Induction of reparative dentine formation in monkeys by recombinant human osteogenic protein-1

Osteogenic protein-1 (OP-1, BMP-7), a member of the transforming growth factor-beta supergene family, induces cartilage and bone formation when implanted in intra- and extraskeletal sites in vivo. The human OP-1 gene has been cloned and biologically active recombinant OP-1 homodimers (hOP-1) produced. The amount of bone induced by hOP-1 in vivo is related to the amount of protein implanted. Dentine possesses bone morphogenetic protein (BMP) activity. Impure material from allogenic bone with BMP activity induced reparative dentine formation in dogs. The objective of this study was to determine if the amount of reparative dentine stimulated by hOP-1 is related to the amount of protein utilized in direct pulp-capping experiments. Freshly exposed molar and premolar pulps were treated with varying amounts of a complex comprising hOP-1 and a carrier matrix of purified bovine type-1 collagen powder (CM) moistened with sterile saline. Reparative dentine was present in all hOP-1/CM treated teeth (12 of 15) that remained sealed for the 6 weeks' healing. Substantially more new dentine was present in teeth treated with hOP-1/CM than in those treated with Ca(OH)2 paste and the amount of reparative dentine formed was proportional to the amount of hOP-1/CM (P < 0.05). No reparative dentine formed in collagen carrier or untreated teeth. The appearances of the new tissue suggested that much of the mass of the hOP-1/CM was replaced first by a pulp-like connective tissue, which mineralized to form reparative dentine.(ABSTRACT TRUNCATED AT 250 WORDS)     

Effects of cementum-impregnated gelatine membrane on early healing following periodontal flap surgery

The purpose of this study was to investigate the initial healing response of surgically flapped periodontal tissues in the presence of gelatine membrane compounded with particles of cementum. Four monkeys with no periodontal disease were used in this experiment. Full thickness flaps were raised and recession type defects were created on the buccal side of the maxillary lateral incisors and second premolars. Exposed root surfaces were thoroughly curetted, and composite cementum-impregnated membranes placed directly onto planed root surfaces. Flaps were then sutured back to the original position. Animals were sacrificed at 2, 4, 7 and 10 post-surgical days, and block specimens including the tooth, gingiva and bone were subsequently processed for light and electron microscopy. The resultant analysis revealed that gelatine membranes were partially resorbed at 2 days post-surgery and completely resorbed by 10 postoperative days. In the early stages of gelatine resorption, most liberated cementum particles accumulated on planed dentin surface but some became demineralized within the surgical wound. Cementoblast-like cells with well-developed rough endoplasmic reticulum appeared on the root surface 7 days following surgery. Newly synthesized collagen fibrils aligned parallel to the root surface were seen at 10 post-surgical days. The results suggest that the newly developed composite membrane enhances the formation of new periodontal attachment.     

Recombinant human osteogenic protein-1 induces dentin formation: an experimental study in miniature swine

It was the aim of the present study to investigate the induction of dentin formation by recombinant human osteogenic protein-1 (hOP-1). In 4 adult miniature pigs a total of 16 teeth with artificially exposed dental pulps were capped with 3 mg of a complex of recombinant hOP-1 in collagen matrix (2.5 micrograms/mg), collagen matrix alone, or calcium hydroxide paste. Teeth were removed in block section after a healing period of 5 weeks. Decalcified sections were processed for light microscopy and histomorphometric analysis. In hOP-1 treated teeth substantial amounts of hard tissue formation (osteodentin and tubular dentin) had consistently led to a complete bridging of the defects. Less dentin formation was seen after calcium hydroxide application. In control defects collagen matrix alone failed to form complete dentin bridges. Recombinant human osteogenic protein-1 in a collagen carrier matrix appeared to be suitable as a bio-active capping agent for surgically exposed dental pulps.     

Analysis of genes implicated in osteogenic signaling by osteogenic protein-1 (OP-1) using differential display method

Bone morphogenetic proteins (BMPs), members of a transforming growth factor-beta (TGF-beta) superfamily, are growth and differentiation factors which induce ectopic bone formation in vivo. Although many studies on osteoinductive properties of BMPs have been conducted, little is known about the downstream components in the signal transduction machinery, beyond the mechanism of BMP receptor activation. In this study, the osteogenic effects by osteogenic protein-1 (OP-1, BMP-7) on osteoblastic cell line MC3T3-E1 and murine stromal cell line ST2 were investigated, especially focusing on differentially expressed genes induced by OP-1 using the differential display method. The major findings were as follows: 1) Alkaline phosphatase specific activities of both MC3T3-E1 and ST2 increased in a dose-dependent manner by OP-1 stimulation. 2) Northern analysis showed a significant increase of osteocalcin mRNA after 7 days of OP-1 treatment. 3) 77 genes, which were differentially expressed in MC 3 T 3-E1 and ST 2 cells, were detected on differential display fingerprints after 16-hour treatment of OP-1. 4) Some of these clones showed high levels of identical to known genes. 5) One clone called ST3v, down-regulated in ST2 cells by OP-1 stimulation, was confirmed with quantitative RT-PCR.     

The integrated processes of hard tissue regeneration with special emphasis on fracture healing

When bone is fractured, a sequence of dynamic events ensue to restore form and therefore function. Many key biologic cell regulators for these events have been identified, expressed through recombinant technology, and their roles posited. Moreover, the availability of recombinantly engineered molecules, such as the bone morphogenetic proteins with their potential to benefit patient care, has ushered in an important era in clinical dentistry that may eliminate either autografting or bank bone allografts. Therefore, in this review article, we have highlighted some of the exciting biologic regulators relevant to bone fracture healing and outlined the dynamic elements in this process.     

Neutrophil attractant protein-1 and monocyte chemoattractant protein-1 in human serum. Effects of intravenous lipopolysaccharide on free attractants, specific IgG autoantibodies and immune complexes

We recently found that normal human sera contain IgG antibodies against two chemoattractants, neutrophil attractant protein-1 (NAP-1/IL-8) and monocyte chemoattractant protein-1 (MCP-1), as well as immune complexes of these proteins. Intravenously administered LPS was reported to cause a sharp rise in serum NAP-1 concentration. Our study was designed to determine if LPS also caused an increase in MCP-1 and to measure associated changes in concentrations of antibody and immune complex. LPS caused a rise to peak within 2 to 3 h in serum concentrations of free NAP-1 and MCP-1, followed by an almost equally rapid fall toward base-line levels by about 5 h postinjection. MCP-1 concentration in sera from the 11 subjects rose to a peak of 330 +/- 52 pM. The peak value for NAP-1 was 80 +/- 11 pM. In 10 of the 11 subjects, free IgG autoantibody to MCP-1 decreased from a mean pre-LPS value of 1820 +/- 660 pM to a mean low of 53% of the respective initial values. Corresponding data for IgG anti-NAP-1 were a pre-LPS concentration of 216 +/- 7 pM, which decreased to a mean low of 44% of the respective initial values. The finding in some subjects of a rapid rise in free antibody after the nadir suggests the possibility of acute regulation of autoantibody secretion rates. Although the results suggested that LPS-induced chemoattractant combined with free antibody, serum concentrations of MCP-1-IgG or NAP-1-IgG did not increase, which points to an as yet unknown mechanism for trapping and elimination of the immune complexes.     

Maxillary sinus augmentation in the non-human primate: a comparative radiographic and histologic study between recombinant human osteogenic protein-1 and natural bone mineral

The posterior maxilla has traditionally been one of the most difficult areas to successfully place dental implants due to poor bone quality and close approximation to the maxillary sinus. Sinus augmentation procedures have become a viable means of assuring adequate bone for the placement of dental implants in this area. However, with the techniques currently employed, a considerable variation in the quality of bone attained with the sinus augmentation procedure exists. The purpose of this in vivo study was to evaluate the healing response and bone formation stimulated by 3 doses of recombinant human osteogenic protein-1 (rhOP-1), 0.25, 0.6, and 2.5 mg OP-1 per gram of collagen matrix; natural bone mineral; or collagen matrix alone (control) placed in the maxillary sinus of adult chimpanzees. Results were assessed using clinical, histologic, and radiographic techniques. Radiographic analysis of the computed tomography scans taken at 1 week, and 2.5, 4.5, and 6.5 months revealed a more rapid mineralization with the 2.5 mg OP-1/g collagen matrix and natural bone mineral treatment groups. The incremental bone mineral density (BMD) increase for these 2 treatments from 1 week to 2.5 months was over 2.5 times the increase found with the collagen matrix alone; these 2 treatments also had a higher BMD at the most superior slices evaluated when compared to the other 3 groups. Biopsy specimens were taken at 3.5, 5.5, and 7.5 months and for all 5 treatment groups bone formation was observed at all time points in the majority of the specimens. At 7.5 months the 2.5 and 0.6 mg OP-1/g collagen matrix treatment groups had an increase in the percent bone area when compared to the matrix alone control. In conclusion, these results demonstrate that sinus augmentation with natural bone mineral or 2.5 mg OP-1/g collagen matrix induce comparable radiographic and histologic evidence of bone formation and that both of these treatments performed superior to the control group of collagen matrix alone based upon all methods of evaluation.     

The effect of prednisolone on the systemic response and wound healing after colonic surgery

To study the effect of preoperative treatment with a single high-dose of glucocorticoid on the systemic and immunological response, wound healing, and convalescence after colonic surgery, thirty patients were double-blind randomized to receive either methylprednisolone 30 mg/kg intravenously 90 minutes prior to induction of anaesthesia (group 1, n = 12), or to receive placebo (group 2, n = 12). Six patients were excluded from the study. Assessments of pain, pulmonary function, convalescence, various injury and wound-healing factors were done until 10 days after surgery. Conventional reduction in pulmonary function and mobilization was improved in group 1. Interleukin-6 and C-reactive protein levels increased significantly less in group 1, as delayed-type hypersensitivity was abolished in group 1. Plasma cascade system activation was significantly less pronounced in group 1. Reduction of collagen turnover was observed in group 1, but without detrimental effect on collagen accumulation. It is concluded that treatment with a single high dose of glucocorticoid before colonic surgery may improve postoperative pulmonary function and mobilization and reduce plasma cascade system activations, the inflammatory response, and immunofunction, but without detrimental effects on wound healing.     

Immunolocalization and messenger RNA expression of bone morphogenetic protein-6 in human benign and malignant prostatic tissue

Skeletal metastases are common in advanced prostate cancer, causing considerable morbidity, and they are usually osteoblastic in nature with no clear explanation for this phenomenon. Bone morphogenetic proteins (BMPs) induce bone formation in vivo, and preliminary work showed a possible association between BMPs and prostatic skeletal metastases; differential expression favors BMP-6 as a potential new marker and mediator of osteosclerotic deposit formation. We investigated BMP-6 mRNA and protein expression by in situ hybridization and immunohistochemistry in malignant and benign prostates from 40 men. BMP-6 mRNA expression was detected exclusively in malignant epithelial cells in 20 of 21 patients (95%) with metastases and in 2 of 11 patients (18%) with localized cancer, and it was absent in 8 benign samples. Immunostaining for BMP-6 was predominantly cytoplasmic and was present in all primary tumors with established metastases and in 4 of 11 (36%) organ-confined cancers. In benign prostatic hyperplasia, basal cells and areas of basal cell hyperplasia were positive for BMP-6 by immunohistochemistry. The results suggest a close association between BMP-6 expression in primary malignant prostatic tissue and skeletal metastases. BMP-6 may be responsible, in part, for the osteoblastic changes in metastatic lesions secondary to prostate cancer.     

Effect of osteogenic protein-1 on the development and mineralization of primary cultures of avian growth plate chondrocytes: modulation by retinoic acid

Osteogenic protein-1 (OP-1), a member of the TGF-beta family of proteins, induces endochondral bone formation. Here we studied the effect of OP-1 on the development of primary cultures of avian growth plate (GP) chondrocytes in either serum-free or serum-containing medium, in the absence or presence of retinoic acid (RA). OP-1 was added on day 7 of culture and continued for 7 days, or until the cultures were harvested, typically on day 21. Alone, OP-1 caused approximately 2-fold increase in proteoglycan synthesis into both the medium and the cell:matrix layer. Additionally, OP-1 caused a dosage-dependent increase in alkaline phosphatase (ALP) activity, and an increase in protein, when given from days 7-14 and examined on day 14. This stimulation was greater in cells grown in serum-free than in serum-containing media (3-5-fold vs. 2-3-fold increase in ALP; approximately 40% vs. approximately 20% increase in protein). Such stimulation of ALP activity and proteoglycan (PG) synthesis in cultured GP cells indicates that OP-1 elicits differentiation of chondrocytes. OP-1 minimally affected cell division (DNA content); however, a slight increase was seen when examined early in the culture. Alone, OP-1 increased mineral (Ca and Pi) content of the cultures by approximately 2-fold in both types of media. As early as day 14, clusters of mineral encircled many of the OP-1 treated cells. Thus, as in vivo, OP-1 strongly promoted mineral formation by the cultured GP chondrocytes. When present together, OP-1 and RA generally blocked the action of the other. Separately OP-1 and RA each stimulated protein synthesis, ALP activity, and Ca2+ deposition; together they were inhibitory to each. Also, RA blocked the stimulation of PG synthesis induced by OP-1; whereas OP-1 decreased cell division engendered by RA. Thus, this GP chondrocyte culture system is a good model for studying factors that influence differentiation and mineral deposition during bone growth in vivo.     

Effects of resorbable fixation on craniofacial skeletal growth: modifications in plate size

On the basis of previous experimental work in young rabbits, modifications in the polymer fixation used (i.e., smaller plate size, change in ratio of polymer components) were studied for potential alleviation of known growth restriction in this model. Ten juvenile rabbits with metallic markers placed at the calvarial sutural junctions were plated across the left coronal suture. At 6 months of age, measurements were taken of both intermarker distances radiographically (growth) and the plates directly. Unlike previous reports, these resorbable plates permitted symmetrical frontal bone development, unaffected growth across the coronal suture, and a histologically normal underlying suture. These changes appear to be the result of elongation of the fixation plate across a growth site. These results indicate that the change in shape of resorbable fixation after initiation of degradation is more important than complete degradation of the material in rapidly growing bone sites.     

Macrophage inflammatory protein-1beta induces migration and activation of human thymocytes

The CC chemokine macrophage inflammatory protein 1beta (MIP-1beta), has been shown to be a chemoattractant preferentially activating CD4(+) CD45RA+ T lymphocytes. Further analysis of chemokine action on lymphocytic cells has shown the potent migration-promoting capacity of MIP-1beta on human thymocytes. The responding cells were the CD4(+) and CD8(+) single-positive (SP), as well as the CD4(+) CD8(+) double-positive (DP) populations, with little if any migratory activity on the double-negative (DN) population. The activation of thymocytes by MIP-1beta appeared to be a direct, receptor-mediated event as evidenced by the rapid mobilization of intracellular calcium, increase in proteins phosphorylated on tyrosine, and activation of the mitogen-activated protein kinase (MAPK) pathway. Radioligand binding analyses showed specific and displaceable binding of MIP-1beta to thymocytes with a Kd of approximately 1 nmol/L, a profile that was comparable with MIP-1beta binding to CCR-5-transfected NIH 3T3 cells. In addition, CCR-5 mRNA was detected in total thymocyte populations indicating that activation of thymocytes by MIP-1beta may occur through binding to CCR-5. Further dissection of the subpopulations showed that only the DP and CD8(+) SP populations expressed CCR-5 and expression data on these two populations was confirmed using anti-CCR-5 monoclonal antibody. These data may be suggestive of a role for MIP-1beta in human thymocyte activation, and show a potential route for HIV infectivity in the developing immune system.     

Effects of quarantine on cats and their owners

The effects of quarantine on 16 cats and their owners were assessed by means of four questionnaires completed by the owners at the beginning of their cat's stay in quarantine, three months later, and two weeks and three months after the cats left quarantine. Changes in body condition were evident in two-thirds of the cats during and at the end of quarantine but not three months later. Mid-way through quarantine, the owners considered their cats were less attached to them, less relaxed, more excitable, more aggressive, more nervous and less playful than before quarantine. When they left quarantine the cats were friendlier, more affectionate and more timid, and three months later they were more affectionate, more nervous and more vocal than before quarantine. When they left quarantine and three months later the cats spent more time with their owners than before quarantine. Most owners visited their cats once or twice a month; the location of the cattery and the limited opening hours restricted the number of visits they made.     

Effects of halothane on the metabolism of human adipose tissue

The metabolism of specimens of human adipose tissue exposed to different concentrations of halothane was studied. Halothane was added to the incubation medium directly or via the gas phase above the medium. The basal lipolysis was significantly increased by low concentrations of halothane. Higher concentrations clearly diminished the lipolysis, but here, in spite of the inhibitory effect on the basal lipolysis, the lipolytic effect of noradrenaline expressed as percent increment was increased. The rate of lipid synthesis from glucose was reduced when halothane was present in the gas phase. The effect of insulin on glucose metabolism was not affected by the presence of halothane, while the antilipolytic action was abolished by high concentrations of halothane. The results show that halothane may exert dual effects on the mobilization of lipids from human adipose tissue; at low concentrations halothane enhances the basal lipolysis, while at higher concentrations it exerts inhibitory effects.     

Effects of irradiation on osseointegration before and after implant placement: a report of three cases

Three patients irradiated as a part of cancer treatment, both before and after placement of endosseous implants, were studied. Total irradiation doses varied from 80 to 195 Gy in the tumor/implant area. Implants failed at a rate of 64.2% during a 3-year follow-up period. All patients developed osteoradionecrosis in the tumor cavities adjacent to the implants. The combined effects of the pre- and postoperative irradiation, in conjunction with the placement of implants, appear to challenge the limits of osseointegration. Until further knowledge is obtained regarding how such highly radiated tissue should be handled, it is recommended that if anchorage of craniofacial prostheses is attempted according to osseointegration principles, it should be performed with the utmost care.     

Plasma levels of monocyte chemoattractant protein-1 but not those of macrophage inhibitory protein-1alpha and RANTES correlate with virus load in human immunodeficiency virus infection

Plasma levels of proinflammatory cytokines, cytokine inhibitors, and the beta chemokines RANTES, macrophage inhibitory protein (MIP)-1alpha, and monocyte chemoattractant protein (MCP)-1 were studied in relationship with virus load in 40 patients exhibiting plasma levels of HIV RNA ranging between undetectable and levels >10(6) copies/mL. Mean plasma levels of MCP-1 were increased in patients with high virus load compared with HIV-seropositive subjects with undetectable plasma viral RNA and healthy controls. MCP-1 levels were directly correlated with plasma levels of HIV RNA. No correlation was observed between virus load and plasma concentrations of MIP-1alpha and RANTES. The results suggest that low rates of viral replication in vivo are not dependent on increased production of the suppressive chemokines RANTES and MIP-1alpha. Since MCP-1 upregulates viral replication in vitro, the results may suggest a role for MCP-1 in triggering viral replication in HIV disease.     

The effect of recombinant human bone morphogenetic protein-2 (rhBMP-2) on the healing of full-thickness defects of articular cartilage

Articular cartilage has a limited capacity for repair. We investigated the effect of rhBMP-2 (recombinant human bone morphogenetic protein-2) on the healing of full-thickness osteochondral defects in adult New Zealand White rabbits. A single defect, three millimeters wide by three millimeters deep, was created in the trochlear groove of the right femur in eighty-nine rabbits. The defect was either left empty, filled with a plain collagen sponge, or filled with a collagen sponge impregnated with five micrograms of rhBMP-2. The animals were killed at four, eight, or twenty-four weeks, and the repair tissue was examined histologically and evaluated with use of a grading scale. The defects also were examined immunohistochemically for the presence of type-II collagen at four and eight weeks. The rate of bone repair was evaluated with fluorescent labeling of bone at two and four weeks and with use of fluorescence microscopy at eight weeks. Treatment with rhBMP-2 greatly accelerated the formation of new subchondral bone and improved the histological appearance of the overlying articular surface. At twenty-four weeks, the thickness of the repair cartilage was 70 per cent that of the normal adjacent cartilage and a new tidemark usually had formed between the repair cartilage and the underlying subchondral bone. The average total scores on the histological grading scale were significantly better (p < 0.01) for the defects treated with rhBMP-2 than for the untreated defects (those left empty or filled with a plain collagen sponge) at all time-points. Immunostaining with an antibody against type-II collagen showed the diffuse presence of this cartilage-specific collagen throughout the repair cartilage in the treated defects. The untreated defects demonstrated minimum staining with this antibody.     

Effects of ketamine on somatosympathetic reflex discharges in cats

Effects of ketamine on somatosympathetic reflex discharges induced from sympathetic trunk with electrical stimulation of superficial peroneal nerve were investigated in 51 cats under anesthesia with urethane and alpha chloralose. These reflex discharges through spinal cord and medulla oblongata consist of two components, A and C reflexes, which are derived from somatic myelinated and unmyelinated afferent fiber respectively. Amplitudes of both A and C reflex potentials were depressed significantly after intravenous injection of ketamine 10 mg.kg-1. The maximum depression was observed 5 min after administration. In decerebrated cats with surgical transection at the midbrain, both A and C reflexes were also depressed after administration of the same dosages, and the maximum level of the depression was more profound than that in brain intact cats. After intrathecal injection of ketamine 1 mg.kg-1 to the lumbar spinal region, a slight depression of C reflex was found, but, dosages of 10 mg.kg-1 significantly depressed both A and C reflexes to the similar levels as those in iv injection to brain intact cats. The maximum depression was observed 30 min after administration. The depressive effects on both reflexes of intravenous ketamine 10 mg.kg-1 were not antagonized by naloxone 0.06 mg.kg-1 in brain intact cats. These results suggest that the suppressive effects of ketamine on somatosympathetic reflexes are caused by direct inhibition of medulla oblongata and spinal cord, whereas supra-midbrain regions may be activated by ketamine, and the effect of ketamine is predominant on medulla oblongata in this situation rather than on the spinal cord.     

Effects of interleukin-1 blockers on ophthalmic wound healing in a rabbit model of trabeculectomy

A trabeculectomy model was used to test the efficacy of some Interleukin-1 blockers on the modulation of ophthalmic wound healing in the pigmented rabbit. The corticosteroid, methyl prednisolone, was used as a positive control and was effective in prolonging the time to failure of the trabeculectomy by 21%. The experimental compounds CK-17, CK-101A, CK-102 and CK-103A were more efficacious than vehicle control at prolonging the number of days before failure of the trabeculectomy by 55%, 55%, 30% and 79%, respectively. The CK- compounds increased the amount of time before failure of the fistula, with no side effects at the doses tested to date.