David C. Buck

Bone Formation with Use of rhBMP-2 (Recombinant Human Bone Morphogenetic Protein-2)*

We examined the effect of rhBMP-2 (recombinant human bone morphogenetic protein-2), delivered in a porous poly(DL-lactic acid) implant, on bone formation in a critical-sized defect in the radial diaphysis in rabbits. A unilateral segmental defect, twenty millimeters long, was created in the radius in ninety-six skeletally mature New Zealand White rabbits. Forty-eight rabbits were evaluated at four weeks and forty-eight, at eight weeks. Six groups were studied at each time-period. The defect was left empty in one group (control), the defect was filled with an autogenous corticocancellous bone graft in one group, and the defect was filled with a porous poly(DL-lactic acid) implant containing zero, seventeen, thirty-five, or seventy micrograms of rhBMP-2 (one group each). Radiographs of the defects were made every two weeks. The percentage of the total area of the defect that was radiopaque was determined with use of computerized radiomorphometry, and this percentage was used as a quantitative measure of the extent of new-bone formation in the defect. There were time and dose-dependent responses to rhBMP-2 for as long as four weeks; thereafter, the effects of seventeen, thirty-five, and seventy micrograms of rhBMP-2 were independent of dose and time (p ⩽ 0.05). The defects that had been treated with either thirty-five or seventy micrograms of rhBMP-2 had a significantly greater (p ⩽ 0.05) area of radiopacity than the defects that had been treated with either zero or seventeen micrograms of rhBMP-2. No significant difference could be found between the defects treated with thirty-five or seventy micrograms of rhBMP-2 and the defects filled with an autogenous graft. Healing and bone formation were examined histologically and histomorphometrically as well. At four weeks, polarized light microscopy revealed remnants of poly(DL-lactic acid) only in the defects that had been filled with an implant containing zero micrograms of rhBMP-2. At eight weeks, regardless of the dose of rhBMP-2, poly(DL-lactic acid) was not visible on histological examination. The presence of multinucleated giant cells was the hallmark of the inflammatory response elicited by poly(DL-lactic acid). At four and eight weeks, macrophages and lymphocytes were also present. The intensity of the cellular response at four weeks suggested an inverse relationship between these cells and the dose of rhBMP-2—that is, there appeared to be more multinucleated giant cells in defects treated with zero micrograms of rhBMP-2 than in defects treated with seventy micrograms of rhBMP-2. At eight weeks, multinucleated giant cells were rare in the defects treated with seventeen, thirty-five, or seventy micrograms of rhBMP-2. Histomorphometric data at four and eight weeks indicated that the amount of bone formation in the defects treated with seventeen, thirty-five, or seventy micrograms of rhBMP-2 was equivalent to the amount in the defects treated with an autogenous graft and was significantly less (p ⩽ 0.05) in the untreated defects and the defects treated with zero micrograms of rhBMP-2 (p ⩽ 0.05). By eight weeks, only thirty-five and seventy micrograms of rhBMP-2 had restored cortices and marrow elements. CLINICAL RELEVANCE: It is a clinical challenge to restore bone lost as a result of trauma, pathological processes, oncological resection, or developmental malformations. Autogenous and banked allogenic bone grafts are used routinely to correct bone defects. However, problems associated with these treatments are well known, and a safe, reliable, and convenient alternative is desirable. In the present study, rhBMP-2 delivered in a porous poly(DL-lactic acid) implant promoted bone formation. Therefore, rhBMP-2 in a poly(DL-lactic acid) delivery system may be suitable to elicit bone formation and healing in segmental bone defects.

Recombinant human bone morphogenetic protein‐2 and collagen for bone regeneration

The study reported describes a combination of recombinant human bone morphogenetic protein-2 (rhBMP-2) and collagen (C) to regenerate bone. Unilateral critical-sized defects (CSDs) were prepared in radii of 32 skeletally mature New Zealand white rabbits. Rabbits were divided evenly among four treatments: autograft, absorbable C (Helistat), 35 microg of rhBMP-2 combined with absorbable C (rhBMP-2/C), and untreated CSDs. The two euthanasia periods were 4 and 8 weeks. Radiographs were taken the day of surgery, every 2 weeks, and at term and the percent of radiopacity was measured. Data analysis revealed a time-dependent increase in the percent radiopacity with rhBMP-2/C. Histological examination revealed the rhBMP-2/C treatment regenerated osseous contour by 8 weeks. According to quantitative histomorphometry, the CSD and C groups had significantly less new bone than either autograft or rhBMP-2/C (p < or = 0.05). The results suggest that rhBMP-2/C could be an effective therapy to restore segmental bone defects.

Tissue-engineered bone biomimetic to regenerate calvarial critical- sized defects in athymic rats

A tissue-engineered bone biomimetic device was developed to regenerate calvaria critical-sized defects (CSDs) in athymic rats. Well-documented evidence clearly confirms that left untreated, CSDs will not spontaneously regenerate bone. To accomplish regeneration, four candidate treatments were assessed: porous poly(D,L-lactide) and type I collagen (PLC), PLC and human osteoblast precursor cells (OPCs) at 2 x 10(5) (PLC/OPCs), PLC and 50 microg of recombinant human bone morphogenetic protein-2 (PLC/rhBMP-2), and PLC/OPCs/rhBMP-2 (the bone biomimetic device). The hypotheses for this study were PLC/OPCs/rhBMP-2 would promote more new bone formation in CSDs than the other treatments and the amount of bone formation would be time dependent. To test the hypotheses, outcomes from treatments were measured at 2 and 4 weeks postoperatively by radiomorphometry for percent radiopacity and by histomorphometry for square millimeters of new bone formation. Data were analyzed by analysis of variance and Fishers protected least significant difference for multiple comparisons with p < or = 0.05. At 2 and 4 weeks, radiomorphometric data revealed PLC/rhBMP-2 and PLC/OPCs/rhBMP-2 promoted significantly more radiopacity than either PLC or PLC/OPCs. Histomorphometry data at 2 and 4 weeks indicated significantly more new bone formation for PLC/rhBMP-2, PLC/OPCs/rhBMP-2, and PLC/OPCs compared to PLC. By 4 weeks, PLC/OPCs/rhBMP-2 and PLC/rhBMP-2 had regenerated the CSDs with more new bone than the other treatments; the quantity of bone at 4 weeks for these treatments was greater than at 2 weeks.

Altered Endothelial Nitric Oxide Synthase Targeting and Conformation and Caveolin-1 Expression in the Diabetic Kidney

Experimental diabetes is associated with complex changes in renal nitric oxide (NO) bioavailability. We explored the effect of diabetes on renal cortical protein expression of endothelial NO synthase (eNOS) with respect to several determinants of its enzymatic function, such as eNOS expression, membrane localization, phosphorylation, and dimerization, in moderately hyperglycemic streptozotocin-induced diabetic rats compared with nondiabetic control rats and diabetic rats with intensive insulin treatment to achieve near-normal metabolic control. We studied renal cortical expression and localization of caveolin-1 (CAV-1), an endogenous modulator of eNOS function. Despite similar whole-cell eNOS expression in all groups, eNOS monomer and dimer in membrane fractions were reduced in moderately hyperglycemic diabetic rats compared with control rats; the opposite trend was apparent in the cytosol. Stimulatory phosphorylation of eNOS (Ser1177) was also reduced in moderately hyperglycemic diabetic rats. eNOS colocalized and interacted with CAV-1 in endothelial cells throughout the renal vascular tree both in control and moderately hyperglycemic diabetic rats. However, the abundance of membrane-localized CAV-1 was decreased in diabetic kidneys. Intensive insulin treatment reversed the effects of diabetes on each of these parameters. In summary, we observed diabetes-mediated alterations in eNOS and CAV-1 expression that are consistent with the view of decreased bioavailability of renal eNOS-derived NO.

Dopamine D2 receptor stimulation of mitogen‐activated protein kinases mediated by cell type‐dependent transactivation of receptor tyrosine kinases

Dopamine D2 receptor activation of extracellular signal‐regulated kinases (ERKs) in non‐neuronal human embryonic kidney 293 cells was dependent on transactivation of the platelet‐derived growth factor (PDGF) receptor, as demonstrated by the effect of the PDGF receptor inhibitors tyrphostin A9 and AG 370 on quinpirole‐induced phosphorylation of ERKs and by quinpirole‐induced tyrosine phosphorylation of the PDGF receptor. In contrast, ectopically expressed D2 receptor or endogenous D2‐like receptor activation of ERKs in NS20Y neuroblastoma cells, which express little or no PDGF receptor, or in rat neostriatal neurons was largely dependent on transactivation of the epidermal growth factor (EGF) receptor, as demonstrated using the EGF receptor inhibitor AG 1478 and by quinpirole‐induced phosphorylation of the EGF receptor. The D2 receptor agonist quinpirole enhanced the coprecipitation of D2 and EGF receptors in NS20Y cells, suggesting that D2 receptor activation induced the formation of a macromolecular signaling complex that includes both receptors. Transactivation of the EGF receptor also involved the activity of a matrix metalloproteinase. Thus, although D2 receptor stimulation of ERKs in both cell lines was decreased by inhibitors of ERK kinase, Src‐family protein tyrosine kinases, and serine/threonine protein kinases, D2‐like receptors activated ERKs via transactivation of the EGF receptor in NS20Y neuroblastoma cells and rat embryonic neostriatal neurons, but via transactivation of the PDGF receptor in 293 cells.

Radiomorphometry and Biomechanical Assessment of Recombinant Human Bone Morphogenetic Protein 2 and Polymer in Rabbit Radius Ostectomy Model

The study objective was to determine the mechanical integrity and radiopacity of regenerated bone within critical-sized defects (CSDs) in radii of rabbits using recombinant human bone morphogenetic protein 2 (rhBMP-2) with a porous, biodegradable poly(D,L-lactic acid) (PDLLA) carrier (designated PLA). Twenty millimeter, unilateral radial ostectomies were created in 96 skeletally mature New Zealand white rabbits. The rabbits were randomly assigned to six treatment groups with two euthanasia periods. Treatment groups included unfilled defect (n = 8), segmental autograft (n = 8), PLA + 0 microg rhBMP-2 (n = 8), PLA + 17 microg rhBMP-2 (n = 8), PLA + 35 microg rhBMP-2 (n = 8), and PLA + 70 microg rhBMP-2 (n = 8). The radiopacity was significantly greater for the 35- and 70-microg rhBMP-2 groups at 4 weeks compared to unfilled controls, PLA only, and 17-microg rhBMP-2 groups and equivalent to the autograft. At 8 weeks all groups receiving rhBMP-2 were equivalent to the autograft and significantly greater than unfilled defects and PLA alone. Similarly, the biomechanical analysis indicated significantly greater torque at failure for the 35-microg rhBMP-2 group compared to all other groups at 4 weeks. By 8 weeks all groups receiving rhBMP-2 and autograft had significantly greater torque than unfilled controls and PLA alone. These radiomorphometric and biomechanical results indicate PLA may be a suitable carrier for rhBMP-2 used for skeletal regeneration.

Novel Interaction of the Dopamine D2 Receptor and the Ca2+-Binding Protein S100B: Role in D2 Receptor Function

S100B is a calcium-binding protein with both extracellular and intracellular regulatory activities in the mammalian brain. We have identified a novel interaction between S100B and the dopamine D2 receptor. Our results also suggest that the binding of S100B to the dopamine D2 receptor enhances receptor signaling. This conclusion is based on the following observations: 1) S100B and the third cytoplasmic loop of the dopamine D2 receptor interact in a bacterial two-hybrid system and in a poly-histidine pull-down assay; 2) immunoprecipitation of the D2 receptor also precipitates FLAG-S100B from human embryonic kidney 293 cell homogenates and endogenous S100B from rat neostriatal homogenates; 3) S100B immunoreactivity was detected in cultured neostriatal neurons expressing the D2 receptor; 4) a putative S100B binding motif is located at residues 233 to 240 of the D2 receptor, toward the amino terminus of the third cytoplasmic loop. D3-IC3, which does not bind S100B, does not contain this motif; and 5) coexpression of S100B in D2 receptor-expressing 293 cells selectively increased D2 receptor stimulation of extracellular signal-regulated kinases and inhibition of adenylate cyclase.

Comparison of expanded polytetrafluoroethylene microvascular grafts to autogenous vein grafts.

The use of 1-mm ID by 1-cm-long expanded polytetrafluoroethylene microvascular grafts in various positions in two experimental animals did not compare favorably with the use of autogenous vein interposition grafts in controls. Light microscopy and scanning electron microscopy showed that early fibrin deposition at the anastomosis lines is followed by fully activated coagulation of the grafts. Use of antiplatelet and anticoagulant drugs, changes in techniques, and alterations in the graft material are possible future directions for improved patency with expanded polytetrafluoroethylene microvascular grafts.

Effectiveness of prophylactic mastectomy in the prevention of breast tumors in C3H mice.

The effectiveness of prophylactic mastectomy in the prevention of breast tumors was studied in spontaneous breast-tumor-forming C3H mice. Prolactin levels were assayed to determine if this hormone was related to the incidence of mammary tumors. Two-hundred and fifty-six 1-month-old C3H mice were divided into four groups (control, 1; sham surgery, 2; mammectomy 50 percent, 3; and mammectomy 100 percent, 4). At the time of sacrifice (0 to 1 year postoperatively) estrus cycles were determined, ventral skin (breast) and ovaries were removed for histology, and serum was collected for prolactin assays. Prolactin levels 24 hours postoperatively were significantly elevated (p less than 0.01) in groups 2 to 4 when compared with group 1. Six months postoperatively, prolactin levels were significantly higher (p less than 0.05) in mice with tumors compared with those without tumors in groups 3 and 4. There were no differences in tumor incidence between the four groups. At 12 months postoperatively, no differences in prolactin levels were noted, but group 2 animals had the highest incidence of mammary tumors (89 percent; p less than 0.01) when compared to groups 3 and 4. Mammary tumor incidence was not decreased by 50 percent or 100 percent mammectomy in C3H mice. Prolactin levels rose in response to surgery and/or anesthesia and remained elevated only in tumor-bearing mice who underwent mammectomy, an occurrence similar to that reported in humans.

Assessment of an experimental bone wax polymer plus TGF‐β1 implanted into calvarial defects

The study reported describes an experimental biodegradable polymer ceramic composite with wax-like handling properties that was combined with 2.0 micrograms of recombinant human transforming growth factor beta (rhTGF-beta(1)). The polymer/rhTGF-beta(1) combination was introduced into standard-sized calvarial defects in rabbits to evaluate biodegradability, biocompatibility, hemostasis control, and bone promotion. The experimental wound model was a standard-size circular calvarial defect 8 mm in diameter. The experimental design included 24 skeletally mature New Zealand white rabbits divided evenly between two time periods (6 and 12 weeks) and among three experimental treatments (untreated defects and defects treated with polymer with or without rhTGF-beta(1)). Evaluations consisted of clinical examinations, standarized radiography, radiomorphometry, as well as histology and histomorphometry. Data were analyzed by an Analysis of Variance (ANOVA) and Fishers Protected Least Significant Difference test at each time period (level of significance p < or = 0.05). Radiomorphometry data indicated that standard-sized defects treated with the wax-like polymer alone and the polymer plus 2.0 micrograms of TGF-beta(1) were significantly more radiopaque than control sites at both 6 and 12 weeks. Histomorphometric data revealed the amount of new bone was significantly greater at 6 weeks in the polymer plus 2.0 micrograms of TGF-beta(1) and in the control group than in the polymer alone. Moreover, at 12 weeks, there was significantly more new bone in the control than in either the polymer alone or the polymer plus 2.0 micrograms of TGF-beta(1). We speculate the incomplete biodegradation of the polymer ceramic composite contributed to the radiopacity and may have retarded osseous regeneration. It is important that the bone wax-like polymer material was biocompatible and acted as a hemostatic agent.