Periosteum-Derived Cells as an Alternative to Bone Marrow Cells for Bone Tissue Engineering Around Dental Implants. A Histomorphometric Study in Beagle Dogs

Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP)

Bone regeneration at implants placed into extraction sockets of maxillary incisors in dogs

AimTo compare the influence of autologous or deproteinized bovine bone mineral as grafting material on healing of buccal dehiscence defects at implants installed immediately into the maxillary second incisor extraction socket in dogs.Material and methodsIn the maxillary second incisor sockets of 12 Labrador dogs, implants were installed immediately following tooth extraction. A standardized buccal defect was created and autologous bone particles or deproteinized bovine bone mineral were used to fill the defects. A collagen membrane was placed to cover the graft material, and the flaps were sutured to fully submerge the experimental areas. Six animals were sacrificed after 2 months, and six after 4 months of healing. Ground sections were obtained for histological evaluation.ResultsAfter 2 months of healing, all implants were osseointegrated. All buccal dehiscence defects were completely filled after 2 months irrespective of the augmentation material (autologous bone or Bio-Oss (R)) applied. Bone-to-implant contact (BIC) on the denuded implant surfaces was within a normal range of 30-40%. However, the newly formed tissue at 2 months was partially resorbed (> 50% of the area measurements) after 4 months.ConclusionsApplying either autologous bone or deproteinized bovine bone mineral to dehiscences at implants installed immediately into extraction sockets resulted in high degree of regeneration of the defects with satisfactory BIC on the denuded implant surface.To cite this article:De Santis E, Botticelli D, Pantani F, Pereira FP, Beolchini M, Lang NP. Bone regeneration at implants placed into extraction sockets of maxillary incisors in dogs.Clin. Oral Impl. Res. 22, 2011; 430-437.

Interference of the blood clot on granulation tissue formation after tooth extraction. Histomorphological study in rats.

The interference of a blood clot in the first postoperative hours of dental extraction wounds was studied in rats. Sixty male albino rats were divided into two groups: Group I, immediately after extraction of right maxillary incisor the gingival mucosa was approximated and sutured; Group II, after 6 to 8 minutes postoperatively the blood clot was removed with saline irrigation and absorbent paper cones. The mucosa was then approximated and sutured. Six animals in each group were sacrificed after 12 hours, 1, 4, 7 and 10 days. There was a profound delay in healing in Group II since, although a new blood clot was later formed, it was not organized. The quality and the constitution, maintenance and retraction of the clot are the regulating factors in connective tissue formation during alveolar healing.

Osseous regeneration in the presence of fibrin adhesive material (Tissucol) and epsilon-aminocaproic acid (EACA).

The effects of Tissucol and Tissucol/EACA on bone healing were evaluated histologically. Experimental defects were made in both tibias of 25 rats. Test materials were placed in defects in right tibias and left tibias served as control. Five animals in each group were killed at 1, 3, 7, 14 and 21 days after surgery. Results showed that: a) Tissucol did not interfere with connective and osseous tissue formation; b) Tissucol allowed new bone formation; c) Tissue residues in Tissucol groups in sections of 21-day specimens did not impair healing; d) Tissucol/EACA was usually completely resorbed and healing was complete 21 days after surgery in the Tissucol/EACA group.

Morphological aspects of muscle regeneration in the Nile tilapia (Oreochromis niloticus)

The aim of our study was to analyze the morphological events in the skeletal muscle of the Nile tilapia (Oreochromis niloticus) after a traumatic lesion. Thirty-two fish were used, on which a small longitudinal incision was made in the muscle. The fish were sacrificed after 7, 14, 21, and 42 days and muscle samples were collected from the lesion and processed for morphological analysis. Muscle regeneration in the tilapia occurred gradually through the analyzed period, possibly due to the proliferation and differentiation of myosatellite cells, which were more morphologically evident 7 and 14 days after lesion.

Bacterial cellulose-hydroxyapatite nanocomposites for bone regeneration

The aim of this study was to develop and to evaluate the biological properties of bacterial cellulose-hydroxyapatite (BC-HA) nanocomposite membranes for bone regeneration. Nanocomposites were prepared from bacterial cellulose membranes sequentially incubated in solutions of CaCl2 followed by Na2HPO4. BC-HA membranes were evaluated in noncritical bone defects in rat tibiae at 1, 4, and 16 weeks. Thermogravimetric analyses showed that the amount of the mineral phase was 40-50 of the total weight. Spectroscopy, electronic microscopy/energy dispersive X-ray analyses, and X-ray diffraction showed formation of HA crystals on BC nanofibres. Low crystallinity HA crystals presented Ca/P a molar ratio of 1.5 (calcium-deficient HA), similar to physiological bone. Fourier transformed infrared spectroscopy analysis showed bands assigned to phosphate and carbonate ions. In vivo tests showed no inflammatory reaction after 1 week. After 4 weeks, defects were observed to be completely filled in by new bone tissue. The BC-HA membranes were effective for bone regeneration. © 2011 S. Saska et al.

Connective tissue grafts in conjunction with implants installed immediately into extraction sockets. An experimental study in dogs

Aim: To evaluate the influence of a sub-epithelial connective tissue graft placed at the buccal aspect of implants installed immediately after tooth extraction on the dimensional changes of hard and soft tissues. Materials and Methods: In six Labrador dogs a bilateral partial- thickness dissection was made buccal to the second mandibular premolar. At the lingual aspect, full-thickness flaps were elevated. The teeth were extracted and implants installed immediately into the distal socket. A connective tissue graft was obtained from the palate and applied to the buccal aspect of the test sites, whereas contra-laterally, no graft was applied. The flaps were sutured to allow a non-submerged installation. After 4 months of healing, the animals were sacrificed, ground sections were obtained and histomorphometric analyses were performed. Results: After 4 months of healing, all implants were integrated (n = 6). Both at the test and at the control sites bone resorption occurred: 1.6 mm and 2.1 mm, respectively. The difference was not statistically significant. The coronal aspect of the peri-implant soft tissue was wider and located more coronally at the test compared with the control sites. The differences were statistically significant. Conclusions: The application of a connective tissue graft placed at the buccal aspect of the bony wall at implants installed immediately after tooth extraction yielded a minimal preservation of the hard tissues. The peri-implant mucosa, however, was significantly thicker and more coronally positioned at the test compared with the control sites. © 2012 John Wiley & Sons A/S.

Guided implant surgery: What is the influence of this new technique on bone cell viability?

Purpose: To evaluate the effect of implant osteotomy on immediate bone cell viability, comparing guided surgery for implant placement with the classic drilling procedure. Materials and Methods: For this study, 20 rabbits were used. The animals were divided into a guided surgery group (GG) and a control group (CG) and were then divided into 4 subgroups - subgroups 1, 2, 3, and 4 - corresponding to drills used 10, 20, 30, and 40 times, respectively. All animals received 5 osteotomies in each tibia, by use of the classic drilling procedure in one tibia and guided surgery in the other tibia. The osteotomized areas were removed and processed immunohistochemically for detection of osteocalcin, receptor activator of nuclear factor κB ligand (RANKL), osteoprotegerin (OPG), and caspase 3. Results: Immunohistochemical analysis showed that osteocalcin expression was initially higher in the CG and remained constant after drill reutilization. Although the expressions of RANKL and OPG were not statistically different for the GG and CG, the RANKL/OPG ratio tended to be higher for the GG. Moreover, caspase 3 expression was elevated in the GG, proportionally to the number of osteotomies, indicating an increase in the apoptosis index in the GG. Conclusions: The classic drilling procedure is more favorable to cell viability than guided surgery.© 2013 American Association of Oral and Maxillofacial Surgeons.

Tissue engineering: Using collagen type i matrix for bone healing of bone defects

Among the many tissues in the human body, bone has been considered as a powerful marker for regeneration and its formation serves as a prototype model for tissue engineering based on morphogenesis. Therefore, collagen type I is one of the most useful biomaterials used in tissue engineering as extracellular matrix components capable to promote bone healing. The literature reveals excellent biocompatibility and safety due to its biological characteristics, such as biodegradability and weak antigenicity, making collagen type I the primary resource in medical applications. Thus, it was also used for tissue engineering including skin replacement, bone substitutes, and artificial blood vessels and valves. The authors describe the treatment of an abscessed apical periodontal cyst and show good outcomes of bone healing, using tissue engineering, as collagen type I matrix. © 2013 by Mutaz B. Habal, MD.

Subcutaneous tissue reaction and cytotoxicity of polyvinylidene fluoride and polyvinylidene fluoride-trifluoroethylene blends associated with natural polymers

Cytotoxicity and subcutaneous tissue reaction of innovative blends composed by polyvinylidene fluoride and polyvinylidene fluoride-trifluoroethylene associated with natural polymers (natural rubber and native starch) forming membranes were evaluated, aiming its applications associated with bone regeneration. Cytotoxicity was evaluated in mouse fibroblasts culture cells (NIH3T3) using trypan blue staining. Tissue response was in vivo evaluated by subcutaneous implantation of materials in rats, taking into account the presence of necrosis and connective tissue capsule around implanted materials after 7, 14, 21, 28, 35, 60, and 100 days of surgery. The pattern of inflammation was evaluated by histomorphometry of the inflammatory cells. Chemical and morphological changes of implanted materials after 60 and 100 days were evaluated by Fourier transform infrared (FTIR) absorption spectroscopy and scanning electron microscopy (SEM) images. Cytotoxicity tests indicated a good tolerance of the cells to the biomaterial. The in vivo tissue response of all studied materials showed normal inflammatory pattern, characterized by a reduction of polymorphonuclear leukocytes and an increase in mononuclear leukocytes over the time (p < 0.05 Kruskal-Wallis). On day 60, microscopic analysis showed regression of the chronic inflammatory process around all materials. FTIR showed no changes in chemical composition of materials due to implantation, whereas SEM demonstrated the delivery of starch in the medium. Therefore, the results of the tests performed in vitro and in vivo show that the innovative blends can further be used as biomaterials. © 2013 Wiley Periodicals, Inc. J Biomed Mater Res Part B: Appl Biomater 101B: 1284-1293, 2013. Copyright © 2013 Wiley Periodicals, Inc.

Tomographic follow-up of bone regeneration after bone block harvesting from the mandibular ramus

Autogenous bone is still considered the gold standard, and the applicability of autogenous bone grafts is well established. However, the possibility of second harvesting from the same donor region remains unclear. The aim of this study was to perform a prospective evaluation of hard tissue deposition in the mandibular ramus after bone block harvesting using cone beam computed tomography (CBCT). Twenty-two patients with indications for augmentation procedures using autogenous bone from the mandibular ramus were selected. Three CBCT scans were performed with a tomographic guide before bone harvesting (T1) and at 14 days (T2) and 6 months (T3) after the surgical procedures. Measurements were obtained in 2D (area, mm(2)) and 3D (volume, mm(3)), and were subsequently compared. In the 2D analysis, the mean bone formation rate was 56%, while for the 3D analysis the mean rate was 9.7%. Despite this difference, there was a significant correlation between area and volume measurements. Our findings demonstrated the presence of hard tissue in the mandibular ramus at 6 months after bone harvesting, which suggests that it would be possible to reuse the same region for a second block harvesting. However, the second bone harvesting would involve less bone for transplantation when compared to the first bone harvesting.

Evaluation of Bone Heating, Drill Deformation, and Drill Roughness After Implant Osteotomy: Guided Surgery and Classic Drilling Procedure

Purpose: This study evaluated and compared bone heating, drill deformation, and drill roughness after several implant osteotomies in the guided surgery technique and the classic drilling procedure. Materials and Methods: The tibias of 20 rabbits were used. The animals were divided into a guided surgery group (GG) and a control group (CG); subgroups were then designated (G0, G1, G2, G3, and G4, corresponding to drills used 0, 10, 20, 30 and 40 times, respectively). Each animal received 10 sequential osteotomies (5 in each tibia) with each technique. Thermal changes were quantified, drill roughness was measured, and the drills were subjected to scanning electron microscopy. Results: Bone temperature generated by drilling was significantly higher in the GG than in the CG. Drill deformation in the GG and CG increased with drill use, and in the CG a significant difference between GO and groups G3 and G4 was observed. In the GG, a significant difference between GO and all other groups was found. For GG versus CG, a significant difference was found in the 40th osteotomy. Drill roughness in both groups was progressive in accordance with increased use, but there was no statistically significant difference between subgroups or between GG and CG overall. Conclusion: During preparation of implant osteotomies, the guided surgery technique generated a higher bone temperature and deformed drills more than the classic drilling procedure. The increase in tissue temperature was directly proportional to the number of times drills were used, but neither technique generated critical necrosis-inducing temperatures. Drill deformation was directly proportional to the number of times the drills were used. The roughness of the drills was directly proportional to the number of reuses in both groups but tended to be higher in the GG group.

Cellular behavior as a dynamic field for exploring bone bioengineering: A closer look at cell-biomaterial interface

Conselho Nacional de Desenvolvimento Científico e Tecnológico (CNPq)

Tissue-engineering-based Strategies for Regenerative Endodontics

Stemming from in vitro and in vivo pre-clinical and human models, tissue-engineering-based strategies continue to demonstrate great potential for the regeneration of the pulp-dentin complex, particularly in necrotic, immature permanent teeth. Nanofibrous scaffolds, which closely resemble the native extracellular matrix, have been successfully synthesized by various techniques, including but not limited to electrospinning. A common goal in scaffold synthesis has been the notion of promoting cell guidance through the careful design and use of a collection of biochemical and physical cues capable of governing and stimulating specific events at the cellular and tissue levels. The latest advances in processing technologies allow for the fabrication of scaffolds where selected bioactive molecules can be delivered locally, thus increasing the possibilities for clinical success. Though electrospun scaffolds have not yet been tested in vivo in either human or animal pulpless models in immature permanent teeth, recent studies have highlighted their regenerative potential both from an in vitro and in vivo (i.e., subcutaneous model) standpoint. Possible applications for these bioactive scaffolds continue to evolve, with significant prospects related to the regeneration of both dentin and pulp tissue and, more recently, to root canal disinfection. Nonetheless, no single implantable scaffold can consistently guide the coordinated growth and development of the multiple tissue types involved in the functional regeneration of the pulp-dentin complex. The purpose of this review is to provide a comprehensive perspective on the latest discoveries related to the use of scaffolds and/or stem cells in regenerative endodontics. The authors focused this review on bioactive nanofibrous scaffolds, injectable scaffolds and stem cells, and pre-clinical findings using stem-cell-based strategies. These topics are discussed in detail in an attempt to provide future direction and to shed light on their potential translation to clinical settings.

Skeletal muscle degeneration and regeneration after injection of bothropstoxin-II, a phospholipase A2 isolated from the venom of the snake Bothrops jararacussu

A myotoxic phospholipase A2, named bothropstoxin II (BthTX-II), was isolated from the venom of the South American snake Bothrops jararacussu and the pathogenesis of myonecrosis induced by this toxin was studied in mice. BthTX-II induced a rapid increase in plasma creatine kinase levels. Histological and ultrastructural observations demonstrate that this toxin affects muscle fibers by first disrupting the integrity of plasma membrane, as delta lesions were the earliest morphological alteration and since the plasma membrane was interrupted or absent in many portions. In agreement with this hypothesis, BthTX-II released peroxidase entrapped in negatively charged multilamellar liposomes and behaved as an amphiphilic protein in charge shift electrophoresis, an indication that its mechanism of action might be based on the interaction and disorganization of plasma membrane phospholipids. Membrane damage was followed by a complex series of morphological alterations in intracellular structures, most of which are probably related to an increase in cytosolic calcium levels. Myofilaments became hypercontracted into dense clumps which alternated with cellular spaces devoid of myofibrillar material. Later on, myofilaments changed to a hyaline appearance with a more uniform distribution. Mitochondria were drastically affected, showing high amplitude swelling, vesiculation of cristae, formation of flocculent densities, and membrane disruption. By 24 hr, abundant polymorphonuclear leucocytes and macrophages were observed in the interstitial space as well as inside necrotic fibers. Muscle regeneration proceeded normally, as abundant myotubes and regenerating myofibers were observed 7 days after BthTX-II injection. By 28 days regenerating fibers had a diameter similar to that of adult muscle fibers, although they presented two distinctive features: central location of nuclei and some fiber splitting. This good regenerative response may be explained by the observation that BthTX-II does not affect blood vessels, nerves, or basal laminae. © 1991.