V. Bienengräber

Nanostructuring of Biomaterials—A Pathway to Bone Grafting Substitute

Background:The bone substitute NanoBone® consists of nanocrystalline hydroxyapatite embedded in a highly porous matrix of silica gel. It promotes the healing of bone defects and is degraded by osteoclasts during bone remodeling. The present study investigates the interactions of NanoBone® with bone tissue.Methods:Granules of NanoBone® were implanted in defects of critical size in the mandible of minipigs. Samples were taken after 5 and 10 weeks and demineralized. The composition of the implanted granules was analyzed by means of transmission and scanning electron microscopy and EDX. Enzymeand immunohistochemistry was used to investigate organic components of NanoBone® granules that arised after implantation in the host.Results:EDX demonstrated that 5 weeks after implantation the silica gel was degraded and replaced by an organic matrix. Ultrastructurally, the matrix appeared amorphous with only single collagen fibrillae.PAS-staining indicated the presence of carbohydrates. Immunohistochemically, the bone proteins osteopontin, osteocalcin and BMP-2 were found as constituents of the new matrix. Alkalic phosphatase activity was located in osteoblasts and newly formed bone on NanoBone® and focally in particles. Osteoclasts with ruffled borders, sealing zones, and acid phosphatase activity were situated in resorption lacunae at granule surfaces not covered by new bone.Conclusions:In vivo, the silica gel of NanoBone® is replaced by bone matrix glycoproteins with known functions in attraction, adhesion, and differentation of bone cells as osteoblasts and osteoclasts. We assume that the deposition of these molecules supports the early phase of NanoBone® degradation by osteoclasts and promotes the production of new bone tissue.

Is It Possible to Prevent Cleft Palate by Prenatal Administration of Folic Acid? An Experimental Study

OBJECTIVE In this study, folic acid was tested for its antiteratogenic effects on experimentally induced cleft palate in animals. DESIGN Eleven pregnant Lew 1 A dams (75 fetuses) received 200 mg/kg procarbazine via gastric tubing on postconception (p.c.) day 14 to induce a cleft palate (CP); seven of the pregnant dams (45 fetuses) were additionally given 4 mg/kg folic acid subcutaneously from the 14th to the 17th day p.c. As a control group, three more pregnant dams (24 fetuses) were not treated with the drugs mentioned above. All fetuses were delivered by Caesarian section on day 20 p.c. OUTCOMES MEASURED All fetuses were weighed and examined macroscopically with a stereomicroscope. Each fetal head was cut into 35 frontal sections and scrutinized histologically. RESULTS None of the control fetuses (n = 24) exhibited a cleft. Without folate administration, 90% of the fetuses (27 of 30) that received procarbazine exhibited a CP. After additional prenatal folate administration, this rate remained virtually unchanged (91%; 41 of 45). However, the proportion of complete (total) CP (4%) was significantly (p <.0001) lower than in the group without folate (53%). Cleft-associated microgenia and microglossia were also significantly less frequent when folate was administered prenatally: microgenia was reduced by 22% (p =.029) and microglossia by 24% (p =.032). CONCLUSIONS On the basis of these results, folate has a partial ameliorating effect on the teratogenicity of procarbazine given to pregnant rats. Additional studies are necessary on the effect of folate in different species, also taking cleft lip and CP into consideration.

Induction and Prevention of Cleft Lip, Alveolus and Palate and Neural Tube Defects with Special Consideration of B Vitamins and the Methylation Cycle

Previous studies on the development of cleft lip, alveolus, palate, and velum and neural tube defects have revealed several shared multifactorial causes. Both anomalies emerge at different times during embryonic development and are not associated with each other unless there is a genetic component to the etiology. Vitamin deficiency disorders are one of several factors contributing to the etiology of these anomalies.Vitamins B6, folic acid and B12 play an essential role in the methylation cycle. A lack of or deficiency in these vitamins thus has severe consequences for the organism, especially the unborn child. Due to its short half-life, vitamin B6 is particularly important for undisturbed embryogenesis and should be taken along with folic acid as a periconceptional supplement to prevent embryonic deformities.This paper is intended to provide the orthodontist (as a member of the interdisciplinary cleft team) with an overview of the issues and etiological significance of vitamin B deficiencies as possible inducers of these embryopathies. This may encourage comprehensive counselling, particularly of parents of children born with deformities who wish to have more children.ZusammenfassungBisherige Untersuchungen zur Entstehung von Lippen-Kiefer-Gaumen-Segel-Spalten und Neuralrohrdefekten brachten eine Reihe gemeinsamer multifaktorieller Ursachen zutage. Beide Fehlbildungen entstehen zu unterschiedlichen Zeitpunkten in der Embryonalentwicklung und treten unter Ausschluss genetischer Ursachen nicht assoziiert auf. Ätiologisch sind für die genannten Fehlbildungen neben anderen Ursachen auch Vitaminmangelstörungen von Bedeutung.Die Vitamine B6, Folsäure und B12 sind maßgeblich am Methylierungszyklus beteiligt. Ein Fehlen oder eine Unterversorgung dieser Vitamine hat somit schwerwiegende Folgen für den Organismus, insbesondere für das ungeborene Kind. Aufgrund einer kurzen Halbwertszeit hat das Vitamin B6 eine besondere Bedeutung für eine reibungslose Embryogenese und sollte deshalb neben Folsäure zusätzlich perikonzeptionell zur Prävention embryonaler Fehlbildungen verabreicht werden.Dem Kieferorthopäden im interdisziplinären Spaltteam soll mit dieser Arbeit eine Übersicht zur Problematik und ätiologischen Bedeutung von Vitamin-B-Mangelzuständen als mögliche Induktoren dieser Embryopathien gegeben werden. Damit bestünde die Möglichkeit einer umfassenden Beratung insbesondere von Eltern fehlgebildeter Kinder mit weiterem Kinderwunsch.

Development of Bioactive Sol-Gel Material Template for In Vitro and In Vivo Synthesis of Bone Material

A novel porous material produced via sol gel process is disclosed which is osteoinductive and biodegradable. Due to the fact that high temperatures are avoided during the production process, the degree of crystallinity is very low and the packing of the crystallites is relatively loose. These are important parameters for the biodegradation properties.Nanoporous silica gel is contained to the support of strength. The low degree of crystallinity and the high degree of porosity (50–70%, pores in the range of few microns) make the material resorbable. A second type of pores designed in the range of few millimetres supports the bone ingrowth.An in vitro formation of bone tissue is shown when the material is exposed to tissue culture medium and inoculated with human osteoblastic cells. Animal tests show the formation of new bone tissue and first steps of biodegradation.

Sex-related differences in procarbazine-induced cleft palate and microgenia and the anti-teratogenic effect of prenatal folic acid supplementation in rats.

Sex-related differences in the frequency of cleft palates and microgenia in rat fetuses prenatally treated with procarbazine (200 mg/kg on day 14 of gestation (GD14), group 1), and the anti-teratogenic effect of prenatal folic acid supplementation (4 mg/kg on GD14 through GD17, group 2) were studied in LEW.1A rats. In group 1, complete clefts were observed in 69% of the male and in 36% of the female fetuses while incomplete clefts (present only in the hard palate) were exhibited by 31% of the males and 43% of the females. Microgenia occurred in all males but only in 64% of the female fetuses. In group 2, the prenatal folic acid supplementation significantly reduced the occurrence frequency of complete clefts to 9% in males and to 0% in females. In contrast, incomplete clefts increased to 82% in males and 91% in females. Microgenias were reduced to 73% and 57% in male and female fetuses, respectively. Since incomplete clefts present in the hard palate are assumed to be residues of spontaneous intra-uterine repair processes of exogenously induced complete palatal clefts, we conclude that prenatal supplementation with folic acid at a dose of 4 mg/kg promotes the intra-uterine repair of cleft palates and offers a partial protection against procarbazine teratogenicity. Furthermore, it is deduced that gender-specific differences exist in the susceptibility to procarbazine and in the anti-teratogenic effect of folic acid on procarbazine-induced microgenia.

Stimulation der Regeneration von Knochendefekten durch Einsatz einer Biokeramik und autologer Osteoblastentransplantation

Fragestellung. Ist es möglich, Critical-size-Defekte durch die artifizielle Stimulation von Osteokonduktion und Osteogenese zur knöchernen Ausheilung zu bringen, sodass spätere Knochentransplantate nicht mehr notwendig werden? Material und Methode. Der knöcherne Durchbau von definierten Critical-size-Defekten im Bereich des vorderen Unterkieferkörpers wurde bei 24 adulten Minischweinen klinisch, histologisch und im Rasterelektronenmikroskop analysiert. Die Defekte wurden mit einer im innovativen Sol-Gel-Verfahren hergestellten Biokeramik, bestehend aus 60% Hydroxylapatit (HA) und 40% β-Trikalziumphosphat (βTCP), allein bzw. kombiniert mit autologen kultivierten Osteoblasten aufgefüllt. In einer Kontrollgruppe wurden die Defekte nur mit Periost gedeckt. Das Beobachtungsintervall betrug 5 Wochen. Dabei wurde die Rolle des Periosts, der Biokeramik und der autolog transplantierten Osteoblasten untersucht. Ergebnisse. Die Biokeramik zeigte eine der Knochenneubildung angepasste Biodegradation und führte mit 72,3% im Vergleich zur Kontrollgruppe (59,3%) zu einer ausgeprägteren Reossifikation der Defekte. Die Defektauffüllung mit autologen, durch Kultivierung vermehrten Osteoblasten in Kombination mit dieser Biokeramik führte zu keiner weiteren Steigerung des Knochendurchbaus. Schlussfolgerung. Die in vivo getestete hochporöse Biokeramik, die sich durch ein lockeres Kristallgefüge auszeichnet, ist als ein geeignetes Knochenersatzmaterial einzuschätzen, welches auch als Matrix für osteoinduktive Substanzen, wie z. B. BMP (bone morphogenic protein), dienen könnte. Question. Does artificial stimulation of osteoconduction and osteogenesis lead to improved bone formation in defects of critical size? Material and methods. Full-thickness, critical-sized defects in the anterior mandible were created in 24 adult mini-pigs. These defects were treated with a new kind of bioactive ceramic (60% hydroxylapatite and 40% β-tricalcium phosphate), applied as a unique sol gel [9]. The bioceramic was tested alone and in combination with autologous osteoblasts. In a control group, periosteum was the only bone-producing source. After 5 weeks, the animals were sacrificed and the defects analyzed clinically, histologically, and by X-ray examination. The effects of periosteum, bioceramics, and osteoblasts were investigated in particular. Results. The new bioceramic was degraded at the same speed as new bone was laid down. The rate of newly formed bone was highest in the bioceramic group at 72.3% (control group with periosteal covering only 59.3%). Additional transplantation of autologous osteoblasts did not result in faster bone production. Conclusion. It seems that this bioactive ceramic is successful as a bone replacement material and will be suitable as a carrier for osteoinductive substances such as bone morphogenetic proteins.

Application of Thiamine in Preventing Malformations, Specifically Cleft Alveolus and Palate, during the Intrauterine Development of Rats

OBJECTIVE Animal experiments were conducted to test the reproducibility of previously documented antiteratogenic effects of thiamine on cleft formation in the craniofacial system. DESIGN Thirteen gravid Wistar rats carrying 98 fetuses were given the hydrazine derivative procarbacine (200 mg/kg BW) on the fourteenth day postconception (PC) to induce malformations, chiefly cleft alveolus and palate (day of determining presence of sperm was called the first day PC). Seven of the treated gravid rats carrying 48 fetuses were additionally given a daily dose of 200 mg/kg thiamine from the thirteenth to the nineteenth day PC. OUTCOME MEASURES A comparative analysis of the fetuses in both experimental groups was conducted externally and, for the skeleton, macroscopically using special staining techniques; the heads were analyzed using successional histologic sections; bodies were examined stereomicroscopically using the razor-blade sectioning technique. RESULTS In 12 of the 16 parameters evaluated, no statistically significant differences were found between experimental groups. In some cases, we even observed an amplifying effect of thiamine on the development of malformations in the rat strain used in our study. CONCLUSIONS Because several previous authors have repeatedly described treatment with thiamine as one of the sufficient prophylactic measures in slowing the development of viscerocranial malformations, especially cleft alveolus and palate, it is of utmost importance that the timing of treatment and dosage of thiamine be taken into consideration not only in animal experiments but also when applying results to humans.

Comparison of a New Kind of Calcium Phosphate Formula Versus Conventional Calciumphosphate Matrices in Treating Bone Defects – A Long-Term Investigation in Pigs

In clinical practice arises an increasing need for bone substitute materials. The main inorganic part of bone is the hydroxyapatite (HA). A new hydroxyapatite formula was created by a sol-gel-process at low temperature level [4]. The aim of this investigation was to test the biodegradation and the induction of bone formation by this new material and to compare these versus conventional fabricated HA and ß-TCP. 30 one-year-old Goettingen minipigs were divided into five groups. Critical size defect (>5 m3) in the mandible was treated differently in all 5 groups:-group I- filling with pure HA, which was fabricated by sol-gel-technique, group II- control, only gelatinous material was given, group III- conventional ß-TCP [Cerasorb®], in group IV- conventional HA [Endobone®] and in group V [Targobone®], a non denatureted bovine collagen matrix was used. Macroscopical and microscopical investigations of the former defects were made eight months postoperatively. The bone formation was superior in the sol-gel-HA-group (group I) in comparison with the control groups (group II) and the conventional fabricated ceramics groups (III and IV). In the sol-gel-HA group, the biodegradation of this new biomaterial was considered to very good with a resorption rate of more than 98%; eight months postoperatively. In this group complete bone formation was seen in former defects. In the control group, only an incomplete bone formation with 48.4% of the defect area was noted. This difference was significant (p<0,001). A less bone formation was also observed in group III and IV with 57.6% and 56.9%. The bovine non-denaturated collagen matrix (group V) leads to only 20% of new formed bone. The new calcium phosphate formula made by a sol-gel method seems to be superior and suitable for filling bone defects.

Differential diagnosis of stafne idiopathic bone cyst with Digital Volume Tomography (DVT)

ObjectivesThe distinction between a pseudocyst and an intervention demanding cyst or process cannot always be found by 2D radiology. The differential diagnosis of a pseudocyst may become more difficult when adjacent processes are present.CaseA symptom free 67-years-old man presented with a periapical radiolucency around the mandibular left second molar as well as at the impacted 3rd molar. A comparison with an older panoramic x-ray showed no expansion during a 27 months period. The new panoramic x-ray and addtional Digital Volume Tomography (DVT) showed asymptomatic separate dental cyst at the impacted wisdom tooth and the missing lingual cortical border in the apical region of the 2nd lower molar. This finding along with clinical vitality of the 1st and 2nd left molars led to the conclusion that the presented pathology was a Stafne Idiopathic Bone Cyst (SIBC) which needed no surgical intervention.DiscussionThe additional use of DVT 3D examination may help in diagnosis of SIBC prior to surgical interventions thus avoiding unwanted surgical intervention.

Disturbances of palatogenesis and their prophylaxis in animal experiments.

The goal of this study using experimental animals was to induce disturbances of palatogenesis which are comparable to human maxillary clefts. Simultaneously, an in vivo method of testing presumed antiteratogenic substances is presented. 13 gravid Wistar rats bearing 98 fetuses received 200 mg/kg of procarbazine on day 14 post conception (p. c.) to induce malformations. 7 of these gravid animals, bearing 48 fetuses, additionally received 200 mg/kg thiamine daily from day 13-19 p. c. to prevent malformations. On day 20 p. c., the fetuses were teratologically screened: all fetuses were externally examined, the skeletons of 1/3 were visualized using cartilage/bone staining methods, and the heads of 2/3 were histologically examined in 24 sequential frontal sections. At birth, the procarbazine-damaged fetuses exhibited a high rate of cleft palate, primarily involving the secondary palate (94%), which was accompanied by retardation and delayed ossification of the viscerocranium. 66% of the fetuses showed pronounced brachygenia. The disturbances of palatogenesis were frequently accompanied by disturbed odontogenesis, which chiefly manifested itself near the cleft of the frontal maxilla as a reduction in size (63%), retardation (38%) or absence (31%) of the tooth germ. In the trial group additionally treated with thiamine, the findings did not differ significantly from these. The animal model presented here proved suitable for studying palatogenesis and localization-specific testing of substances presumed to have antiteratogenic effects. A prophylactic effect of thiamine initially tested as a highly-dosed monotherapy was not verifiable.