Site-Specific Variations in Bone Mineral Density under Systemic Conditions Inducing Osteoporosis in Minipigs.

Osteoporosis is a systemic bone disease with an increasing prevalence in the elderly population. There is conflicting opinion about whether osteoporosis affects the alveolar bone of the jaws and whether it poses a risk to the osseointegration of dental implants. The aim of the present study was to evaluate the effects of systemic glucocorticoid administration on the jaw bone density of minipigs. Thirty-seven adult female minipigs were randomly divided into two groups. Quantitative computed tomography (QCT) was used to assess bone mineral density BMD of the lumbar spine as well as the mandible and maxilla, and blood was drawn. One group of minipigs initially received 1.0 mg prednisolone per kg body weight daily for 2 months. The dose was tapered to 0.5 mg per kg body weight per day thereafter. The animals in the other group served as controls and received placebo. QCT and blood analysis were repeated after 6 and 9 months. BMD was compared between the two groups by measuring Hounsfield units, and serum levels of several bone metabolic markers were also assessed. A decrease in BMD was observed in the jaws from baseline to 9 months. This was more pronounced in the prednisolone group. Statistically significant differences were reached for the mandible (p < 0.001) and the maxilla (p < 0.001). The administration of glucocorticoids reduced the BMD in the jaws of minipigs. The described model shows promise in the evaluation of osseointegration of dental implants in bone that is compromised by osteoporosis.

Coating with artificial matrices from collagen and sulfated hyaluronan influences the osseointegration of dental implants.

Dental implants are an established therapy for oral rehabilitation. High success rates are achieved in healthy bone, however, these rates decrease in compromised host bone. Coating of dental implants with components of the extracellular matrix is a promising approach to enhance osseointegration in compromised peri-implant bone. Dental titanium implants were coated with an artificial extracellular matrix (aECM) consisting of collagen type I and either one of two regioselectively low sulfated hyaluronan (sHA) derivatives (coll/sHA1Δ6s and coll/sHA1) and compared to commercial pure titanium implants (control). After extraction of the premolar teeth, 36 implants were inserted into the maxilla of 6 miniature pigs (6 implants per maxilla). The healing periods were 4 and 8 weeks, respectively. After animal sacrifice, the samples were evaluated histomorphologically and histomorphometrically. All surface states led to a sufficient implant osseointegration after 4 and 8 weeks. Inflammatory or foreign body reactions could not be observed. After 4 weeks of healing, implants coated with coll/sHA1Δ6s showed the highest bone implant contact (BIC; coll/sHA1Δ6s: 45.4%; coll/sHA1: 42.2%; control: 42.3%). After 8 weeks, a decrease of BIC could be observed for coll/sHA1Δ6s and controls (coll/sHA1Δ6s: 37.3%; control: 31.7 %). For implants coated with coll/sHA1, the bone implant contact increased (coll/sHA1: 50.8%). Statistically significant differences could not be observed. Within the limits of the current study, aECM coatings containing low sHA increase peri-implant bone formation around dental implants in maxillary bone compared to controls in the early healing period.

OH⁻ deficiency in dental enamel, crown and root dentine as studied by ¹H CRAMPS.

High-resolution solid-state NMR based on combined rotation and multipulse spectroscopy (CRAMPS) has been applied to study chemical structures of dental tissues. The samples of human enamel, crown dentine and root dentin studied in this work were used without chemical pre-treatment. The quantitative ¹H NMR spectra permit an assignment to different structures and a quantification of the content of hydroxyl groups. While there is 40% hydroxyl content in the enamel, there is significantly less in the dentin, 14% in the crown and 9% in the root. Thus this study provides the direct evidence of OH⁻ ion deficiency in all dental tissues supporting earlier findings that bone and dental mineral apatite is poorly hydroxylated.

Influence of different modifications of a calcium phosphate cement on resorption and new bone formation: an in vivo study in the minipig.

Calcium phosphate cements (CPCs) are established in surgery as temporary bone replacement materials. The most common and important class of CPC, transformed into nanocrystalline hydroxyapatite after setting, is characterized by good biocompatibility and osteoconductivity. However, acceleration of remodelling is in the focus of ongoing research. In the present study, the bone healing efficacy of Biocement D (BioD) modified with mineralized collagen alone (BioD/coll) or in combination with osteocalcin (BioD/coll/OC), O-phospho-L-serine (BioD/coll/PS), sodium citrate (BioD/coll/cit), and polylactide (BioD/coll/PL), respectively, was evaluated in a large animal model. Resorption of the bone substitutes and new bone formation were studied in cyst-like jaw defects of minipigs after filling with the unmodified BioD and the modified BioD variants, respectively. Histomorphometric analysis revealed small differences between the different cement types with respect to resorption. However, new bone formation was improved in case of defects repaired with BioD/coll/OC and BioD/coll/PS and slightly improved in case of BioD/coll and BioD/coll/PL.

Histological behaviour of zirconia implants: an experiment in rats.

New biomaterials including surface modifications should undergo in vitro and in vivo evaluation before clinical trials. The objective of our in vivo study was to evaluate the biocompatibility of one of the newly fabricated zirconia implant surfaces, called "mds". For this purpose, the osseointegration of these implants was analyzed after implantation in surgically created defects in the cranium of adult male rats. After a healing period of 28 and 56 days, respectively, bone tissue specimens containing the implants were processed and histologically analyzed. For this purpose, sections were stained with haematoxylin/eosin and Masson Goldner trichrome. No signs of cellular inflammatory infiltrate were found in any of the animals. After 28 days, slices showed pronounced development of blood vessels and bone regeneration. After 56 days of healing, direct bridging of the bone defects was detectable with distinctly visible kit lines. There were cell rich areas of connective tissue/bone marrow between zirconia discs and bearing bone. Histomorphometric analysis presented a regenerated bone mean value of 36.3% after 28 days of healing. After 56 days of healing, a 1.6 fold increased bone mean value was observed (58.2%). Using the same analysis, 1% and 39.9% of bone-implant-contact was visible after both healing periods, respectively. On average, connective tissue/marrow spaces occupied 99% of implant-contact-area after 28 days of healing. This area was reduced to 60.1% after 56 days. Within the limits of the animal investigation presented, it was concluded that the tested surface modification of zirconia implants were biocompatible and osseoconductive.

Characterization of the osseointegration of Algipore and Algipore modified with mineralized collagen type I.

OBJECTIVE: Algipore is a clinically established bone substitute. The present study evaluated the osseoconductive and resorptive characteristics of Algipore modified with collagen type I (ACI). STUDY DESIGN: Three defects of 10 × 3 mm were set in the frontal bone of 10 adult female minipigs. One cavity was filled with commercially available Algipore, and the second with ACI. The third cavity was left unfilled and served as reference. After 4 months of healing, the animals were humanely killed. Bone formation and resorption characteristics of the substitutes were evaluated histomorphologically and histomorphometrically using Donath's sawing and grinding technique. RESULTS: Neither material caused inflammatory reactions. Compared with controls, both substitutes showed significantly higher fractions of trabecular bone (control: 42.2%; Algipore: 58.7%, [P < .001]; ACI: 53.6%, [P = .013]). After 4 months, the remaining fraction of Algipore was 42.2% and the fraction of ACI was 47.9% (P = .016). CONCLUSIONS: The present study demonstrates that the modification of Algipore with collagen I does not show any benefits compared with pure Algipore in small calvarial bone defects in minipigs.

Ultrasound-aided resorbable osteosynthesis of fractures of the mandibular condylar base: an experimental study in sheep.

We evaluated the osteosynthesis of condylar fractures using resorbable mini plates and ultrasound-aided insertion of pins clinically and histologically. Stability was greater than that with resorbable screws because of the fusion of pin and plate. Long term evaluation showed complete resorption of the polymeric osteosynthesis material.

Biological functionalization of dental implants with collagen and glycosaminoglycans-A comparative study.

Biological implant surface coatings are an emerging technology to increase bone formation. Such an approach is of special interest in anatomical regions like the maxilla. In the present study, we hypothesized that the coating of titanium implants with components of the organic extracellular matrix increases bone formation and implant stability compared to an uncoated reference. The implants were coated using collagen-I with either two different concentrations of chondroitin sulfate (CS) or two differentially sulfated hyaluronans. Implant coatings were characterized biochemically and with atomic force microscopy. Histomorphometry was used to assess bone-implant contact (BIC) and bone-volume density (BVD) after 4 and 8 weeks of submerged healing in the maxilla of 20 minipigs. Further, implant stability was measured by resonance frequency analysis (RFA). Implants containing the lower CS concentration had significantly more BIC, compared to the uncoated reference at both times of interest. No significant increase was measured from week 4 to 8. Differences in BVD and RFA were statistically not significant. A higher concentration of CS and the application of sulfated hyaluronans showed no comparable increase in BIC. This study demonstrates a positive effect of a specific collagen-glycosaminoglycan combination on early bone formation in vivo.

Orthodontic tooth movement into jaw regions treated with synthetic bone substitute.

INTRODUCTION: Orthodontic tooth movement is fraught with risks and complications. Root resorption is the most frequent and important outcome which may arise due to inappropriate force magnitude or tooth movement into dense or altered bone. This study was aimed to demonstrate histologic changes in tooth roots following movement into a jaw region treated with synthetic bone substitute. Another objective was to evaluate the method of experimental tooth movement using an animal model. MATERIAL AND METHODS: One mandibular premolar was extracted in each of three pigs (Sus scrofa domesticus). The extraction alveolus was filled with synthetic bone substitute material and a orthodontic appliance was attached for 90 days. The force for tooth movement was in the range of 1-2 N. Subsequently, specimens were collected using segmental osteotomy and were prepared histologically. Unmoved teeth served as controls. RESULTS: Histological analysis showed clear lacuna-like lesions in the root surface area of all specimens. The lesions were largest in the apical area, while the mid-root region was less affected. CONCLUSIONS: Due to deformations of the orthodontic tooth moving appliance, the force could not be exactly defined. Therefore, marked resorption lesions of the root surfaces cannot be unequivocally attributed to the synthetic bone substitute. However, the type of lesions on root surfaces permits the assumption that orthodontic tooth movement into areas filled with synthetic bone substitute may be associated with an increased risk of root resorption.

Surface-conditioned dental implants: an animal study on bone formation.

AIM: The aim of this study was to determine whether bone formation around surface-conditioned implants is enhanced compared with non-surface-conditioned sandblasted acid-etched titanium implants. MATERIALS AND METHODS: One hundred and forty-four implants were placed in the mandible of 18 minipigs. Before placement, implants were either surface conditioned in a solution containing hydroxide ions (conSF) or assigned to controls. Animals were euthanized after 2, 4 and 8 weeks of submerged healing, the 8-week group receiving polyfluorochrome labelling at week 2, 4, 6 and 8. One jaw quadrant per animal was selected for histological and histomorphometrical evaluation of mineralized bone-implant contact (mBIC), osteoid-implant contact (OIC) and bone volume (BV) analysis. RESULTS: Polyfluorochrome labelling showed no general differences in bone dynamics. mBIC showed the most pronounced differences after 2 weeks, reaching 65.5% for conSF compared with 48.1% for controls, p=0.270. Differences levelled out after 4 weeks (67.4% control, 65.7% conSF) and 8 weeks (64.0% control, 70.2% conSF). OIC levels were initially comparable, showing a slower decline for conSF after 4 weeks. BV was higher for conSF at all times. No significant differences could be found. CONCLUSION: A tendency towards increased mBIC was shown for surface-conditioned implants after short-term healing.

Increased bone formation around coated implants.

AIM: We hypothesized that coating threaded, sandblasted acid-etched titanium implants with collagen and chondroitin sulphate (CS) increases bone formation and implant stability, compared with uncoated controls. MATERIALS AND METHODS: Three different implant surface conditions were applied: (1) sandblasted acid-etched (control), (2) collagen/chondroitin sulphate (low-dose--CS1), (3) collagen/chondroitin sulphate (high-dose--CS2). Sixty 9.5 mm experimental implants were placed in the mandible of 20 minipigs. Bone-implant contact (BIC) and relative peri-implant bone-volume density (rBVD--relation to bone-volume density of the host bone) were assessed after 1 and 2 months of submerged healing. Implant stability was measured by resonance frequency analysis (RFA). RESULTS: After 1 month, coated implants had significantly more BIC compared with controls (CS1: 68%, p<0.0001, CS2: 63%, p=0.009, control: 52%). The rBVD was lower for all surface conditions, compared with the hostbone. After 2 months, BIC increased for all surfaces. No significant differences were measured (CS1: 71%, p=0.016, CS2: 68%, p=0.67, control: 63%). The rBVD was increased for coated implants. RFA values were 71-77 at implantation, 67-73 after 1 month and 74-75 after 2 months. Differences in rBVD and RFA were not statistically significant. CONCLUSIONS: Data analysis suggests that collagen/CS has a positive influence on bone formation after 1 month of endosseous healing.

Prefabricated bony radial forearm flap for secondary mandible reconstruction after radiochemotherapy.

BACKGROUND: Primary reconstruction of the mandible is the golden standard of surgical treatment after ablative tumor surgery. Many different microvascular bone grafts are used to reduce wound healing complications at the severely compromised recipient site. The loss of primary grafts due to radiotherapy or osteoradionecrosis can make secondary mandibular reconstruction necessary. To address this problem, we developed the technique of the prefabrication of a radial forearm flap with cancellous bone. The aims were to establish these techniques into the clinical routine and to create a safe and reliable flap with low donor site morbidity. METHODS: In patients who had undergone ablative tumor surgery radiochemotherapy, and primary reconstruction, prefabricated bony radial forearm flaps (PBRFFs) were applied for secondary reconstruction of the mandible. Cylinders of cancellous bone taken from the iliac crest were implanted in the lower forearm to allow the necessary vascularization. After a healing period of 4 weeks, the PBRFF was elevated and grafted into the mandibular defect. RESULTS: All grafts healed uneventfully. However, 1 case required revision of the venous anastomosis after 2 days. The transplants improved the contour of the lower face enabling a good correction of the facial asymmetry. During the follow-up of up to 4 years, the radiographic controls showed good bony consolidation between the graft and the stumps of the mandible as well as formation of cortical bone around the cancellous bone cylinders. CONCLUSION: These results demonstrate that the PBRFF is a safe and reliable graft which provides alternate solution in which other microvascular bone grafts have already failed. In the future, the iliac bone graft may be replaced with scaffold seeded with stem cells for further reduction of donor site morbidity.

Immunohistochemical comparison of markers for wound healing on plastic-embedded and frozen mucosal tissue.

Immunohistologic investigations of wound healing in human oral mucosa require specific cell biological markers as well as consecutive small biopsies. Small specimens are ideally embedded in plastic (methylmethacrylate, MMA) resin due to their miniature size. This limits the use of antibodies for these markers. In this immunohistochemical study, the distribution of wound healing markers, e.g. cytokeratin (CK), laminin, collagen IV, vimentin, vinculin and fibronectin, were compared between semithin sections of plastic-embedded tissue and frozen sections of mucosal tissue in order to assess their use for future investigations. The antibodies against laminin, collagen IV and CK 1/2/10/11, 5/6, 13, 14, 17, 19 gave comparable staining patterns on cryostat sections of attached mucosa and on semithin sections of MMA-embedded attached mucosa. In the epithelial cell layers, the following distribution of CK immunostaining was observed: The basal cell layer was positive for CK 5/6, CK 14 and CK 19; the intermediate cell layer for CK 13, CK 17 and CK 1/2/10/11, and the superficial cell layer for CK 13 and CK 1/2/10/11. For most of these antibodies, enzyme digestion with 0.1% trypsin was adequate for demasking the antigens, except for anti-CK 14, anti-CK 17 and anti-laminin; predigestion with 0.4% pepsin in 0.01 N HCl gave similar staining results. The antibodies against vimentin, vinculin, fibronectin and CK 4 showed no affinity or a reciprocal reaction on the semithin sections. Therefore, the antibodies against CK 1/2/10/11; 5/6; 13; 14; 17, and 19, as well as the basement proteins laminin and collagen IV are deemed markers suitable on semithin sections of plastic-embedded attached oral mucosa.

Mechanical loading of orthodontic miniscrews - significance and problems: an experimental study.

Orthodontic miniscrews are exposed to three mechanical loading phases during clinical use: torsional loading upon insertion, flexural loading during anchorage function, and torsional loading upon removal. The aim of this study was to simulate clinical loading conditions for different types of orthodontic miniscrews in vitro to quantify the effects of combined torsional and bending stress. Various orthodontic miniscrew systems (Lomas, Dual-top, Aarhus anchorage, Tomas-pin and T.I.T.A.N.-pin) comprising 10 samples each were subjected to the following loading sequences in vitro: a torsional load corresponding to manual insertion with limited torque; and flexural loading at two different insertion depths. For all screw systems with torsional pre-loading (simulating insertion), subsequent flexural loading (simulating anchorage) yielded permanent deformations of approximately 0.15-0.25 mm, depending on the insertion depth. Since EDX analysis revealed comparable elemental compositions for the different screw systems, the differences in mechanical properties are attributed to screw design. Torsional loading during screw insertion may cause premature mechanical weakening and needs to be minimized. Unless fully inserted, screws show pronounced plastic deformation and hence fracture risk under subsequent flexural loading.

O-phospho-L-serine: a modulator of bone healing in calcium-phosphate cements.

Bone substitution materials are seen as an alternative to autogenous bone transplants in the reconstruction of human bone structures. The aim of the present animal study was to evaluate the clinical handling and the conditions of bone healing after the application of a phosphoserine and collagen-I-modified calcium-phosphate cement (Biozement D). The application of phosphoserine is supposed to influence the texture of the extracellular matrix. Standardised bone defects were created in the lower jaw of 10 adult minipigs. These defects were reconstructed with a pasty calcium-phosphate cement mixture. After a healing time of 4 months, the animals were sacrificed. The mandibles of all animals were resected and non-decalcified histological sections of the areas of interest were prepared. The experiment was evaluated by means of qualitative histology and histomorphometry. The hydroxyapatite cement entirely hardened intraoperatively. Modelling and handling of the cement was facile and the margin fit to the host bone was excellent. Histology showed that resorption started in the periphery and proceeded exceptionally fast. The bony substitution, especially in phosphoserine-endowed cements, was very promising. After a healing period of 4 months, phosphoserine cements showed a bone regeneration of nearly two-thirds of the defect sizes. In the applied animal experiment, the newly developed hydroxyapatite collagen-I cement is well suited for bone substitution due to its easy handling, its excellent integration and good resorption characteristics. The addition of phosphoserine is very promising in terms of influencing resorption features and bone regeneration.

Finite element representation of bone substitute remodelling in the jaw bone.

The finite element (FE) method was originally developed on a physical basis for the computation of structure-mechanical problems. Meanwhile, it has been widely applied to medical issues. This study sought a suitable method to build a FE model for remodelling processes in osseous defects supplemented with bone substitute material. For this purpose, the second right premolars were extracted from four pig mandibles (Sus scrofa domesticus) and the extraction alveoli were provided with synthetic bone substitute material. After 70 days, a segmented osteotomy of this area was performed for specimen collection. Radiographs of the specimens were taken in defined planes before and after fixation and embedded with Technovit 9100. Fixation-related shrinkage was quantified from the radiographs using reference lines. Computer tomographic (CT) and microCT images of the fixed and embedded specimens were obtained. From these data, a FE model was built. The construction of a FE model is sufficient to represent bone remodelling after supply of bone substitute material. The use of microCT data permits building a clearly more precise model.

The biomaterial influences the ossification after sinus floor elevation using tissue-engineered bone grafts.

Sinus floor elevation is the standard procedure that allows dental implant insertion in the atrophic posterior maxilla. Instead of autogenous bone, tissue-engineered bone grafts can be used, but clear comparative clinical studies also assessing the influence of the biomaterial are missing. In six patients, tissue-engineered bone grafts were used in eight sinus floor elevations. After culturing osteoblast-like cells from biopsies of the maxilla, they were seeded on scaffolds made either from demineralised bovine bone matrix (DBBM) or from solvent-dehydrated mineralised bone (SDBB), and grafted. In all patients primary wound healing was without complications, except for one patient in the SDBB group. After 12 months, implant insertion was possible only in the SDBB group; in the DBBM group, fibrous connective tissue was found in an attempt of implant insertion. After 5 months, implant placement was performed in one patient of each group. However, the two implants inserted in the DBBM group were lost after 6 weeks. Histology of the bone cores in the DBBM group at 5 months showed lamellar bone and osteoid, and at 12 months showed fibrous connective tissue. Inflammation and some resorption of the scaffold was found 5 months after SDBB grafting, and after 12 months cancellous bone formation encapsulating SDBB remnants were observed. These preliminary data suggest that the preparation method of the bovine bone matrix, in particular the mineral content, and therefore the mechanical stability may have some influence on the generation of new bone.

Suitability of differently designed matrix-based implant surface coatings: an animal study on bone formation.

INTRODUCTION: The aim of the present study was to assay how bone formation around dental implants is influenced by differently composed collagen matrices and RGD peptide as implant surface coatings compared to a sandblasted titanium surface. MATERIAL AND METHODS: Five different implant surface coatings were designed: titanium (sandblasted), collagen type I, collagen type I&III, RGD-peptide, and mineralized collagen. Sixty experimental implants of a square-shaped design were inserted into the mandibles of 12 minipigs, 3 months following extraction of the premolar teeth. During the 6-month study period, sequential polyfluorochrome labeling was performed. After sacrifice, bone implant contact (BIC) was evaluated using histologic and histomorphometric methods. RESULTS: New bone formation was observed against all implant surfaces. Polyfluorochrome labeling showed that bone growth started from the host bone in the majority of samples. The highest BIC was measured for collagen I and collagen I/III coated implants; however, significant differences between the coatings could not be found. CONCLUSION: Osseointegration was achieved for all implant surfaces. Although a statistically significant increase in BIC could not be demonstrated for the experimental coatings after the 6 months study period, there was also no discernible detrimental effect of the coatings in comparison to the uncoated titanium surfaces.

Histologic study of incorporation and resorption of a bone cement-collagen composite: an in vivo study in the minipig.

OBJECTIVE: Calcium phosphates are clinically established as bone defect fillers. They have the capability of osseoconduction and are characterized by a slow resorption process. The present study evaluated the suitability of a newly developed calcium phosphate cement modified with collagen type I. STUDY DESIGN: The modified cement paste was inserted in differently designed defects of 10 minipigs. Further, an alveolar ridge augmentation was performed, applying the cement paste. The cement hardened in situ during the operation, forming a hydroxyapatite collagen composite. Animals were sacrificed after 1, 3, 6, 12, and 18 months. The tissue integration and resorption process was then evaluated using nondecalcified microsections. All animals were evaluated for histology. RESULTS: The implanted material showed osseoconductive characteristics. Resorption started from the edge of the defect zone, and bone substitution followed rapidly. Twelve months after placement of the cement, complete remodeling was observed. CONCLUSION: It can be concluded that the applied hydroxyapatite-collagen cement composite shows good resorption and bone integration.

Cryopreserved tissue engineered mucosa.

Single cells suspensions used for grafting in the clinical setting may be reliably cryopreserved by established protocols. However, for tissue engineered constructs which now also get used as grafts in the clinic such established protocols and assays which indicate graft viability and their function as graft do not exist. a) The purpose was to develop a cryoprotocol and an animal model to test the efficacy of tissue engineered to act as graft after cryopreservation. b) Therefore, tissue engineered mucosa grafts consisting of keratinocytes and fibroblasts grown in a collagen sponge were cryopreserved and grafted in the nude rat to test its efficacy to function as mucosa graft. At different points after cryopreservation the mucosa was grafted into the nude rats. Healing of grafts was allowed for one or three weeks. c) Sufficient cells survived the cryopreservation allowing for the development of epithelial-fibroblast tissue in the collagen sponge. After three weeks of healing the formation of mucosa tissue was more complete and more collagen sponge had disappeared. d) The nude rat model is suitable to assess the efficacy of tissue engineered mucosa to function as graft after cryopreservation. The formation of human epithelial-fibroblast tissue in vivo has to be interpreted as proof of principle that the approach of cryopreservation of tissue engineered grafts is working.