Evaluation of bone healing enhancement by lyophilized bone grafts supplemented with platelet gel: a standardized methodology in patients with tibial osteotomy for genu varus.

Orthopedic practice may be adversely affected by an inadequate bone repair that might compromise the success of surgery. In recent years, new approaches have been sought to improve bone healing by accelerating the rate of new bone formation and the maturation of the matrix. There is currently great interest in procedures involving the use of platelet gel (PG) to improve tissue healing, with satisfactory results both in vitro and in maxillofacial surgery. Otherwise, to our knowledge, only a preliminary clinical study was undertaken in the orthopedic field [Kitoh et al., Bone 2004;35:892-898] and the efficacy of PG is still controversial. Our paper focuses on the effect on bone regeneration by adding PG to lyophilized bone chips used for orthopedic applications. The clinical model and the laboratory methodology were standardized. As a clinical model, we employed the first series of patients of a randomized case-control study undergoing high tibial osteotomy (HTO) for genu varus. Ten subjects were enrolled: in 5 patients lyophilized bone chips supplemented with PG were inserted during tibial osteotomy (group A); 5 patients were used as a control (group B) and lyophilized bone chips without gel were applied. Forty-five days after surgery, computed tomography scan guided biopsies of grafted areas were obtained and the bone maturation was evaluated by a standardized methodology: the osteogenic and angiogenic processes were semi-quantitatively characterized by using histomorphometry, and the mineral component of the lyophilized and host bone was analyzed by using X-ray diffraction technique with sample microfocusing and microradiography. Lyophilized bone with PG seems to accelerate the healing process, as shown by new vessel formation and deposition of newly formed bone, with no evidence of inflammatory cell infiltrate, when compared with lyophilized bone without gel. On the contrary, lyophilized bone undergo a resorption process, and a fibrous tissue often fills the spaces between chips. A histiocytic/giant-cell reaction is sometimes present. Otherwise, no differences have been found concerning microstructure. Our findings show the reliability of the methodology used to monitor early bone repair. The completion of the study and the evaluation of the ultimate clinical outcome are necessary in order to verify PG in vivo effects in orthopedic surgery.

A preliminary in vitro and in vivo study of the effects of new anthraquinones on neutrophils and bone remodeling.

Osteolysis, that is, progressive periprosthetic bone loss, is responsible for approximately 70% of aseptic loosening and implant failure. Usually, it is due to a granulomatous reaction wear-induced, leading to macrophage and osteoclast-mediated bone resorption. At present, there is no established prophylaxis or treatment for this process. For this purpose, as a preliminary investigation, we aimed to study the effects in two directions, inhibition of proinflammatory signals, and bone remodeling activity, of two newly synthesized anthraquinone molecules [N,N'-Diethylamino-2,6-anthraquinone-disulfonamide (GR375) and N,N'-(p-ethoxyphenyl)-2,6-anthraquinone-disulfon amide (GR377)]. Among the pro-inflammatory signals, the ability of the two anthraquinones to interfere with the production of superoxide anion (O(2) (-)), which was assumed as a marker of reactive oxygen species (ROS), was evaluated in an in vitro cell model by testing phagocytes, such as human neutrophils, challenged by the chemotactic agent N-formylmethionyl-leucyl-phenylalanine (FMLP). Both compounds inhibited O(2) (-) production, in a dose-dependent way, without exerting scavenger effects. An in vivo model was applied to investigate their effect on bone remodeling. Fifty-four female Wistar rats were divided into eight groups of six animals each, and a 4-week treatment was applied in two phases. A 25 mg/kg/os dose in the first phase and 12.5-6.25 mg/kg/os doses in the second one were employed. The tibia trabecular bone at the secondary spongiosa level was analyzed, and trabecular bone volume (%TBV), trabecular thickness (TbTh), and apatite lattice parameters were measured. At the highest doses of GR375 and GR377 the %TBV and the TbTh increased by 33.2, 34.6%, and 3.6 and 9.1%, respectively, whereas crystallographic parameters were not significantly different from the untreated group. Our results suggest a simultaneous antiinflammatory and antiosteoclastic activity of both drugs that encourages to perform further research. If it will be confirmed, they could be proposed in a variety of bone diseases, in particular, when acute inflammation is associated to osteolytic processes and, eventually, in the prevention and treatment of periprosthetic osteolysis.

Diagnosis of infection after total hip replacement.

Subclinical infection in patients with pain following total hip replacement (THR) is an underestimated condition that needs consideration because it mimics aseptic loosening, contributes to periprosthetic osteolysis, and necessitates proper treatment. We aimed to define the reliability of diagnostic parameters that are routinely used before revision surgery for the assessment of infection. A continuous series of 26 subjects who underwent THR revision surgery was considered, including 21 cases diagnosed as aseptic loosening (group A) and 5 hip revisions with a clinical diagnosis for infection (group B). Seven subjects at the time of the primary arthroplasty were used as negative controls (group C). Technetium-99m labeled hydroxymethylene diphosphonate [(99m)Tc-HDP]- and technetium-99m hexamethylpropyleneamine oxide [(99m)Tc-HMPAO)]-labeled granulocyte scintigraphy, histology of peri-implant tissues, laboratory tests for inflammation, and microbiology were performed. Scintigraphy was positive for loosening [positive (99m)Tc-HDP scan] but negative for infection [negative (99m)Tc-HMPAO-labeled granulocyte scan] in all group A patients, whereas in 11 cases (52%) a positive culture was unexpectedly obtained. Histology showed conflicting results: Polymorphonuclear cells (PMNs) were found only in 5 of 11 culture-positive patients, whereas in 2 cases the presence of PMNs did not correspond to a positive culture. In group B patients, both isotope scans and microbiology were found to be positive. All control subjects (group C) had negative cultures. In our opinion, smoldering infection could be present in a significant proportion of cases of failed hip implants currently diagnosed as "nonseptic." The inflammatory response to wear debris and the presence of superimposed, slowly growing bacteria could act synergically, both contributing to the pathogenesis of periprosthetic osteolysis.

Ability of restorative and fluoride releasing materials to prevent marginal dentine demineralization.

The study aimed to define the in vitro secondary caries inhibiting potential of restorative materials currently used in dental practice. Class V restorations were prepared in extracted human third molars and immersed in a demineralizing solution (lactic acid, pH 4.5) at 37 degrees C for 2 days to simulate secondary caries formation. The bonding and the restorative systems tested in the study were: Scotchbond 1+Z 250 (Group A), Scotchbond 1+F 2000 (Group B), ABF+APX (Group C), ABF+F2000 (Group D). Perimarginal dentine, immediately close to the margin of the restoration, and exposed dentine, at approximately 0.5 mm from the margins of the restoration, after exposure to the acid solution, were investigated; protected dentine, at approximately 4 mm from the margin in a varnish-covered area, was analysed as control. Polarized light microscopy and contact transverse microradiography (TMR) were employed. The output parameters were lesion shape and size (depth in microm) of the exposed dentine, dentine mineral volume%, and integrated mineral loss (Delta Z, in %volmicrom) of the lesions. Compomers (Groups B and D) showed a thinner demineralization of the outer lesions, a less demineralization along the perimarginal dentine (inner lesion) and more caries inhibition zones or CIZs (Delta Z positive values) compared to composites (Groups A and C). In conclusion, Groups B and D materials seemed to partially counteract the marginal demineralization induced by an acid solution and favourably influence the formation of CIZs along the restorations. On the contrary, composites did not show a protective effect, probably due to an insufficient marginal seal and the lack of fluoride release.

Enamel microhardness after in vitro demineralization and role of different restorative materials.

This study aimed to evaluate the extension of enamel demineralization around the margin of restorations after immersion in cariogenic solution, in an attempt to define the role of new restorative materials in preventing secondary caries formation. For this purpose, enamel microhardness was measured. Twelve class V restorations in human extracted third molars were prepared in vitro and immersed in a demineralizing solution (lactic acid, pH 4.5) at 37 degrees C for 3 days to simulate the formation of secondary caries and its effect on the marginal integrity of composite restorations. The restorative systems tested in the study were Scotchbond 1 + Z 250 (group A), ABF + APX (group B), Fuji IX (group C), SE Bond + APX (group D), and Scotchbond 1 + F 2000 (group E). The microhardness was measured close to the margin of restoration (marginal exposed enamel), at 2.0 mm from the margin (exposed enamel), and at approximately 4 mm from the margin in a varnish-covered enamel area (protected enamel). Five measurements were made on each site at 20, 40, 60, 80, and 100 microm depth from the external enamel surface. Exposed enamel and marginal exposed enamel were greatly affected by the cariogenic solution, as confirmed by the high rate of demineralization. The marginal exposed enamel showed a higher rate of demineralization than the exposed enamel, as demonstrated by the lowest microhardness values. The materials that claimed fluoride release did not prevent any type of enamel marginal alteration. This study revealed that enamel close to the margin of restoration may be rapidly affected by secondary caries formation when immersed in a demineralizing-cariogenic solution and that fluoride-releasing materials are unable to reduce the marginal demineralization processes. These demineralization processes may be responsible for marginal secondary caries and for restoration failures.

[Beta-endorphin and stress hormones in patients affected by osteoarthritis undergoing thermal mud therapy]. / Beta-endorfina e ormoni dello stress in pazienti affetti da artrosi e sottoposti a fangoterapia.

BACKGROUND: Thermal mud is a therapeutic agent widely used in the treatment of painful arthritic processes. The mechanism by which mud therapy works is still not well known. Its effect continues for months after completion of treatment. In order to verify whether thermal mud treatment brings about changes in the production of hormone peptides from proopiomelanocortin, the levels of plasma beta-endorphin and some hormones of the pituitary-adrenal glands (ACTH and cortisol) were determined in patients affected by osteoarthritis undergoing thermal mud therapy. METHODS: The levels of plasma beta-endorphin and some hormones of the pituitary-adrenal glands (ACTH and cortisol) were assessed by radiometric methods in seventeen males affected by osteoarthritis. The patients underwent a cycle of twelve sessions of thermal mud therapy. The tests were carried out immediately before thermal treatment, immediately after the first session, twelve days after the start of treatment, and again one month after completion of the treatment. RESULTS: beta-endorphin levels decreased significantly twelve days after the start of treatment. The level was still lower, although not significantly, even thirty days after completion of the treatment. Plasma ACTH also decreased during treatment. The decrease of this hormone was progressive and persisted after completion of treatment. Significant variations compared to baseline were found only thirty days after completion of treatment. Plasma cortisol decreased significantly after only one session of mud therapy. This hormone did not decrease any further during treatment, however, after twelve days it was still significantly lower than baseline. After completion of treatment, cortisol slightly increased, but thirty days later it was still lower, although not significantly, than baseline. CONCLUSIONS: It may be suggested that thermal treatment, by reducing inflammation, reduced pain and therefore diminished the cause of stress.

[Cytokines of bone turnover in postmenopause and old age]. / Citochine del turnover osseo in età postmenopausale e senile.

BACKGROUND: The aim of the study was the assessment of the influence of cytokines on bone ageing by measuring their level in serum and their secretion in vitro by monocytes from women of different age. METHODS: The levels of cytokines in 34 postmenopausal subjects and 14 old subjects were compared to those measured in 13 cycling subjects who were considered as control group. Subjects suffering from diseases inducing secondary osteoporosis, subjects taking medications that affect bone metabolism and alcohol- or tobacco-consumers were excluded from the study. The levels in serum of (i) the bone stimulating peptide insulin-like growth factor-I (IGF-I), (ii) the inhibitor of bone resorption interferon-gamma (IFN gamma), (iii) the stimulators of bone resorption interleukin-1 beta (IL-1 beta) tumor necrosis factor alpha (TNF alpha) and interleukin 6 (IL-6) were evaluated by immunoassay. IL-1 beta, TNF alpha and IL-6 secreted by monocytes (MO) cultured in vitro from peripheral blood of the same subjects were measured, too. Bacterial endotoxin (LPS) was used as stimulator for cytokine secretion by monocytes. RESULTS: Unlike IFN gamma, which was unaltered, circulating IGF-I level was found significantly diminished in postmenopausal and old subjects compared to control group. Among stimulators of bone resorption, IL-6 was greatly increased in postmenopausal and old subjects, while TNF alpha was reduced in postmenopausal group. In the supernatants of unstimulated monocytes the level of IL-1 beta was consistently decreased in old subjects; TNF alpha was found to be decreased in postmenopausal and old subjects. The stimulation index (SI), calculated as the ratio between the level of cytokines secreted by LPS-stimulated MO and the level of cytokines secreted by unstimulated MO, was found to be significantly increased for IL-1 beta and TNF alpha in postmenopausal subjects vs control group. In the old subjects the SI for IL-6 was enhanced. CONCLUSIONS: The data collected suggest that the measurement of cytokines in serum and supernatants from monocytes may give a picture of the mechanisms regulating bone aging.

Cd 34+ cells and clonogenicity of peripheral blood stem cells during chemotherapy treatment in association with granulocyte colony stimulating factor in osteosarcoma.

The introduction of aggressive chemotherapy in the treatment of osteosarcoma has improved the long-term outcome for these patients. With the increasing aggressiveness of chemotherapy protocols, hematopoietic growth factors have emerged as useful adjuncts involving, in some cases, rescue by peripheral blood stem cell (PBSC) infusion to assist faster recovery and maintain relative dose intensity. To evaluate the number of PBSCs needed, we analyzed the number of CD34+ cells and hematopoietic progenitor cells in the peripheral blood of 16 patients with osteoblastic, condroblastic and fibroblastic osteosarcoma enrolled in an Istituto Ortopedico Rizzoli-Scandinavian Sarcoma Group (IOR-SSG) pilot study, consisting of two cycles of preoperative high dose chemotherapy. The blood samples were studied at different times. The CD34+ cells were analyzed by flow cytometry and the hematopoietic progenitor cells were analyzed by tissue culture clonogenic assay. In comparing the two courses of chemotherapy, we observed that modification of the mean values of WBC, CD34+ and CFU-GM were very similar. The second course of chemotherapy seemed to induce greater hematological toxicity. All three parameters showed good correlation. The results demonstrated that the best time to collect PBSC by means of leukapheresis is post G-CSF used as rescue after ifosfamide treatment. We verified the ability of G-CSF to mobilize PBSCs in patients with osteosarcoma through cytofluorimetric analysis of CD34+ cells and their clonogenic capability. Moreover, during this preoperative treatment, we identified the best time to collect a sufficient number of PBSCs, that is after 9-10 days of G-CSF treatment following the first cycle of ifosfamide.

Globalization in the pharmaceutical industry, Part II.

This is the second of a two-part report on the pharmaceutical industry. Part II begins with a discussion of foreign direct investment and inter-firm networks, which covers international mergers, acquisitions, and minority participation; market shares of foreign-controlled firms; international collaboration agreements (with a special note on agreements in biotechnology); and licensing agreements. The final section of the report covers governmental policies on health and safety regulation, price regulation, industry and technology, trade, foreign investment, protection of intellectual property, and competition.

Globalization in the pharmaceutical industry, Part I.

This report on the pharmaceutical industry will be published in two parts. Part I begins with a summary of the study and its conclusions. The authors then provide an overview of the characteristics of the industry and current trends in its growth and structure: production and consumption, employment, research and development, capital investment, firm and product concentration and product competition, and pricing. A discussion of international trade follows, covering intra- and inter-regional, intra-firm, and intra-industry trade. The report will continue in the next issue of the Journal (Part II) with a look at foreign direct investment, inter-firm networks, and governmental policies.

Evaluation of cytokines regulating bone turnover in the serum of post-menopausal and senile women.

The authors evaluated the Insulin-like Growth Factor-1 (IGF-1) stimulating the formation of bone tissue, and interferon gamma (IFN gamma), inhibiting bone resorption, in the serum of women, 13 of fertile age, 34 of post-menopausal age, and 14 of senile age. Values for IGF-1 in the serum were considerably low in patients of post-menopausal and senile age, and presented highly significant differences with values for subjects of fertile age. The values for IFN gamma did not present significant differences between different age groups. It may be assumed that post-menopause and during senile age physiological osteopenia may be favored by a decrease in the secretion of IGF-1.

[The capability of silicone prostheses to promote postoperative infections. An in-vitro study]. / Capacità delle protesi in silicone di favorire le infezioni post-chirurgiche. Studio in vitro.

The authors have quantitatively evaluated and compared in vitro the Staphylococcus aureus and Staphylococcus epidermidis strain adhesiveness on various types of silicone used in surgery. The results show that there are some adhesiveness differences between the two assayed strains (S. epidermidis adhesiveness > S. aureus adhesiveness) and among silicone types (adhesiveness on soft silicones > adhesiveness on hard silicones). However all silicones are suitable substrata for in vitro bacterial adhesion.

Corneal endothelium cell adhesion on intraocular lenses in vitro.

An in vitro evaluation was conducted of the adhesion capabilities of rabbit corneal endothelial cells on intraocular lenses (IOLs) made of heparin-coated polymethylmethacrylate (HSM-PMMA). The concave endothelial surfaces of albino rabbit corneas were placed in contact in vitro with the convex surfaces of the optical side of HSM-PMMA IOLs in 4-day cultures. PMMA IOLs served as controls. After an incubation period, the preparations were examined via phase-contrast microscopy and via inverted microscopy after staining with vital dye (neutral red), both with and without the cornea in place. After fixation and staining with Giemsa, the cells adherent on the lens were counted on five different microscopic fields. It was observed that the corneal endothelial cells adhered equally to heparin-coated and untreated PMMA IOLs.

Cellular reactions in hip prosthesis loosening.

Based on the results obtained in histological examinations carried out on periprosthetic tissues in a large series of cases of prosthetic hip joint explants, the authors analyze the cellular events that may occur in loosening phenomena as compared to what occurs in the paraphysiological repair process that is observed in the stable prosthesis. Like other authors, they believe that the principal role in the mechanism of loosening is played by macrophages which are recalled in a large number, at times together with multinucleate giant cells, at the bone-implant interface, after micromovements of the prosthesis and the formation of wear particles have occurred. The macrophages would be capable of favoring resorption of the periprosthetic bone tissue, producing areas of osteolysis in which the transmission of the mechanical stress of loading is modified. The ensuing prosthetic instability increases wear phenomena, causes a greater amount of osteolysis, and, in a vicious cycle, loss of the relationship between bone and implant, and, thus, prosthetic loosening. Finally, the authors report a hypothesis on the pathogenesis of the phenomenon, based on which non-physiological stress, associated with wear and eventually infection, leads to loosening.

Results of histological grading on 100 cases of hip prosthesis failure.

One hundred cases of hip prosthesis failure were classified on the basis of the different types of tissue reaction occurring around alloplastic material. The results revealed infectious phlogosis in 32% of the cases, phlogosis due to wear in 42%, phlogosis due to allergy in 1% and mixed phlogosis in 25%. The distribution of the type and degree of intensity of the phlogosis, in relation to the duration of the implant, is also highlighted. This new grading technique yields reproducible results.

The effect of injection of powdered biomaterials on mouse peritoneal cell populations.

The authors suggest an experimental model for the assaying, before implantation, of the tissue reaction that wear particles from artificial joints can cause in the human body. Mice were intraperitoneally injected with biomaterial powders in suspension in saline. The materials used were chromium, chromium oxide, nickel, nickel oxide, aluminum, alumina, alumina-titania, cobalt, molybdenum, titanium, silver, zirconium oxide, iron oxide, and stainless steel. Peritoneal lavage was performed a week after injection. A thorough morphological and quantitative analysis of the cell suspension thus obtained was made both immediately after collection and after a 24 h culture.