Effects of a synthetic peptide of a parathyroid hormone-related protein on calcium homeostasis, renal tubular calcium reabsorption, and bone metabolism in vivo and in vitro in rodents.

A synthetic peptide corresponding to the first 34 amino acids of the parathyroid hormone-related protein (PTH-rP) produced by a human tumor associated with hypercalcemia was examined for skeletal and renal effects on calcium metabolism in vivo and in vitro. These effects were compared with those of human parathyroid hormone (1-34), hPTH (1-34). Equal doses of PTH-rP(1-34) and hPTH(1-34) produced equivalent stimulation of adenylate cyclase in vitro in bone cells and kidney cells and tubules. Subcutaneous injection of PTH-rP(1-34) in mice caused a significant dose-related increase in blood ionized calcium similar to that seen with hPTH(1-34) at equivalent doses. Repeated injections of equal doses of both peptides caused sustained hypercalcemia which was significantly greater in PTH-rP(1-34)-treated mice, although each induced comparable increases in histomorphometric indices of osteoclastic bone resorption. PTH-rP(1-34) and hPTH(1-34) also caused similar increases in bone resorption when incubated with fetal rat long bones in organ culture. Infusion of either peptide into thyroparathyroidectomized rats suppressed urinary calcium excretion and increased urinary excretion of cyclic AMP. PTH-rP appears to have similar effects to those of PTH on the skeleton, the kidney, and overall calcium homeostasis.

Occurrence of hypercalcemia and leukocytosis with cachexia in a human squamous cell carcinoma of the maxilla in athymic nude mice: a novel experimental model of three concomitant paraneoplastic syndromes.

Hypercalcemia and leukocytosis may occur in conjunction as paraneoplastic syndromes associated with malignant disease. Here we describe a human squamous cell carcinoma of the maxilla that was associated with hypercalcemia and leukocytosis, and also cachexia. The primary tumor was surgically removed and established in permanent cell culture. When either primary tumors or cultured tumor cells were inoculated into nude mice, the nude mice developed the same paraneoplastic syndromes as those which occurred in the patient from whom the tumor was originally derived. The plasma calcium was increased two and one-half-fold and the WBC count 30-fold, and the body weight was decreased by 45% in tumor-bearing animals. Each of these paraneoplastic syndromes was alleviated by surgical excision of the tumor, indicating that the paraneoplastic syndromes were due to a factor or factors produced by the primary tumor. The development of each of these paraneoplastic syndromes in nude mice correlated positively with the other two syndromes. We examined the organs of tumor-bearing mice and found striking histopathologic abnormalities in the bones, spleen, and liver, but no infiltration with tumor cells. The bones showed marked evidence of osteoclastic bone resorption. This model of a human tumor associated with the hypercalcemia-leukocytosis paraneoplastic syndrome, together with cachexia, should make it possible to determine the mechanisms responsible for these paraneoplastic syndromes and their relationship to each other.

Diagnostic tools and biologic markers: animal models in the study of osteoporosis and oral bone loss.

Recent data have suggested that there may be a relationship between osteoporosis and oral bone loss. Both diseases are major public health problems of enormous magnitude, but their relationship is poorly understood. This is due to their complex pathogenesis and the fact that coexisting periodontal disease, loss of the dentition, site-specific variability in anatomy and bone density, as well as aging and many other factors complicate the development and progression of bone loss relative to each. The focus of this article is to review the characteristics of animals that are suited for studies of the relationship of osteoporosis and oral bone loss to better understand their relationship.

In vivo induction of bone by recombinant human transforming growth factor beta 1.

A single application of recombinant human transforming growth factor beta 1 (rhTGF-beta 1) adjacent to cartilage was found to induce bone formation in rabbit ear full-thickness skin wounds. At doses that optimally promote soft tissue healing, 25-100 ng rhTGF-beta 1 per wound caused osseous tissue formation starting 21 days after wounding to reach a peak incidence and area of bone formation at day 42. Bone formation was followed by active remodeling, resulting in lower incidence and area of bone formation at days 56 and 70. The early phase of bone formation was located overlying the cartilage and involved perichondrial cells that appeared to differentiate directly into osteoblasts forming bone matrix without a cartilage precursor. Cartilage was replaced with bone at later time points. rhTGF-beta 1 was able to increase the ratio of osteoblasts to osteoclasts lining the trabecular surface of bone and thus increase the net amount of bone formation. The present studies suggest a potential therapeutic role for rhTGF-beta 1 in hard tissue repair.

Expression of human transforming growth factor alpha by Chinese hamster ovarian tumors in nude mice causes hypercalcemia and increased osteoclastic bone resorption.

Transforming growth factor alpha (TGF-alpha) is a polypeptide regulator of cell growth produced by many malignant tumors. It stimulates osteoclastic resorption in bone organ culture and osteoclast-like cell formation in marrow culture. To determine whether tumor production of TGF-alpha can cause hypercalcemia in vivo, we used Chinese hamster ovarian (CHO) cells transfected with the human TGF-alpha gene (TCHO), which stably express and secrete TGF-alpha. We used nontransfected CHO cells as controls (CCHO). TCHO and CCHO were inoculated intramuscularly into one hindlimb of nude mice and grew as local solid tumors. After 4 weeks of TCHO tumor growth, plasma ionized calcium (Ca2+) increased to reach 1.48 +/- 0.03 mM (mean +/- SEM), whereas mice bearing similarly sized CCHO tumors and non-tumor-bearing mice (NTB) remained normocalcemic (normal range for Ca2+, 1.15-1.30 mM). Plasma TGF-alpha was undetectable by an ELIFA assay in all NTB mice, was markedly increased in all TCHO mice (5.75 +/- 0.78 ng/ml), and was slightly increased in CCHO mice (0.50 +/- 0.22 ng/ml). Quantitative bone histomorphometry showed a prominent increase in osteoclastic bone resorption in TCHO mice. These data suggest that TGF-alpha is a mediator of hypercalcemia and increased osteoclastic bone resorption in tumors that produce it in sufficient quantity.

Effects of interleukin-1 on bone turnover in normal mice.

Interleukin-1 (IL-1) is a potent stimulator of osteoclastic bone resorption in vitro and causes hypercalcemia and increased osteoclastic resorption when infused into normal mice for 72 h. However, its longer term or local effects on bone turnover in vivo are unknown. To study these, we injected IL-1 alpha once daily for 3 days into the sc tissue over the calvariae of normal mice and examined its effects on calvarial bone morphology during the subsequent 4 weeks using quantitative histomorphometry. Increased bone resorption inside the calvariae and elevated plasma calcium concentrations were present 24 h after the last IL-1 injection. These early systemic effects were not prevented by indomethacin. During the following 3-4 weeks most of the bone on the injected side of the calvariae was resorbed by osteoclasts and was subsequently replaced by increased amounts of new bone. These longer term local effects on bone turnover were prevented by indomethacin. However, indomethacin did not prevent the formation of new bone inside the calvariae at sites of resorption induced by IL-1 independent of prostaglandin production. These findings indicate that IL-1 stimulates bone turnover systemically, independent of prostaglandin production, and that it has profound long term local effects on bone turnover that are mediated through prostaglandins.

Analysis of transforming growth factor beta and other cytokines in autoimmune exocrinopathy (Sjögren's syndrome).

Cytokines play a major role in tissue destruction caused by autoimmune dysregulation. In Sjögren's syndrome (SS) patients, salivary glands are the target organs for autoimmune tissue damage. In the present study, reverse transcriptase-polymerase chain reaction (RT-PCR) was used to look for cytokine mRNA expressed in SS salivary glands. Focus score was used to determine the severity of the lesions. Cytokine production in supernatants of the salivary gland cell culture was measured by enzyme-linked immunosorbent assay (ELISA). Immunohistochemical staining was used to identify the local presence of transforming growth factor beta (TGF-beta). Interleukin (IL)-2, IL-6, and IL-10 mRNA were expressed in moderate to severe SS salivary gland lesions. TGF-beta mRNA was constitutively expressed in normal and SS salivary glands. In SS salivary gland cell cultures, IL-6 and IL-10 proteins were produced. TGF-beta production was reduced in high focus score SS glands. Normal and minimally involved SS salivary gland ductal epithelium and acinar cells were found to produce TGF-beta by immunostaining. In conclusion, an excess production of IL-2, IL-6, and IL-10 and a reduced production of the immunosuppressive cytokine, TGF-beta, may be responsible for the progression of the salivary gland lesion in SS. Specific immunotherapy can now be designed based on mechanisms to correct this cytokine imbalance and benefit patients with autoimmune diseases, such as SS.

Guided tissue fabrication from periosteum using preformed biodegradable polymer scaffolds.

A successful tissue engineering method for bone replacement would imitate natural bone graft by providing the essential elements for new bone formation using synthetic scaffolds, osteogenic cell populations, and bone induction factors. This is a study of the suitability of various formulations of poly(DL-lactic-co-glycolic acid) (PLGA) foams to provide a tissue conducting scaffold in an ovine model for bone flap fabrication. Three formulations were used of different copolymer ratio and molecular weight. Porous wafers of PLGA were stacked into rectangular chambers (volume 4 cm3) enclosed on five sides. Some chambers also contained autologous morcellized bone graft (MBG). The chambers were inserted with the open face adjacent to the cambium layer of the periosteum in rib beds of seven sheep and harvested after 8 weeks in vivo. Gross and histologic examination of the resulting tissue specimens demonstrated molded units of vascularized tissue generally conforming to the shape of the chambers and firmly attached to the periosteum. Polymer degradation appeared to occur by varying degrees based on polymer formulation. New bone formation was observed only in areas containing MBG. There was no evidence of significant inflammatory reaction or local tissue damage at 8 weeks. We conclude that a PLGA foam scaffold is (1) an efficient conductor of new tissue growth but not osteoinductive, (2) contributes to the shape of molded tissue, and (3) biocompatible when used in this model. Further studies are warranted to develop practical methods to deliver bone induction factors to the system to promote osseous tissue generation throughout the synthetic scaffold.

The ingrowth of new bone tissue and initial mechanical properties of a degrading polymeric composite scaffold.

Trabecular bone deficiency causes a dilemma at surgery in a variety of clinical situations, including trauma, tumor resection, and reconstruction. A synthetic material to replace trabecular bone would be biocompatible, provide temporary mechanical strength to the reconstructed region, and serve as a scaffold upon which new bone could grow (i.e., osteoconduction). In addition, it should serve as a carrier for osteoinductive biomolecules, degrade into nontoxic materials that the body can excrete via normal metabolic pathways, and allow the new bone to remodel along lines of local stress. A particulate filled composite based on an unsaturated linear polyester was designed as a candidate material for this application. The components are mixed with a monomer that cross links the double bonds of the unsaturated polyester. Degradation occurs via hydrolytic degradation of the backbone polymer's ester linkages. This strategy of prepolymer synthesis via condensation polymerization in the laboratory followed by cross linking the unsaturated prepolymer via radical polymerization at surgery offers design flexibility. The radical polymerization allows curing during surgery to facilitate reconstruction of various shaped defects. The laboratory synthesis of the prepolymer allows alterations of its composition and physical properties to effect desired properties in the resulting composite. This study investigates the effect of several composite material formulations on the in vitro mechanical properties and the associated in vivo histologic characteristics of the resulting material. The prepolymer molecular weight, presence of a leachable salt, and amount of cross linking monomer had strong effects on the resulting strength and modulus of the composite. These strengths were on the order of 5 MPa, a magnitude appropriate for consideration of the material as a temporary trabecular bone substitute. The in vivo studies in a rat proximal tibia model demonstrated progressive growth of new bone against the receding surface of the degrading material, and ingrowth of new bone trabeculae into the interior of the degrading specimen. The specimen was also well integrated with the surrounding bone, with no internal fibrosis. There was an absence of a foreign body inflammatory response to the presence of this material over a 5-week time span. This material may thus be an attractive candidate for temporary replacement of trabecular bone, facilitating both osteoconduction and osteoinduction.

Human Curvularia infections. Report of five cases and review of the literature.

Curvularia lunata is a saprobic dematiaceous mould that resides primarily in soil (Ellis, 1966). Reports of human disease caused by this organism are rare but include: endocarditis, brain abscess, skin infections, onychomycosis, keratitis, pneumonia, disseminated disease, mycetoma, allergic bronchopulmonary disease, and one case of sinusitis. Since 1983, we have encountered five cases of paranasal sinusitis due to C. lunata. None of the patients suffered from known immunologic disorders or underlying debilitating diseases. These five cases are presented and the literature of human phaeohyphomycosis caused by Curvularia spp. is reviewed.

Immunoperoxidase characterization of extramedullary plasmacytoma of the head and neck.

A retrospective analysis of 18 cases of extramedullary plasmacytoma (EMP) was done to characterize the type of immunoglobulin being synthesized by the neoplastic cells. The immunoperoxidase stain can be performed on previously fixed tissue and offers a means of predicting the likelihood of progression to multiple myeloma (MM). Tumors producing IgG make up the majority of cases of EMP in the literature and only 9% have progressed to MM. Smaller numbers of tumors producing other immunoglobulin classes have been studied, but it is clear that their prognosis is much more grave.

Novel formulation of fibroblast growth factor-2 in a hyaluronan gel accelerates fracture healing in nonhuman primates.

Recent advances in understanding the biology of fracture healing and the availability of specific macromolecules has resulted in the development of novel treatments for injuries to bone. Fibroblast growth factor-2 or basic fibroblast growth factor (4 mg/ml), a potent mitogen, and hyaluronan (20 mg/ml), an extracellular matrix component, were combined into a viscous gel formulation intended for direct, percutaneous injection into fresh fractures. In an experimental primate fracture model, a bilateral 1-mm-gap osteotomy was surgically created in the fibulae of baboons. A single direct administration of this hyaluronan/fibroblast growth factor-2 formulation to the defect site significantly promoted local fracture healing as evidenced by increased callus formation and mechanical strength. Radiographic analysis showed that the callus area was statistically significantly larger at the treated sites than at the untreated sites. Specimens treated with 0.1, 0.25, and 0.75 ml hyaluronan/fibroblast growth factor-2 demonstrated a 48, 50, and 34% greater average load at failure and an 82, 104, and 66% greater energy to failure than the untreated controls, respectively. By histologic analysis, the callus size, periosteal reaction, vascularity, and cellularity were consistently more pronounced in the treated osteotomies than in the untreated controls. These results suggest that hyaluronan/fibroblast growth factor-2 may provide a significant advance in the treatment of fractures.

The relation of keratinization to bacterial colonization on the baboon tongue as demonstrated by scanning electron microscopy.

Scanning electron microscopic observations of the baboon tongue demonstrating specificity of microbial distribution as related to epithelial keratinization are presented. The number of bacteria inhabiting a surface was related to the degree of keratinization. Orthokeratotic dorsal tongue surfaces were most heavily colonized by bacteria. The parakeratotic and non-keratinized surfaces were less heavily populated, with non-keratinized areas showing the fewest numbers of bacteria.

The use of bone morphogenetic protein in the treatment of non-union in a canine model.

A non-union model was established in the mid-part of the radial diaphysis in dogs. The non-union was treated with operative implantation of a carrier (guanidine-extracted, demineralized bovine bone or a polylactic acid polymer), alone or in combination with fractions that had been enriched in bone morphogenetic protein. All sites of treatment were examined radiographically and histomorphometrically at twelve weeks after implantation. Guanidine-extracted, demineralized bovine bone, alone or combined with fifteen milligrams of canine bone morphogenetic protein, failed to induce any healing of the non-union. When polylactic acid alone had been implanted, a small amount of reparative new bone was found in the defect at three months. When polylactic acid combined with fifteen milligrams of canine bone morphogenetic protein had been implanted, a significant increase in new bone formation was seen (p less than 0.03), compared with that seen in control animals. Trabecular bone bridged the gap between the proximal and distal fragments in all four specimens from the dogs that had received that treatment. In contrast, when polylactic acid combined with bovine bone morphogenetic protein had been implanted, significant reparative new bone was not found in the defect at three months.

An intraosseous device for studies of bone-healing. The effect of transforming growth-factor beta.

A novel implantable device, the analytic bone implant, was used in order to establish a model for studies of bone-healing and the evaluation of factors that augment the process, such as transforming growth-factor beta (TGF-beta). This device was implanted into the tibiae of four baboons. After healing, bone was removed from the center chamber. Recombinant human TGF beta-1 was then delivered to the core of the device. After twenty-two days of healing, the device was disassembled and the newly formed bone was removed from the core of the implant for histomorphometric analysis. An analysis of the bone revealed a substantial effect of TGF-beta on osteoblastic activity and proliferation compared with that seen in control and placebo groups. However, despite increased osteoblastic activity, trabecular bone volumes at twenty-two days were equivalent among the groups. The number of osteoclasts and the erosion of the surface were also increased, although not significantly so. Substantial endochondral formation of bone was seen in the supraperiosteal tissues directly over the implants that contained TGF-beta but not over the implants in the control and placebo groups. These data demonstrate the utility of this bone-implant model for studies of bone-healing with minimally invasive methods. In addition, use of the device provided the first in vivo data on the effects of TGF-beta at an intermediate (twenty-two-day) time-point in the healing process in a non-human primate.

Bone morphogenetic protein but not transforming growth factor-beta enhances bone formation in canine diaphyseal nonunions implanted with a biodegradable composite polymer.

BACKGROUND: The purpose of the present study was to create an effective bone-graft substitute for the treatment of a diaphyseal nonunion. METHODS: A standardized nonunion was established in the midportion of the radial diaphysis in thirty mongrel dogs by creating a three-millimeter segmental bone defect (at least 2 percent of the total length of the bone). The nonunion was treated with implantation of a carrier comprised of poly(DL-lactic acid) and polyglycolic acid copolymer (50:50 polylactic acid-polyglycolic acid [PLG50]) containing canine purified bone morphogenetic protein (BMP) or recombinant human transforming growth factor-beta (TGF-beta1), or both, or the carrier without BMP or TGF-beta1. Five groups, consisting of six dogs each, were treated with implantation of the carrier alone, implantation of the carrier with fifteen milligrams of BMP, implantation of the carrier with 1.5 milligrams of BMP, implantation of the carrier with fifteen milligrams of BMP and ten nanograms of TGF-beta1, or implantation of the carrier with ten nanograms of TGF-beta1. At twelve weeks after implantation, the radii were examined radiographically and the sites of nonunion were examined histomorphometrically. RESULTS: We found that implantation of the polylactic acid-polyglycolic acid carrier alone or in combination with ten nanograms of TGF-beta1 failed to induce significant radiographic or histomorphometric evidence of healing at the site of the nonunion. The radii treated with the carrier enriched with either 1.5 or fifteen milligrams of BMP showed significantly increased periosteal and endosteal bone formation on histomorphometric (p < 0.05) and radiographic (p < 0.02) analysis. CONCLUSIONS: Bone formation in a persistent osseous defect that is similar to an ununited diaphyseal fracture is increased when species-specific BMP incorporated into a polylactic acid-polyglycolic acid carrier is implanted at the site of the nonunion. TGF-beta1 at a dose of ten nanograms per implant did not induce a similar degree of bone formation or potentiate the effect of BMP in this model. CLINICAL RELEVANCE: The biodegradable implant containing BMP that was used in the present study to treat diaphyseal nonunion is an effective bone-graft substitute.

An IgD extramedullary plasmacytoma involving the sphenoid sinus at onset: an immunohistochemical study.

A 60-year-old man developed a left VIth-nerve paralysis and underwent biopsy of an extramedullary plasmacytoma in the sphenoid sinus. Immunohistochemistry demonstrated cytoplasmic IgD and lambda determinants. Subsequent specimens from left clavical and thoracic epidural lesions also showed a plasma cell neoplasm with IgD and lambda determinants. No abnormal plasma cells were found in bone marrow specimens. Lambda Bence-Jones protein and monoclonal IgD-lambda protein in the serum were detected several months after onset. The patient died with disseminated plasmacytoma 1 year after diagnosis. The mode of presentation, pattern of metastasis, and secretory features were typical of extramedullary plasmacytoma arising in the upper airway. Immunohistochemistry was more productive than serum electrophoresis and immunoelectrophoresis for following the course of this neoplasm.

Autoradiographic evidence of sex steroid receptors in laryngeal tissues of the baboon (Papio cynocephalus).

A number of studies have implicated the gonadal steroids as significant factors in laryngeal development and disease. In addition, antiandrogens are receiving limited trials in the treatment of laryngeal carcinoma. However, there is little experimental data to document the presence, and more specifically the location of receptors for the sex steroid hormones in the larynx. The purpose of this study is to provide such data. Utilizing an autoradiographic technique, tissues from baboons injected with tritiated estradiol (3H-E2) or dihydrotestosterone testosterone (3H-DHT) were examined and analyzed. The data gathered confirmed that the larynx is rich in receptors for these steroids and that there are specific patterns of distribution of receptor positive cells. The vocalis muscle and other mesenchymal tissues contained the largest number of receptors, while ciliated columnar and stratified squamous epithelium were negative. The significance of these findings is discussed.

Fungal maxillary sinusitis caused by Curvularia lunata.

Recently, reports have surfaced in which saprobic fungi, as well as fungi pathogenic for plants, seem to be evolving as human pathogens. While the bulk of infected patients are immunocompromised, many individuals appear to be immunocompetent. To our knowledge, this is the second known published case of maxillary sinusitis caused by Curvularia lunata in an immunocompetent patient. An interesting feature of this case is that the patient was treated with only surgery (débridement and irrigation) with complete resolution and no recurrence.

Nuclear uptake of sex steroids in gingiva of the baboon.

Influences of sex steroid hormones on the periodontium are documented. Confirmation of the presence and location of receptors for these hormones is important in achieving a more complete understanding of their role in periodontal disease. Biochemical studies have provided evidence of sex steroid receptors in cytosols prepared from gingival tissues, however, no morphologic data are available on the location or specific cell types with receptors. For this study, three mature male and three female baboons received tritiated 5-alpha-dihydrotestosterone (3H-DHT) while three similar males and three females received tritiated estradiol-17 beta (3H-E2). A control animal from each group of three was given an excess of the corresponding unlabeled hormone in addition to the tritiated dose. Gingival specimens were obtained at necropsy as part of a total body study of receptor distribution, and autoradiographs were prepared on emulsion-coated slides. Heavy nuclear uptake was observed in periosteal fibroblasts and scattered fibroblasts of the lamina propria in male given 3H-E2. Females had moderate uptake of E2 in similar but fewer sites than males. There was minimal uptake of 3H-DHT in one male and two females. These data provide additional evidence of steroid receptors in the periodontium and contribute to our understanding of responses of the periodontium to sex steroids.