enzymatically to the more potent androgen, dihydrotestosterone.

The role of proteoglycans in idiopathic carpal tunnel syndrome.

Decompression of the carpal canal is the most common hand surgery performed in the United States. Hand surgeons perform 460,000 carpal tunnel releases (CTR) each year, which cost the medical industry in excess of two billion dollars per year. The focus of this investigation was to identify the changes, which occur in the flexor tenosynovium of patients undergoing CTR at the connective tissue level. The connective tissues determine the amount and arrangement of macromolecules (fibers, proteoglycans, and glycoproteins) in the extracellular matrix. The proteoglycans are soluble macromolecules that have both structural and metabolic roles. Glycoproteins help to form the interstitial space, basement membrane and function as cell surface receptors. The mechanical function of the proteoglycans includes stabilization of the collagen fibers as well as function in the hydration of the tissues. It has been previous shown that changes in the oxygen concentration at the tissue level can alter the proteoglycans profile of the tissue. During periods of hypoxia, such as those obtained during repetitive motion CTS; the glycolytic pathway acts as the energy source for the tissue. Productions of chondroitin sulfates are a process consumes NAD and would be potentially toxic to the cells under anaerobic conditions. Production of keratan sulfate is NAD sparing product, and may act as a survival pathway for cells under adverse conditions. The disruption in the proteoglycan balance will allow for alterations in the ECM and changes in hydration status of the tissues may have serious implication in CTS because the carpal canal is anatomically very narrow and increases in volume within the canal can result in further compression of the nerve. Flexor tenosyioum was obtained from patients undergoing CTR and compared with control tissue for dermatan, keratan and chondroitin sulfate. The results show a greater density of keratan reactivity in CTS tissues identified by immunostaining. In addition to changes in proteoglycan content there was also an increase in new vessel formation in the CTS tissues. The data obtained suggests that the shifts in the proteoglycan ratios may render the tissues less able to withstand the compressive forces and therefore allow for more force to be placed on the median nerve within the carpal canal.