Comparison of ready-made and self-setting alginate membranes used as a barrier membrane for guided bone regeneration

In order to understand the requirements of guided bone regeneration (GBR) involving alginate base self-setting barrier membranes, GBR was performed in the case of bicortical bone defects formed at the tibiae of experimental animals employing self-setting and ready-made alginate membranes. Connective tissue ingress into the bone defects at the skin side of the tibia was observed when GBR was generated utilizing ready-made alginate membrane. In contrast, bone defects were reconstructed with bone tissue when GBR was generated with self-setting alginate membrane formed from aqueous 3% sodium alginate and 3% CaCl₂ solutions. The unreacted aqueous sodium alginate solution inherent to self-setting alginate membrane did not inhibit bone tissue regeneration. Rather, callus bone was formed using sodium alginate as the nucleus. However, when GBR was effected with self-setting alginate membrane formed from aqueous 10% CaCl₂ solution, membrane was too thick and thus regeneration of bone tissue in the bone cavity was prevented. Therefore, we concluded that self-setting alginate membrane is very useful as a barrier membrane for GBR upon appropriate adjustment of conditions with respect to preparation of alginate membrane.     

Plasmid DNA-loaded asymmetrically porous membrane for guided bone regeneration

Although bone defects can be restored spontaneously,bone reconstruction with sufficient strength and volume continues to be a challenge in clinical practices.In recent years,the use of a variety of biomaterials with bioactivity has been attempted to compensate for this limitation.Herein,we fabricated a pDNA(encoding for BMP-2)-loaded asymmetrically porous polycaprolactone(PCL)/Pluronic F127 membrane as a bioactive guided bone regeneration(GBR)membrane,using a modified immersion-precipitation method.It was observed that the GBR membrane allows continuous release of pDNA for more than20 weeks.The pDNA was sufficiently transfected into human bone marrow stem cells(h BMSCs)without significant cytotoxicity and the gene-transfected cells showed prolonged synthesis of BMP-2.From in vitro osteogenic differentiation and in vivo animal studies,the effective induction of osteogenic differentiation of h BMSCs and enhanced bone regeneration by the pDNA-loaded asymmetrically porous PCL/Pluronic F127 membrane was observed,suggesting that the pDNA-loaded membrane as a bioactive GBR membrane can be an alternative therapeutic technique for effective bone regeneration.     

Reconstruction of bone fenestration on mandiblar by the guided bone regeneration methods with beta-TCP/PLGC membranes

Guided Bone Regeneration (GBR) is a method for bone tissue regeneration. In this method, membranes are used to cover bone defects and to block the invasion of the surrounding soft tissues. It would provide sufficient time for the osteogenic cells from bone marrow to proliferate and form new bony tissues. In spite of the potential usefulness of this method, no appropriate materials for the GBR membrane have been developed. Here we design the ideal mechanical properties of the GBR membranes and created novel materials, which is the composite of beta-tricalcium phosphate (beta-TCP) and block copolymer of L-lactide, glycolide, and epsilon-caplolactone (PLGC). In the animal experiments with the use of the GBR membranes for large bone defects, we observed significant enhancement in the bone regeneration after 12 weeks implantation and proved the effectiveness of the materials.     

Preparation of electrospun siloxane-poly(lactic acid)-vaterite hybrid fibrous membranes for guided bone regeneration

A nonwoven fabric scaffold for guided bone regeneration (GBR) consisting of siloxane-poly(lactic acid) (PLA)-vaterite hybrid material (SiPVH) was prepared by an electrospinning method. To improve the cellular compatibility of the fabric, the fibers were completely coated with hydroxyapatite (HA) by soaking in simulated body fluid. The HA-coated SiPVH nonwoven fabric contained large-sized spaces and showed the ability of releasing soluble silica and calcium species, which have previously been reported to stimulate osteogenic cells at the genetic level. A new type of GBR membrane was prepared by bonding SiPVH fabric with PLA nonwoven fabric with small-sized pores for preventing the intrusion of soft tissue. The resultant bi-layered membrane was expected to be effective not only for having an open structure for bone formation and a barrier to soft tissue, but also for enhancing bone growth by the release of ionic species.     

Human amniotic membrane for guided bone regeneration of calvarial defects in mice

Due to its biological properties, human amniotic membrane (hAM) is widely studied in the field of tissue engineering and regenerative medicine. hAM is already very attractive for wound healing and it may be helpful as a support for bone regeneration. However, few studies assessed its potential for guided bone regeneration (GBR). The purpose of the present study was to assess the potential of the hAM as a membrane for GBR. In vitro, cell viability in fresh and cryopreserved hAM was assessed. In vivo, we evaluated the impact of fresh versus cryopreserved hAM, using both the epithelial or the mesenchymal layer facing the defect, on bone regeneration in a critical calvarial bone defect in mice. Then, the efficacy of cryopreserved hAM associated with a bone substitute was compared to a collagen membrane currently used for GBR. In vitro, no statistical difference was observed between the conditions concerning cell viability. Without graft material, cryopreserved hAM induced more bone formation when the mesenchymal layer covered the defect compared to the defect left empty. When associated with a bone substitute, such improved bone repair was not observed. These preliminary results suggest that cryopreserved hAM has a limited potential for GBR.

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Preparation and characteristics of a binderless carbon nanotube monolith and its biocompatibility

A carbon nanotube (CNT) monolith without any binders was obtained by spark plasma sintering (SPS) treatment at 1100 °C under 40 MPa pressure. Transmission electron microscope results confirmed that this material maintained the nanosized tube microstructure of raw CNT powder after SPS treatment. The density and mechanical properties of this material were similar to cancellous bone. The material was implanted in subcutaneous tissue and femurs of rats and tissue samples were analyzed by histopathology at 1 and 4 weeks after surgery. Although some foreign body giant cells were seen around the CNTs, no severe inflammatory response such as necrosis was observed, and CNT implants were surrounded by newly formed bone in the femur. The study provides the first in vivo testing evidence that pure bulk carbon nanotubes are not a strongly inflammatory substance and have no toxicity for bone regeneration. Our study is the first successful experiment to consolidate CNTs without binders, and may provide an effective method for CNT monolith synthesis, as well as demonstrating that a binderless carbon nanotube material with a strength matching that of bone could be a candidate bone substitute material and a bone tissue engineering scaffold material.     

Analysis of bone formation and membrane resorption in guided bone regeneration using deproteinized bovine bone mineral versus calcium sulfate

Guided Bone Regeneration (GBR) is a technique based on the use of a physical barrier that isolates the region of bone regeneration from adjacent tissues. The objective of this study was to compare GBR, adopting a critical-size defect model in rat calvaria and using collagen membrane separately combined with two filling materials, each having different resorption rates. A circular defect 8?mm in diameter was made in the calvaria of Wistar rats. The defects were then filled with calcium sulfate (CaS group) or deproteinized bovine bone mineral (DBBM group) and covered by resorbable collagen membrane. The animals were killed 15, 30, 45 and 60 days after the surgical procedure. Samples were collected, fixed in 4% paraformaldehyde and processed for paraffin embedding. The resultant sections were stained with H&E for histological and histomorphometric study. For the histomorphometric study, the area of membrane was quantified along with the amount of bone formed in the region of the membrane. Calcium sulfate was reabsorbed more rapidly compared to DBBM. The CaS group had the highest percentages of remaining membrane at 15, 30, 45 and 60 days, compared to the DBBM group. The DBBM group had the highest amount of new bone at 45 and 60 days compared to the CaS group. Based on these results, it was concluded that the type of filling material may influence both the resorption of collagen membrane and amount of bone formed.

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Tyrosine-derived polycarbonate membrane in treating mandibular bone defects. An experimental study.

This study was designed to evaluate the suitability of a novel bioabsorbable material in treating bone defects. A poly(desaminotyrosyl-tyrosine-ethyl ester carbonate) (PDTE carbonate) membrane (thickness 0.2-0.3 mm) was implanted into the mandibular angle of 20 New Zealand White rabbits to cover a through-and-through defect (12 x 6 mm). In group 1, the defects were left unfilled but covered with membrane and in group 2 the defects were filled with bioactive glass mesh and covered with membrane, too. Controls were left uncovered and unfilled. The animals were followed for 6, 12, 24 and 52 weeks, respectively. The material was evaluated by qualitative analysis of histological reactions and newly formed bone.We found that PDTE carbonate elicited a modest foreign body reaction in the tissues, which was uniform throughout the study. New bone formation was seen in all samples after six weeks. Group 1 had more new bone formation until 24 weeks and after this the difference settled. Based on findings of this study it was concluded that PDTE carbonate membranes have good biocompatibility and are sufficient to enhance bone growth without additional supportive matrix.     

Tyrosine derived polycarbonate membrane is useful for guided bone regeneration in rabbit mandibular defects

Standardized bilateral through-and-through defects (12 × 6 mm) were created extraorally in the mandibular angle of 18 New Zealand White rabbits. Animals were divided in to three groups (n = 6) according to the intended healing time. On the left side, defects were covered with a poly(desaminotyrosyl-tyrosine-ethyl ester carbonate) (PDTE carbonate) membrane wrapped around the inferior border of the mandible and fixed with bioabsorbable sutures. On the right side, the defects were filled with a mesh made of bioactive glass 13–93 and 3 wt% chitosan. The defects were covered with the same membranes. Periosteal flap was sutured over the membrane. Radiographically, bone ingrowth was seen in all specimens at 12 weeks postoperatively. At 24 weeks, completely ossified area remained approximately at the same level as at 12 weeks, but the non-ossified area decreased to almost zero. However, the bioactive glass mesh did not improve the results. Nevertheless, enveloping the defect with PDTE carbonate membrane seemed to play a crucial role in new bone formation. Based on these results, we conclude that tyrosine polycarbonate is a promising new material for guided bone regeneration.     

Influence of the porosity of hydroxyapatite ceramics on in vitro and in vivo bone formation by cultured rat bone marrow stromal cells

The in vitro and in vivo osteoblastic differentiation of rat bone marrow stromal cells (MSCs) was assessed on hydroxyapatite disks with 3 different porosities: 30%, 50%, and 70% (HA30, HA50, and HA70, respectively). MSCs obtained by 10-day culture of fresh bone marrow cells were subcultured for 2 weeks on 3 kinds of porous HA disks in the presence and absence of dexamethasone (Dex). After 2 weeks of subculture, alkaline phosphatase (ALP) activity and osteocalcin production of MSCs/HA composites with Dex were higher than those without, and increased with increasing porosity. The resultant bone tissue grafts “cultured-bone/HA constructs” were implanted subcutaneously into the backs of syngeneic rats, and harvested 1, 2, and 4 weeks after implantation. At 1 week, only cultured-bone/HA70 constructs exhibited expanded bone formation. At 2 and 4 weeks, active osteoblasts and progressive bone formation were observed morphologically in both cultured-bone/HA50 and HA70 constructs. At 4 weeks, bone tissue was observed even in cultured-bone/HA30 constructs. ALP activity and osteocalcin production also increased with increasing porosity and time after implantation. In this in vivo model, different scaffold porosity with similar crystal morphology of the apatite phase demonstrated marked differences in ability to support osteogenesis by implanted rat MSCs.     

Periodontal regeneration in experimentally-induced alveolar bone dehiscence by an improved porous biphasic calcium phosphate ceramic in beagle dogs

Regeneration of lost periodontium is the focus of periodontal therapy. To achieve the effective regeneration, a number of bone graft substitute materials have been developed. This study aimed to investigate the histological response in alveolar bone dehiscences which were filled with an improved biphasic calcium phosphate (BCP) ceramic with more reasonable pore diameter, pore wall thickness and porosity. Twenty-four alveolar bone dehiscences were made surgically in twelve beagle dogs by reflecting mucoperiosteal flaps on the buccal aspect of bilateral lower second premolars and removing alveolar bone. The left dehiscences were treated with BCP ceramic and the contralaterals were cured with the open flap debridement (OFD) as controls. Three dogs were used at week 4, 12, and 24 respectively. Histological observations were processed through three-dimensional micro-computed tomographic imaging, fluorescence and light microscopy. The histological study indicated that the biphasic ceramic was biocompatible, and regeneration was achieved more effectively through the BCP treatment. There were also arrest of epithelial migration apically and formation of new bone and cementum, as well as proliferation of fibrous connective tissues that became attached to the newly formed cementum at week 24, while there was no significant periodontal regeneration in the OFD group only with epithelial tissue migrating into the dehiscence regions. Clinically speaking, though the surgical location formed a limitation to the application of the improved BCP on the periodontal regeneration, the actual result was positive. It proved that the BCP had biocompatibility and was able to act as a stable scaffold to induce periodontal regeneration effectively.     

Poly(Vinylidene Fluoride-Trifluorethylene)/barium titanate membrane promotes de novo bone formation and may modulate gene expression in osteoporotic rat model

Osteoporosis is a chronic disease that impairs proper bone remodeling. Guided bone regeneration is a surgical technique that improves bone defect in a particular region through new bone formation, using barrier materials (e.g. membranes) to protect the space adjacent to the bone defect. The polytetrafluorethylene membrane is widely used in guided bone regeneration, however, new membranes are being investigated. The purpose of this study was to evaluate the effect of P(VDFTrFE)/BT [poly(vinylidene fluoride-trifluoroethylene)/barium titanate] membrane on in vivo bone formation. Twenty-three Wistar rats were submitted to bilateral ovariectomy. Five animals were subjected to sham surgery. After 150 days, bone defects were created and filled with P(VDF-TrFE)/BT membrane or PTFE membrane (except for the sham and OVX groups). After 4 weeks, the animals were euthanized and calvaria samples were subjected to histomorphometric and computed microtomography analysis (microCT), besides real time polymerase chain reaction (real time PCR) to evaluate gene expression. The histomorphometric analysis showed that the animals that received the P(VDF-TrFE)/BT membrane presented morphometric parameters similar or even better compared to the animals that received the PTFE membrane. The comparison between groups showed that gene expression of RUNX2, BSP, OPN, OSX and RANKL were lower on P(VDF-TrFE)/BT membrane; the gene expression of ALP, OC, RANK and CTSK were similar and the gene expression of OPG, CALCR and MMP9 were higher when compared to PTFE. The results showed that the P(VDF-TrFE)/BT membrane favors bone formation, and therefore, may be considered a promising biomaterial to support bone repair in a situation of osteoporosis.     

Extracorporeal hydroxyapatite-chamber for bone and biomaterial studies

Hydroxyapatite (HA) spherules and autologous bone (AB) with a central vascular pedicle were housed inside an HA-chamber to form the skeletal segment of specific shape. Experimental chambers were then inserted in a pocket between medial thigh muscles in 13 New Zealand male rabbits for 3 months. Three graft group were scheduled: (A) HA and AB without vascular pedicle, (B) HA with vascular pedicle, (C) HA and AB with vascular pedicle. At term, histology showed tissue and cellular degeneration in group A chambers. Due to spherules coalescence, fibrous tissue is formed in group B chambers. Group C chambers contained living osteocytes in the implanted bone, several newly formed vessels in soft tissue, bone and partial hydroxyapatite erosions. New bone was formed in apposition to both autologous bone and hydroxyapatite. Our study suggests that this experimental model could be used to grow adequately sized vascularized skeletal segments.     

Nanocrystalline hydroxyapatite for bone repair: an animal study

Hydroxyapatite has become the most common material to replace bone or to guide its regeneration. Nanocrystalline hydroxyapatite suspension had been introduced in the clinical use recently under the assumption that small dimension of crystals could improve resorption. We studied the resorption and osteointegration of the nanocrystalline hydroxyapatite Ostim^? in a rabbit model. The material was implanted either alone or in combination with autogenic or allogenic bone into distal rabbit femora. After survival time of 2, 4, 6, 8 and 12 weeks the implants had been evaluated by light and electron microscopy. We observed a direct bone contact as well as inclusion into soft tissue. But we could observe no or only marginal decay and no remarkable resorption in the vast majority of implants. In situ the nanocrystalline material mostly formed densely packed agglomerates which were preserved once included in bone or connective tissue. A serious side effect was the initiation of osteolysis in the femora far from the implantation site causing extended defects in the cortical bone.     

Repair of rabbit femoral condyle bone defects with injectable nanohydroxyapatite/chitosan composites

Repair of massive bone loss remains a challenge to the orthopaedic surgeons. Autologous and allogenic bone grafts are choice for bone reconstructive surgery, but limited availability, risks of transmittable diseases and inconsistent clinical performances have prompted the development of tissue engineering. In the present work, the bone regeneration potential of nanohydroxyapatite/chitosan composite scaffolds were compared with pure chitosan scaffolds when implanted into segmental bone defects in rabbits. Critical size bone defects (6 mm diameter, 10 mm length) were created in the left femoral condyles of 43 adult New Zealand white rabbits. The femoral condyle bone defects were repaired by nanohydroxyapatite/chitosan compositions, pure chitosan or left empty separately. Defect-bridging was detected by plain radiograph and quantitative computer tomography at eight and 12 weeks after surgery. Tissue samples were collected for gross view and histological examination to determine the extent of new bone formation. Eight weeks after surgery, more irregular osteon formation was observed in the group treated with nanohydroxyapatite/chitosan composites compared with those treated with pure chitosan. 12 weeks after surgery, complete healing of the segmental bone defect was observed in the nanohydroxyapatite/chitosan-group, while the defect was still visible in the chitosan-group, although the depth of the defect had diminished. These observations suggest that the injectable nanohydroxyapatite/chitosan scaffolds are potential candidate materials for regeneration of bone loss.     

Guided bone regeneration using osteopatite® granules and polytetrafluoroethylene membranes

Granules of a modified hydroxyapatite, Osteopatite®, were implanted in the right posterior tibiae of adult rabbits. We studied the extent of bone regeneration in bone holes. In the right tibiae, that were filled with granules of this biomaterial covered with a polytetrafluoro-ethylene (PTFE) membrane using, as a control, uncovered granules. In the left tibia, an empty hole was covered with PTFE membrane and a second hole was left empty to be used as a control. A histomorphometric study was carried out using light microscopy, four and eight weeks after the surgery. The covered granules presented a higher percentage of bone contact than the uncovered ones, and it was also possible to observe a better bone tissue organization, mainly produced by the immobilization action of the PTFE membrane. Empty bone defects covered with PTFE membranes, two months after implantation, presented large areas of Haversian bone and direct bone contact to the PTFE membrane.     

Bone regeneration using an injectable calcium phosphate/autologous iliac crest bone composites for segmental ulnar defects in rabbits

Background Treatment of segmental bone loss remains a challenge in skeletal repair. A major therapeutic goal is the development of implantable materials that will promote bone regeneration. Objective We evaluate bone regeneration in grafts containing different concentrations autologous iliac crest bone (ACB) particles, carried in a new injectable calcium phosphate cement (CPC), in ulnar bone defects in rabbits. Methods Large upper-mid-diaphyseal defects (10 mm) were created in the left ulnae of 60 skeletally mature New Zealand white rabbits. ACB concentrations of 0, 25, 50, 75, and 100% (by volume) in CPC were used to fill operated sites. Defect bridging was monitored by serial radiography at 4, 8, and 12 weeks post-operation. Samples were then examined histologically and by manual palpation to determine the extent of new bone formation. Results At 4 weeks, we observed more elaborate structures and extensive absorption in ulnae treated with mixtures containing low concentrations of ACB (such as 0% and 25% volume of ACB/CPC), compared with those treated with mixtures containing high concentrations of ACB (such as 75% and 100% volume of ACB/CPC). At 8 weeks, histomorphometry revealed increased trabecular area and volume in the group treated with high ACB concentrations compared with those treated with low ACB concentrations. At 12 weeks, complete cortical bridging and regeneration of marrow space were detected in groups treated with high concentrations of ACB, and the amount of new bone regeneration was greater in these groups than in those treated with low ACB concentrations. Conclusions Treatment of rabbit ulnar defects with injectable CPC carrying an optimized concentration of ACB particles can lead to cortical bridging and bone marrow regeneration within 12 weeks.     

Fabrication of transparent hydroxyapatite and application to bone marrow derived cell/hydroxyapatite interaction observation in-vivo

Transparent hydroxyapatite (HA), 99.6% relative density, was prepared by hot isostatic pressing. Optical transmittance of 1 mm thick HA was greater than 60% at 700 nm. Twenty transparent HA plates (10×7×1 mm) were implanted into the medullary cavities of two dogs' femora. At 1 and 2 weeks after implantation the bone marrow derived cells that adhered to the HA surfaces were histochemically examined by light microscopy and scanning electron microscopy. Four types of enzymatic reactions were employed to identify cell types. Alkaline phosphatase was used as a marker for osteoblasts, acid phosphatase and tartrate-resistant acid phosphatase as a marker for osteoclasts and non-specific esterase as a marker for macrophages. Cells adhered to the HA surfaces were a mixed population of osteoblasts, osteoclasts and macrophages. By 2 weeks, the number of osteoblasts increased and formed a bone-like matrix. A small number of osteoclasts formed resorption lacunae on the HA after 2 weeks. By utilizing the transparency of the HA, whole-view of bone matrix formation by osteoblasts and simultaneous resorption of HA by osteoclasts was observed simply and dynamically by light microscopy.     

Effect of bioactive glass granules and polytetrafluoroethylene membrane on repair of cortical bone defect

The effect of bioactive glass (BG) granules and nonresorbable polytetrafluoroethylene (PTFE) membrane on the repair of cortical bone defects was studied. Monocortical holes (diameter 3.0 mm) were drilled in rabbit tibia. Sixteen holes were filled with BG granules (diameter 630–800 m). Twelve holes were left empty and covered with PTFE membrane. No material was used at ten control holes. All experiment areas were covered with periosteum attached to the soft tissue flap. Histomorphometric evaluation of resection specimens showed that new bone and glass particles formed a continuous bridge in the BG group at the upper part of the hole, occupying 73.6% and 61.7% of the defect at 6 and 12 weeks, respectively. If only the amount of bone but not glass particles was included in the measurements the corresponding figures were 31.4% and 41.5%. The bone repair in the PTFE group was 12.1% and 11.3% and in the control group 25.1% and 23.3% at 6 and 12 weeks, respectively. The results indicate that BG granules improve repair of cortical bone defects and PTFE membrane seems to impair bone formation in these defects.     

Histological evaluation of xenogeneic bone treated by supercritical CO2 implanted into sheep

Untreated xenogeneic bone is known to be rejected when implanted into human or animal bone. It contains bone marrow tissue which is highly antigenic. To be used as an alternative to auto and allografts, this antigenic material must be entirely removed. Removal of this soft tissue contained in the bone structure was performed using a delipidation process with supercritical CO₂ followed by a deproteination with hydrogen peroxide or protease extraction. Such prepared materials were implanted into sheep bone for periods of 3 weeks, 2 months and 4 months. Bone whose organic matrix was destroyed by sintering, and untreated xenogeneic bone were used as controls. Qualitative histology and histomorphometry, measuring the percentage of the material in contact with newly formed, bone were performed on implant sections. This showed that the osseointegration of the supercritical CO₂-treated bone samples was equivalent to that of bone made anorganic by sintering while the untreated bone was embedded into an inflammatory tissue made up of macrophages, giant cells and plasmocytes.