The osteogenesis of Ginsenoside Rb1 incorporated silk/micro-nano hydroxyapatite/sodium alginate composite scaffolds for calvarial defect / 国际口腔科学杂志·英文版

Ginsenoside Rb1, the effective constituent of ginseng, has been demonstrated to play favorable roles in improving the immunity system. However, there is little study on the osteogenesis and angiogenesis effect of Ginsenoside Rb1. Moreover, how to establish a delivery system of Ginsenoside Rb1 and its repairment ability in bone defect remains elusive. In this study, the role of Ginsenoside Rb1 in cell viability, proliferation, apoptosis, osteogenic genes expression, ALP activity of rat BMSCs were evaluated firstly. Then, micro-nano HAp granules combined with silk were prepared to establish a delivery system of Ginsenoside Rb1, and the osteogenic and angiogenic effect of Ginsenoside Rb1 loaded on micro-nano HAp/silk in rat calvarial defect models were assessed by sequential fluorescence labeling, and histology analysis, respectively. It revealed that Ginsenoside Rb1 could maintain cell viability, significantly increased ALP activity, osteogenic and angiogenic genes expression. Meanwhile, micro-nano HAp granules combined with silk were fabricated smoothly and were a delivery carrier for Ginsenoside Rb1. Significantly, Ginsenoside Rb1 loaded on micro-nano HAp/silk could facilitate osteogenesis and angiogenesis. All the outcomes hint that Ginsenoside Rb1 could reinforce the osteogenesis differentiation and angiogenesis factor's expression of BMSCs. Moreover, micro-nano HAp combined with silk could act as a carrier for Ginsenoside Rb1 to repair bone defect.

Alveolar bone repair with strontium- containing nanostructured carbonated hydroxyapatite

ABSTRACT Objective: This study aimed to evaluate bone repair in rat dental sockets after implanting nanostructured carbonated hydroxyapatite/sodium alginate (CHA) and nanostructured carbonated hydroxyapatite/sodium alginate containing 5% strontium microspheres (SrCHA) as bone substitute materials. Methods: Twenty male Wistar rats were randomly divided into two experimental groups: CHA and SrCHA (n=5/period/group). After one and 6 weeks of extraction of the right maxillary central incisor and biomaterial implantation, 5 μm bone blocks were obtained for histomorphometric evaluation. The parameters evaluated were remaining biomaterial, loose connective tissue and newly formed bone in a standard area. Statistical analysis was performed by Mann-Withney and and Wilcoxon tests at 95% level of significance. Results: The histomorphometric results showed that the microspheres showed similar fragmentation and bio-absorbation (p>0.05). We observed the formation of new bones in both groups during the same experimental periods; however, the new bone formation differed significantly between the weeks 1 and 6 (p=0.0039) in both groups. Conclusion: The CHA and SrCHA biomaterials were biocompatible, osteoconductive and bioabsorbable, indicating their great potential for clinical use as bone substitutes.

Osseointegration aspects of placed implant in bone reconstruction with newly developed block-type interconnected porous calcium hydroxyapatite

ABSTRACT Artificial bone has been employed to reconstruct bone defects. However, only few reports on implant placement after block bone grafting exist. Objectives The purpose of this study was to evaluate the osseointegration of dental implant in bone reconstructions with interconnected porous calcium hydroxyapatite (IP-CHA). Material and Methods The IP-CHA cylinders (D; 4.3 mm, H; 10.0 mm) were placed into bone sockets in each side of the femurs of four male dogs. The IP-CHA on the right side was a 24-week sample. Twelve weeks after placement, a titanium implant was placed into a socket that was prepared in half of the placed IP-CHA cylinder on the right side. On the left side, another IP-CHA cylinder was placed as a 12-week sample. After another 12 weeks, the samples were harvested, and the bone regeneration and bone-implant contact (BIC) ratios were measured. Results New bone formation area was superior in the 24-week IP-CHA compared with the 12-week IP-CHA. BIC was not significantly different between IP-CHA and the parent sites. Osseointegration was detected around the implant in IP-CHA-reconstructed bone. Conclusion Our preliminary results suggest that IP-CHA may be a suitable bone graft material for reconstructing bones that require implant placement.

Effect of addition of nano-hydroxyapatite on physico-chemical and antibiofilm properties of calcium silicate cements

ABSTRACT Objective Mineral Trioxide Aggregate (MTA) is a calcium silicate cement composed of Portland cement (PC) and bismuth oxide. Hydroxyapatite has been incorporated to enhance mechanical and biological properties of dental materials. This study evaluated physicochemical and mechanical properties and antibiofilm activity of MTA and PC associated with zirconium oxide (ZrO2) and hydroxyapatite nanoparticles (HAn). Material and Methods White MTA (Angelus, Brazil); PC (70%)+ZrO2 (30%); PC (60%)+ZrO2 (30%)+HAn (10%); PC (50%)+ZrO2 (30%)+HAn (20%) were evaluated. The pH was assessed by a digital pH-meter and solubility by mass loss. Setting time was evaluated by using Gilmore needles. Compressive strength was analyzed by mechanical test. Samples were radiographed alongside an aluminum step wedge to evaluate radiopacity. For the antibiofilm evaluation, materials were placed in direct contact with E. faecalis biofilm induced on dentine blocks. The number of colony-forming units (CFU mL-1) in the remaining biolfilm was evaluated. The results were submitted to ANOVA and the Tukey test, with 5% significance. Results There was no difference in pH levels of PC+ZrO2, PC+ZrO2+HAn (10%) and PC+ZrO2+HAn (20%) (p>0.05) and these cements presented higher pH levels than MTA (p<0.05). The highest solubility was observed in PC+ZrO2+HAn (10%) and PC+ZrO2+HAn (20%) (p<0.05). MTA had the shortest initial setting time (p<0.05). All the materials showed radiopacity higher than 3 mmAl. PC+ZrO2 and MTA had the highest compressive strength (p<0.05). Materials did not completely neutralize the bacterial biofilm, but the association with HAn provided greater bacterial reduction than MTA and PC+ZrO2 (p<0.05) after the post-manipulation period of 2 days. Conclusions The addition of HAn to PC associated with ZrO2 harmed the compressive strength and solubility. On the other hand, HAn did not change the pH and the initial setting time, but improved the radiopacity (HAn 10%), the final setting time and the E. faecalis antibiofilm activity of the cement.

Combined effects of melatonin and FGF-2 on mouse preosteoblast behavior within interconnected porous hydroxyapatite ceramics - in vitro analysis

ABSTRACT Objective Biocompatible materials such as interconnected porous hydroxyapatite ceramics (IP-CHA) loaded with osteogenic cells and bioactive agents are part of an evolving concept for overcoming craniofacial defects by use of artificial bone tissue regeneration. Amongst the bioactive agents, melatonin (MEL) and basic fibroblast growth factor (FGF-2) have been independently reported to induce osteoblastic activity. The present in vitro study was undertaken to examine the relationship between these two bioactive agents and their combinatory effects on osteoblastic activity and mineralization in vitro. Material and Methods Mouse preosteoblast cells (MC3T3-E1) were seeded and cultured within cylindrical type of IP-CHA block (ø 4x7 mm) by vacuum-assisted method. The IP-CHA/MC3T3 composites were subjected to FGF-2 and/or MEL. The proliferation assay, alkaline phosphatase enzyme activity (ALP), mRNA expressions of late bone markers, namely Osteocalcin (OCN) and Osteopontin (OPN), and Alizarin Red staining were examined over a period of 7 days. Results FGF-2 mainly enhanced the proliferation of MC3T3-E1 cells within the IP-CHA constructs. MEL mainly induced the mRNA expression of late bone markers (OCN and OPN) and showed increased ALP activity of MC3T3 cells cultured within IP-CHA construct. Moreover, the combination of FGF-2 and MEL showed increased osteogenic activity within the IP-CHA construct in terms of cell proliferation, upregulated expressions of OCN and OPN, increased ALP activity and mineralization with Alizarin Red. The synergy of the proliferative potential of FGF-2 and the differentiation potential of MEL showed increased osteogenic activity in MC3T3-E1 cells cultured within IP-CHA constructs. Conclusion These findings indicate that the combination of FGF-2 and MEL may be utilized with biocompatible materials to attain augmented osteogenic activity and mineralization.

Cytocompatibility and biocompatibility of nanostructured carbonated hydroxyapatite spheres for bone repair

ABSTRACT Objective The aim of this study was to investigate the in vitro and in vivo biological responses to nanostructured carbonated hydroxyapatite/calcium alginate (CHA) microspheres used for alveolar bone repair, compared to sintered hydroxyapatite (HA). Material and Methods The maxillary central incisors of 45 Wistar rats were extracted, and the dental sockets were filled with HA, CHA, and blood clot (control group) (n=5/period/group). After 7, 21 and 42 days, the samples of bone with the biomaterials were obtained for histological and histomorphometric analysis, and the plasma levels of RANKL and OPG were determined via immunoassay. Statistical analysis was performed by Two-Way ANOVA with post-hoc Tukey test at 95% level of significance. Results The CHA and HA microspheres were cytocompatible with both human and murine cells on an in vitro assay. Histological analysis showed the time-dependent increase of newly formed bone in control group characterized by an intense osteoblast activity. In HA and CHA groups, the presence of a slight granulation reaction around the spheres was observed after seven days, which was reduced by the 42nd day. A considerable amount of newly formed bone was observed surrounding the CHA spheres and the biomaterials particles at 42-day time point compared with HA. Histomorphometric analysis showed a significant increase of newly formed bone in CHA group compared with HA after 21 and 42 days from surgery, moreover, CHA showed almost 2-fold greater biosorption than HA at 42 days (two-way ANOVA, p<0.05) indicating greater biosorption. An increase in the RANKL/OPG ratio was observed in the CHA group on the 7th day. Conclusion CHA spheres were osteoconductive and presented earlier biosorption, inducing early increases in the levels of proteins involved in resorption.

Repair of Surgical Bone Defects Grafted with Hydroxylapatite + ß-TCP and Irradiated with λ=850 nm LED Light

The treatment of bone loss due to different etiologic factors is difficult and many techniques aim to improve the repair, including a wide range of biomaterials and recently, photobioengineering. This work aimed to assess by histological analysis the repair of bone defects grafted with biphasic synthetic micro-granular HA + β-TCP associated with LED phototherapy. Forty rats were divided into 4 groups (Clot, LED, Biomaterial and LED + Biomaterial) each subdivided into 2 subgroups according to the time of animal death (15 and 30 days). Surgical bone defects were prepared on the femur of each animal with a trephine drill. In animals of the Clot group the defect was filled only by blood clot, in the LED group the defect filled with the clot was further irradiated. In the animals of Biomaterial and LED + Biomaterial groups the defect was filled by biomaterial and the last one was further irradiated (λ=850±10 nm, 150 mW, Φ ~ 0.5 cm2, 20 J/cm2 - session, 140 J/cm2- treatment) at 48-h intervals for 2 weeks. Following animal death, samples were taken and analyzed by light microscopy. Using the degree of maturation of the bone by assessment of the deposition/organization of the basophilic lines in the newly formed bone tissue, the LED + Biomaterial group was the one in a more advanced stage of bone repair process at the end of the experiment. It may be concluded that the use of LED phototherapy was effective in positively modulating the process of bone repair of bone defects in the femur of rats submitted or not to biomaterial grafting.

O tratamento de perdas ósseas devido a diferentes fatores etiológicos é difícil e muitas técnicas têm por objetivo melhorar o reparo incluindo o uso de uma ampla gama de biomateriais e, recentemente, a fotobioengenharia. Este trabalho teve como objetivo avaliar, por meio de análise histológica, o reparo de defeitos ósseos enxertados com HA bifásica micro-granular sintética + β -TCP associada à fototerapia LED. Quarenta ratos foram divididos em quatro grupos (Clot, LED, Biomaterial e LED + Biomaterial), subdivididos no dois subgrupos de acordo com o momento da morte (15 e 30 dias). Defeitos ósseos cirúrgicos foram criados em um fêmur de cada animal com uma broca trefina. Em animais do grupo coágulo, o defeito foi preenchido apenas pelo coágulo sanguíneo, no grupo de LED o defeito foi preenchido pelo coágulo e irradiado. Nos animais dos grupos do biomaterial e LED + biomaterial, os defeitos foram preenchidos com biomaterial e o último foi adicionalmente irradiado (λ=850±10 nm, 150 mW, Φ ~ 0.5 cm2, 20 J/cm2 - session, 140 J/cm2 -tratamento) a cada 48 h por duas semanas. Após a morte dos animais, amostras foram colhidas e analisadas por microscopia de luz, usando o grau de maturação do osso como marcador (deposição/organização das linhas basofílicas) no tecido ósseo neoformado. O grupo de LED + biomaterial apresentou processo de reparação mais avançado ao fim do experimento. Pode-se concluir que o uso da fototerapia LED foi eficaz na modulação positiva do processo de reparo ósseo de defeitos ósseos no fêmur de ratos submetidos ou não a enxerto com biomaterial.

Bacteriology of deep carious lesions underneath amalgam restorations with different pulp-capping materials - an in vivo analysis

Microorganisms remaining in dentin following cavity preparation may induce pulp damage, requiring the use of pulp-capping agents with antimicrobial activity underneath permanent restorations. OBJECTIVE: The aims of this study were to analyze the bacteriological status of carious dentin and to assess the efficacy of different base underneath silver amalgam restorations. MATERIAL AND METHODS: This study was conducted on 50 patients aged 13 to 30 years. Sterile swabs were used to take samples after cavity preparation, which was assessed by microbiological culture to identify the microorganisms present. Following this, cavities were restored with silver amalgam, using one of the materials being investigated, as the base: calcium hydroxide (Group II), polyantibiotic paste (Group III), a novel light-cured fluoride-releasing hydroxyapatite-based liner (Group IV) and mineral trioxide aggregate - MTA (Group V). In Group I, the cavities were restored with silver amalgam, without any base. After 3 months, the amalgam was removed and samples taken again and analyzed for the microbial flora. RESULTS: Lactobacilli were the most commonly isolated microorganisms in the samples of carious dentin. Groups IV and V showed negative culture in the 3-month samples. There was no statistically significant difference between Groups I, II and III. There was no significant difference between Groups IV and V (p>0.05). Both Groups IV and V showed significantly better results when compared to Groups I, II and III (p<0.05). CONCLUSIONS: The hydroxyapatite-based liner and MTA performed significantly better in terms of antibacterial activity than the other materials.

Ação da matriz óssea bovina desmineralizada na neoformação óssea em ratos submetidos ao alcoolismo experimental: avaliação histológica e histométrica / Action of demineralized bovine bone matrix on bone neoformation in rats submitted to experimental alcoholism: histological and histometric evaluation

O etanol inibe a proliferação de células osteoblásticas, gerando baixa massa óssea e aumento na prevalência de fraturas na população alcoólatra. A quantidade de defeitos ósseos criados cirurgicamente, e pelos vários tipos de acidentes, tem aumentado atualmente e existe uma preocupação muito grande na descoberta de substâncias que acelerem a neoformação óssea que preencham essas cavidades. Baseado no exposto anteriormente resolveu-se realizar este trabalho com o objetivo de observar se a matriz óssea bovina desmineralizada (Genox ®) altera a neoformação óssea em ratos submetidos ao alcoolismo experimental, usando para isso análise histológica e histométrica. Para isso foram utilizados 40 ratos machos (Rattus norvegicus), separados em 2 grupos de 20 animais cada, assim distribuídos: Grupo E1, que recebeu álcool etílico a 25%, diluído em água de torneira, e cavidade cirúrgica preenchida somente por coágulo sanguíneo, e Grupo E2, que recebeu álcool etílico a 25%, diluído em água de torneira, e cavidade cirúrgica preenchida por Gen-ox®. Após um período de 3 semanas de adaptação gradativa ao álcool, os animais receberam dieta alcoólica de 25% por um período de 90 dias. Decorrido esse período, a tíbia esquerda de todos os animais foi submetida a uma cirurgia onde se produziu uma cavidade cirúrgica experimental, que no Grupo E1 ficou preenchida por coágulo sanguíneo, e no Grupo E2 preenchida por Gen-ox®. Cinco animais de cada grupo foram sacrificados em períodos de 10, 20, 40 e 60 dias contados a partir do dia da cirurgia experimental, para retirada de parte da tíbia, onde a cavidade cirúrgica foi realizada. Os blocos retirados foram processados histologicamente e submetidos à coloração por Tricrômico de Masson, para estudo histomorfológico e histométrico da área total do defeito, quantidade de tecido conjuntivo presente e quantidade de tecido ósseo neoformado. Os resultados mostraram que a reorganização da medula óssea e reparação total da...

Ethanol inhibits the proliferation of osteoblastic cells, leading to low bone mass and increased prevalence of fractures in the alcoholic population. The amount of bone defects surgically created, and various types of accidents has increased and there is currently a great concern in the discovery of substances that accelerate new bone formation to fill those cavities. Based on the foregoing it was decided to undertake this work in order to see whether demineralized bovine bone (Gen-ox®) alters bone formation in rats subjected to experimental alcoholism, using it for histological and histometric analysis. For this we used 40 male rats (Rattus norvegicus) separated in two groups of 20 animals each one, distributed as follows: Group E1, which received 25% ethanol, diluted in tap water, and the surgical cavity filled only by a blood clot, and Group E2, which received 25% ethanol, diluted in tap water, and the surgical cavity filled with Gen-ox®. After a period of three weeks of gradual adaptation to alcohol, the animals received 25% alcohol diet for a period of 90 days. After this period, the left tibia of all animals underwent a surgery where it produced an experimental surgical cavity, which in Group E1 was filled by blood clot, and in Group E2 filled with Gen-ox®. Five animals from each group were sacrificed on days 10, 20, 40 and 60 days from the day of experimental surgery to remove part of the tibia, where the sinus surgery was done. The blocks were removed and processed histologically stained by Masson trichrome, for histomorphological and histometric study of the total area of the defect, amount of connective tissue and amount of new bone. The results showed that the reorganization of the bone marrow and full repair of the surgical cavity in Group E1 had occurred in a shorter time than in Group E2. It was also noted that in the final period, the animals in Group E2 showed areas of connective tissue and thick bone...

Ação da matriz óssea bovina desmineralizada na neoformação óssea em ratos submetidos ao alcoolismo experimental: avaliação histológica e histométrica / Action of demineralized bovine bone matrix on bone neoformation in rats submitted to experimental alcoholism: histological and histometric evaluation

O etanol inibe a proliferação de células osteoblásticas, gerando baixa massa óssea e aumento na prevalência de fraturas na população alcoólatra. A quantidade de defeitos ósseos criados cirurgicamente, e pelos vários tipos de acidentes, tem aumentado atualmente e existe uma preocupação muito grande na descoberta de substâncias que acelerem a neoformação óssea que preencham essas cavidades. Baseado no exposto anteriormente resolveu-se realizar este trabalho com o objetivo de observar se a matriz óssea bovina desmineralizada (Genox ®) altera a neoformação óssea em ratos submetidos ao alcoolismo experimental, usando para isso análise histológica e histométrica. Para isso foram utilizados 40 ratos machos (Rattus norvegicus), separados em 2 grupos de 20 animais cada, assim distribuídos: Grupo E1, que recebeu álcool etílico a 25%, diluído em água de torneira, e cavidade cirúrgica preenchida somente por coágulo sanguíneo, e Grupo E2, que recebeu álcool etílico a 25%, diluído em água de torneira, e cavidade cirúrgica preenchida por Gen-ox®. Após um período de 3 semanas de adaptação gradativa ao álcool, os animais receberam dieta alcoólica de 25% por um período de 90 dias. Decorrido esse período, a tíbia esquerda de todos os animais foi submetida a uma cirurgia onde se produziu uma cavidade cirúrgica experimental, que no Grupo E1 ficou preenchida por coágulo sanguíneo, e no Grupo E2 preenchida por Gen-ox®. Cinco animais de cada grupo foram sacrificados em períodos de 10, 20, 40 e 60 dias contados a partir do dia da cirurgia experimental, para retirada de parte da tíbia, onde a cavidade cirúrgica foi realizada. Os blocos retirados foram processados histologicamente e submetidos à coloração por Tricrômico de Masson, para estudo histomorfológico e histométrico da área total do defeito, quantidade de tecido conjuntivo presente e quantidade de tecido ósseo neoformado. Os resultados mostraram que a reorganização da medula óssea e reparação total da...

Ethanol inhibits the proliferation of osteoblastic cells, leading to low bone mass and increased prevalence of fractures in the alcoholic population. The amount of bone defects surgically created, and various types of accidents has increased and there is currently a great concern in the discovery of substances that accelerate new bone formation to fill those cavities. Based on the foregoing it was decided to undertake this work in order to see whether demineralized bovine bone (Gen-ox®) alters bone formation in rats subjected to experimental alcoholism, using it for histological and histometric analysis. For this we used 40 male rats (Rattus norvegicus) separated in two groups of 20 animals each one, distributed as follows: Group E1, which received 25% ethanol, diluted in tap water, and the surgical cavity filled only by a blood clot, and Group E2, which received 25% ethanol, diluted in tap water, and the surgical cavity filled with Gen-ox®. After a period of three weeks of gradual adaptation to alcohol, the animals received 25% alcohol diet for a period of 90 days. After this period, the left tibia of all animals underwent a surgery where it produced an experimental surgical cavity, which in Group E1 was filled by blood clot, and in Group E2 filled with Gen-ox®. Five animals from each group were sacrificed on days 10, 20, 40 and 60 days from the day of experimental surgery to remove part of the tibia, where the sinus surgery was done. The blocks were removed and processed histologically stained by Masson trichrome, for histomorphological and histometric study of the total area of the defect, amount of connective tissue and amount of new bone. The results showed that the reorganization of the bone marrow and full repair of the surgical cavity in Group E1 had occurred in a shorter time than in Group E2. It was also noted that in the final period, the animals in Group E2 showed areas of connective tissue and thick bone...

Effect of hydroxyapatite and bovine serum albumin on the antibacterial activity of MTA

The purpose of this study was to compare the inhibitory effect of bovine serum albumin [BSA] and hydroxyapatite [HA] on the antibacterial activity of white-colored MTA [WMTA] against Staphylococcus [S.] aureus and Streptococcus [S.] mutans after 24 and 72 hours. All materials were prepared according to the manufacturer's directions immediately before testing. The antibacterial effect of each group [WMTA, WMTA+BSA and WMTA+HA] was determined by measuring the diameter of zone of inhibition in millimeters after incubation at 37[degree sign] C for 24 and 72 hours in a humid atmosphere. Each test was repeated three times. Data were analyzed using ANOVA and Tukey's test. In the 24 hours samples as well as in 72 hours samples, the antibacterial activity of MTA+HA group was significantly greater than two other groups against S. aureus [P<0.05]. However, the antibacterial activity of MTA+HA group against S. mutans was not significantly different from the MTA group in 24 hours as well as 72 hours samples. BSA reduced the antibacterial activity of MTA against both tested bacteria in the 24 and 72 hour samples [P<0.05]. The products studied exhibited antibacterial activity. However, in both time intervals, the MTA+HA group exerted the greatest activity against S. mutans and S. aureus

In vitro antimicrobial activity of phramongkutklao hydroxyapatite antibiotic pellet.

OBJECTIVE: Assess the antimicrobial activity of Phramongkutklao antibiotic hydroxyapatite pellets in vitro. MATERIAL AND METHOD: The selected bacteria used in the present study were the standard and drug-resistant strains of Staphylococcus aureus and Pseudomonas aeruginosa, which are the common organisms causing infection in Orthopedics. Phramongkutklao antibiotic hydroxyapatite pellets developed by the Orthopedics Department, Phramongkutklao Hospital contained either Vancomycin, Fosfomycin, or Gentamycin. Each preparation was placed on an agar plate inoculated with each bacterium. The inhibition zones were monitored in 24 hours. Then the pellets were moved onto the new inoculated plate every day for 28 days. RESULTS: Compared with the control group, Phramongkutklao antibiotic hydroxyapatite pellets had good inhibitory effect against S.aureus. Vancomycin hydroxyapatite pellets were able to maintain their activity for 28 days whereas Gentamycin hydroxyapatite pellets was effective for only three days. Fosfomycin hydroxyapatite pellets could inhibit MSSA for 13 days and MRSA for 25 days. On the contrary, all three Phramongkutklao antibiotic hydroxyapatite pellets had very low efficacy against Paeruginosa. CONCLUSION: Phramongkutklao antibiotic hydroxyapatite pellets were able to inhibit the growth of S. aureus both MSSA and MRSA while the activity against P. aeruginosa needs to be developed.

effect of osteogenic medium on the adhesion of rat bone marrow stromal cell to the hydroxyapatite

To investigate the adhesive properties of bone marrow stromal cell [BMSC] on the hydroxyapatite [HA] particles and analyze their behavior. The study took place in the Department of the Histology and Embryology, Celal Bayar University, Manisa and in the Department of Bioengineering, Ege University, Izmir, Turkey between 2004 and 2005. We cultured BMSC from the mature rat tibia and differentiated to the osteoblasts by osteogenic medium. The BMSCs were subcultured and were taken to the HA substrate. We measured their proliferation capacity and viability with MTT assay using the spectrophotometric method. Furthermore, we identified the osteoblast-like cells by immunohistochemical staining of osteonectin and osteocalcin and we analyzed the behavior of the cells on different sized HA particles by SEM at the end of 3 days incubation. Osteogenic medium caused the proliferation capacity of BMSC to speed up and the effects appeared earlier. We confirmed the osteoblastic differentiation by staining of most cells with osteoblastic markers. Subcultured cells were similarly adhesive to the HA particles and the osteogenic medium did not alter this behavior. They spread on the substrate similarly. Most of the cells demonstrated the cytoplasmic protrusion. Morphology of the cells did not change much with or without osteogenic medium. Different sizes of HA particles did not affect the adhesive properties of these cells except HA gel. The spreading and attachment ratios of the cells on HA gel were more than the others. We found that there was heterogeneity in BMSC on differentiation capacity to the osteoblast, which was a sign of a subpopulation. Adhesive cells showed similar morphology and behavior under the effect of osteogenic medium. The only difference was the spreading capacity on the HA gel where cell used this substrate more effectively for adhesion

Clinical study of efficacy of hydroxyapatite.

The cleaved part of collagen during collagen formation was secreted into the circulation. The present study used this remnant part of collagen called procollagen type 1 propeptide at carboxyl terminal (PICP) as a bone formation marker Clinical study of efficacy of hydroxyapatite tablet (800 mg) in 47 menopausal women with bone resorption marker, (CTx) below 0.330 ng/ml. and their general condition is healthy. All cases were free of medication at least one month before the present study. After one month, the mean of PICP was 1452.28 ng/ml SD = 625.58 which is significantly higher than the mean of baseline control, 930.98 ng/ml SD=477.44 (p=0.0001).

Comparison of tissue reaction and osteointegration of metal implants between hydroxyapatite/Ti alloy coat: an animal experimental study.

BACKGROUND: One important clinical application of hydroxyapatite (HA) is coating on metal implants to stimulate osteo-integration thus enhancing fixation of the implant to bone, especially plasma-sprayed HA coating applied on Ti alloy substrate. The poor bonding strength between HA and Ti alloy has been of great concern to orthopedists. The biocomptable coat such as Ti alloy (TiO2) coat is one method to improve adhesive strength. OBJECTIVE: The objective of this study was to detect and analyze possible differences in bone formation, bone integration and tissue reaction between group I (uncoated Titanium), group II (Hydroxyapatite coated Titanium), and group III (Hydroxyapatite/TiO2 coated Titanium) implant specimens when embedded into bony hosts. METHOD: Rectangular specimens were implanted into the femoral bone of adult dogs in randomly different sites including: proximal left, proximal right, distal left, distal right. The tailor-made implant specimens were inserted in 5 x 5 mm preprepared sockets. Radiographic evaluation was taken at 0, 1, 3 and 6 months. All animals were sacrificed at 3 and 6 months post implantation. The femoral bone containing implants were dissected and then prepared to be further investigated. The bone-implant interface was analyzed by H&E surface staining, radiography and scanning electron microscopy. Data concerning percentage of osteointegration and adhesiveness of hydroxyapatite layer from different kinds of implants along the entire length of each implants were collected and analyzed for evaluation of any significant differences. RESULTS: No osteo-integration was noted in Group I, but there was 25.57 per cent osteointegration in Group II and 28.63 per cent in Group III. No statistically significant differences were observed between Group II and Group III. However, the coating layer in Group II was found to have detached, in some area, from the metal substrate. Histologically, no adverse tissue reaction was found around any kind of implant. CONCLUSION: Biocompatable bond coat is one of the methods to improve adhesive strength of hydroxyapatite coated implants. In the present study it could be concluded that, besides the improvement in adhesiveness, the intervening TiO2 coating layer had no negative effect concerning bone formation and integration and also showed no adverse surrounding soft tissue reaction.

Comparative evaluation of protective antigen produced from Bacillus anthracis & Escherichia coli.

BACKGROUND & OBJECTIVE: Anthrax has been reported from almost every country and India is endemic for this disease. There is considerable under reporting of the disease because of lack of microbiological facilities and diagnostic reagents. In India only conventional methods which have limitations, are being used to diagnose the disease. Hence the aim of this study was to isolate and purify protective antigen (PA) using different protocols and to use this PA for detection of anti-PA antibodies from sera samples. METHODS: Protective antigen was isolated and purified from the Sterne strain of Bacillus anthracis. B. anthracis lacking pXO1 and pXO2 transformed with pYS5 (B. anthracis pYS5) and recombinant Escherichia coli transformed with pQE30 containing PA gene using hydroxyapatite (HA), Q-sepharose fast protein liquid chromatography (FPLC) and nickel-nitrilotriacetic acid (Ni-NTA) chromatographic methods, respectively. A mixture of PA and edema factor (EF) was injected subcutaneously into rabbits to test the biological activity of PA. The immunogenicity of PA was tested by inoculating the protein into rabbits along with adjuvant. Using this PA, 20 bovine sera samples (pre- and post-vaccinated) were tested by Western blotting (WB) for the presence of anti-PA antibodies. RESULTS: The 83 kDa PA protein was obtained from all the bacteria with the yields of 13, 50 and 9.0 mg/l from Sterne B. anthracis, B. anthracis pYS5 and recombinant Esch. coli, respectively. Formation of edematous ulcers at the site of PA+EF injection clearly confirmed the retention of biological activity of the proteins. Of the 10 post-vaccination sera tested, 9 showed clear positive by WB whereas none of the pre-vaccination sera showed the reaction. INTERPRETATION & CONCLUSION: The purified PA preparations obtained in the present study may possibly be utilized for detection of anti-PA antibodies in the sera of anthrax patients for timely diagnosis of the disease and, might also be tested for their efficacy and use as human anthrax vaccine.

Efeito hidroxiapatita sintética no processo de reparo e na populaçäo de mastócitos em ratos Wistar / Effect of sinthetic hidroxyapatite in healing of wounds and mast cells in Wistar rats

Foram realizadas, utilizando-se microscopia de luz, análise morfológica e quantitativa da populaçäo de mastócitos no tecido conjuntivo subcutâneo de ratos Wistar frente à açäo da hidroxiapatita sintética em pó, além da análise morfológica da evoluçäo do processo de reparo tecidual nos intervalos de tempo de 2, 7, 15 e 30 dias de pós-operatório. Morfologicamente, os mastócitos se apresentaram de duas formas: nas camadas mais superficiais destacam-se os de forma alongada, enquanto que nas camadas mais profundas predominavam as formas arredondadas ou ovaladas. A análise quantitativa demonstrou que a populaçäo de mastócitos no grupo experimental era menor aos dois dias pós-incisäo e que se elevava até o 30§ dia. No grupo controle, os números médios de mastócitos foram mais elevados nos intervalos de 7 e 15 dias. Os testes estatísticos demonstraram que houve diferença estatisticamente significante no número de mastócitos no grupo experimental de 30 dias quando comparado com o grupo de controle. Evidenciou-se que a hidroxiapatita provocou reaçäo tipo corpo estranho, caracterizada pela presença de inúmeras células gigantes multinucleadas, inespecíficas. O processo de reparo por cicatrizaçäo evoluiu normalmente em ambos os grupos através das fases de inflamaçäo, fibroplasia e maturaçäo

X-Ray diffraction analysis of healed bone defect after intraosseously application hydroxylapatite.

The study was carried out on 10 male guinea pigs with an average weight 250 grams. The Surgical bony defect was performed under general anaesthesia using ether inhalation, so that, the bone cavity was prepared on both sides of midline of the mandible. The right one was used for the implanted material and the left one was used as control. The animals were sacrificed after eight weeks, the specimens were dried in oven, then grinded and finally placed in the special carrier of the x-ray diffractometer, the results showed the following: The specimens of both normal and control bone composed of the same constituent such as, Basic calcium phosphate mainly, calcium carbonate and alpha-calcium phosphate as a minor constituent. On the other hand, the specimens of healed bony defects in which Hydroxylapatite was placed, composed of the same constituent and Hydroxylapatite was clearly identified. In View of the evidence presented, it is reasonable to suggest that, the role of Hydroxylapatite as implanted material was osteaimductive and osteoconductive