Optimisation des matériaux hybrides à base de cellules souches pour la réparation de grands défauts osseux

The limitations imposed to both autogenous and allogenous bone grafts led to the development of new strategies for the treatment of large bone defects. The approach of bone tissue engineering aims to restore damaged bone tissue by combining osteocompetent cells such as mesenchymal stromal cells (MSC), and material scaffolds like ceramics. However, the therapeutic effectiveness of cell constructs has not yet met that of autologous bone grafts, in part due to the high death rate of cells (loaded onto the material scaffold) upon their implantation into the injured site. In order to improve the therapeutic functionality of these cell constructs, different strategies can be implemented. In this context, the Glassbone project aimed to optimize the conditions for preparation of tissue engineered products by approaching three aspects: identification of optimal ceramic scaffold relevant to bone formation; survival of implanted cells post-implantation, and finally cell preconditioning to promote cell viability in vivo. Such project will pave the way for the development of new “pro-survival” tissue engineered materials for optimal tissue regeneration.     

Comparison of contamination of femoral heads and pre-processed bone chips during hip revision arthroplasty

With bone impaction grafting, cancellous bone chips made from allograft femoral heads are impacted in a bone defect, which introduces an additional source of infection. The potential benefit of the use of pre-processed bone chips was investigated by comparing the bacterial contamination of bone chips prepared intraoperatively with the bacterial contamination of pre-processed bone chips at different stages in the surgical procedure. To investigate baseline contamination of the bone grafts, specimens were collected during 88 procedures before actual use or preparation of the bone chips: in 44 procedures intraoperatively prepared chips were used (Group A) and in the other 44 procedures pre-processed bone chips were used (Group B). In 64 of these procedures (32 using locally prepared bone chips and 32 using pre-processed bone chips) specimens were also collected later in the procedure to investigate contamination after use and preparation of the bone chips. In total, 8 procedures had one or more positive specimen(s) (12.5 %). Contamination rates were not significantly different between bone chips prepared at the operating theatre and pre-processed bone chips. In conclusion, there was no difference in bacterial contamination between bone chips prepared from whole femoral heads in the operating room and pre-processed bone chips, and therefore, both types of bone allografts are comparable with respect to risk of infection.     

Cranial reconstruction for metastatic breast cancer

All women with advanced breast cancer who are medically stable despite their disease are candidates for tumor extirpation and reconstruction. Advanced breast cancer today is incurable, and many prognostic factors can be used to try to predict a clinical course and response to therapy; however, no guidelines are available. Our case report most likely represents a metastasis to the calvarium with intracranial extension, reported to occur in about 3 percent of primary breast cancer patients. As demonstrated here, tumor ablation with immediate, one-stage reconstruction of large scalp defects is possible without the need for free tissue transfer or a delay in adjuvant therapy. Local tissue rearrangement has been employed for coverage of defects up to 50 percent of the cranium. The resulting donor defects can be closed with split-thickness skin grafts over pericranium. Serial tissue expansion and rearrangement can be used secondarily to replace skin grafts with hair-bearing scalp. Bony defects can be managed with either autogenous or alloplastic materials. Split-calvarial bone grafts can be harvested from the same operative field and cover small to medium-sized defects. Other sources of autogenous grafts include split ribs and iliac bone. Metals, calcium ceramics, and polymers such as methylmethacrylate can be used to cover intracranial contents and restore calvarial contour when defects are large or when autogenous material is not available. Palliation from tumor burden, prevention of pathologic fracture and oncologic emergencies, controlling pain, and enhancing quality of life are the goals of the oncologic and reconstructive surgeons in cases of advanced breast cancer. These goals are becoming even more important as new forms and combinations of chemotherapy, radiation, and gene therapy are extending the life expectancy of women with breast carcinoma.     

Novel technology to provide an enriched therapeutic cell concentrate from bone marrow aspirate

Current strategies to repair fractures rely on orthopaedic surgeons harvesting bone from one area of the body, typically pelvis and transferring it to the fracture site. The amount of tissue available is therefore limited, requiring a second surgical procedure and often causing the patient long term pain. An alternative approach is utilise therapeutic cells contained within bone marrow aspirate during the primary procedure. The number of therapeutic cells within a fresh aspirate is insufficient to provide clinically acceptable bone healing in a timescale that is satisfactory to the surgeon and the patient. Therefore methods to efficiently concentrate bone marrow in the clinical setting are required. Centrifugation is the current method of choice but has limitations in that it requires large capital equipment, servicing and there are potential issues of tissue contamination. We have developed a novel, acoustically‐assisted filtration device that addresses these limitations, delivering a concentrated bone marrow in a point of care, single use, fully disposable, compact device. An additional advantage is that the level of concentration required can be specified by the end user. The resulting bone marrow concentrate has been characterised in terms of cell number, viability and osteogenic potential using flow cytometry and alkaline phosphatase assay. When compared to recent clinical studies using bone marrow to repair non‐union fractures, the findings from our work suggest that the bone marrow concentrate is likely to be highly therapeutic and clinically efficacious as a bone fracture repair strategy. A product concept for use in the clinical setting is presented. © 2010 American Institute of Chemical Engineers Biotechnol. Prog., 2010     

Augmentation rhinoplasty: dorsal onlay grafting using shaped autogenous septal cartilage

We describe our experience with autogenous septal cartilage onlay grafts for augmentation of the nasal dorsum in primary and secondary rhinoplasty cases. After careful nasofacial analysis, the grafts are custom-shaped into inverted-V-frame, A-frame, or inverted-U-frame grafts, depending on the type and degree of augmentation desired. The dorsal elevation is thus tailored to fit the imperfection at hand, resulting in a smooth, natural-looking nasal contour. The indications for each type of graft are reviewed, and the surgical technique of graft harvesting and carving is detailed and illustrated.     

Use of the cylinder osteotome for cancellous bone grafting

An instrument is described for removing iliac crest cancellous bone grafts under local anesthesia. The device has widespread applications for plastic and orthopedic surgery. The convenience of obtaining bone grafts with this device broadens the use of bone-grafting techniques in patients in whom the need for bone graft is unexpected.     

Human saphenous vein and coronary bypass surgery: ultrastructural aspects of conventional and "no-touch" vein graft preparations

Coronary artery bypass graft surgery (CABG) is routinely used to restore blood flow to diseased cardiac muscle due to coronary artery disease. The patency of conventional grafts decreases with time, which is due to thrombosis and formation of neointima. A primary cause of graft failure is the mechanical damage inflicted to the graft during harvesting, including removal of surrounding tissue accompanied by high pressure saline distension to overcome vasospasm (both causing considerable mechanical trauma). The aim of this study was to compare the ultrastructural features of human saphenous vein (SV) grafts harvested conventionally and grafts prepared using an atraumatic 'no-touch' harvesting technique introduced by Souza (1996). The results of this study showed a better preservation of the lumenal endothelium and medial vascular smooth muscle (SM) in 'no-touch' versus conventional grafts. A 'fast' (within 30 min) response of SM cells to conventional harvesting was noted where features of both SM cell division and apoptosis were observed. It is concluded that the 'preserved' nature of the 'no-touch' aortocoronary SV grafts renders them less susceptible to thrombotic and atherosclerotic factors than grafts harvested conventionally. These features are suggested to contribute to the improved early patency rate described using the no-touch technique of SV harvesting.     

New biomaterials and methods for craniofacial bone defect: chondroid bone grafts in maxillary alveolar clefts

The purpose of this study was to evaluate autogenous osteogenic marrow within chondroid bone grafts in simulated alveolar defects of mice in order to determine the ability of the graft material to effectively close the cleft from an osseous standpoint and to observe the effect of the grafting procedure. Critical-sized defects were made in the premaxillary bones of male mice using a surgical trephine and a low-speed dental engine as a model of the maxillary alveolar cleft for testing bone-inductive agents. Premaxillary trephine defects were not repaired by fibrous tissue or bone formation 30 days after operation. This nonhealing bony wound of the premaxilla in mice may be useful as a model for studying the effect of bone-inductive agents on the healing of alveolar clefts. Distraction osteogenesis is a recently advanced principle of bone lengthening in which a long bone separated by osteotomy is subjected to slow progressive distraction using an external fixation device. The osteotomy site was surrounded by an external callus consisting of hyaline cartilage. The callus contained a lot of chondroid bone. The transplant bone within chondroid bone was characterized by bone formation and remodeling 30 days after transplantation. Throughout the experiment, our findings demonstrated, for the first time, that the transplant bone that contains chondroid bone may be used clinically in relation to craniofacial bone defects to improve the treatment of bone grafts.     

Assessment of cryopreserved donor skin viability: the experience of the regional tissue bank of Siena

Skin allografts from cadaver donors are an important resource for treating extensive burns, slow-healing wounds and chronic ulcers. A high level of cell viability of cryopreserved allografts is often required, especially in burn surgery, in Italy. Thus, we aimed to determine which conditions enable procurement of highly viable skin in our Regional Skin Bank of Siena. For this purpose, we assessed cell viability of cryopreserved skin allografts procured between 2011 and 2013 from 127 consecutive skin donors, before and after freezing (at day 15, 180, and 365). For each skin donor, we collected data concerning clinical history (age, sex, smoking, phototype, dyslipidemia, diabetes, cause of death), donation process (multi-tissue or multi-organ) and timing of skin procurement (assessment of intervals such as death-harvesting, harvesting-banking, death-banking). All these variables were analysed in the whole case study (127 donors) and in different groups (e.g. multi-organ donors, non refrigerated multi-tissue donors, refrigerated multi-tissue donors) for correlations with cell viability. Our results indicated that cryopreserved skin allografts with higher cell viability were obtained from female, non smoker, heartbeating donors died of cerebral haemorrhage, and were harvested within 2 h of aortic clamping and banked within 12 h of harvesting (13–14 h from clamping). Age, cause of death and dyslipidaemia or diabetes did not appear to influence cell viability. To maintain acceptable cell viability, our skin bank needs to reduce the time interval between harvesting and banking, especially for refrigerated donors.     

Effects of bone morphogenetic protein-7 stimulation on osteoblasts cultured on different biomaterials

The objective of the present study was to investigate the effects of an in vitro stimulation of human osteoblasts by recombinant human bone morphogenetic protein-7 (rhBMP-7) on the collagen types and the quantity of the collagen cross-links synthesized in a three-dimensional culture on various biomaterials for bone replacement. Trabecular bone chips were harvested from human iliac crests, and cell cultures were established at standard conditions. One hundred and fifty nanograms per milliliter of rhBMP-7 was added. For the second passage a cell scraper was used to bring the cells into suspension, and 100 microl osteoblasts (at a density of 3.3 x 10(5)) were transferred onto nine blocks of either Bio-Oss, Tutoplast, or PepGen p-15. Blocks incubated with cells that were not treated with rhBMP-7 served as controls. Cell colonization of the biomaterials was observed by scanning electron microscopy (SEM) and transmission electron microscopy (TEM) after a period of 2, 4, and 6 weeks. Throughout the experiment medium, supernatants were collected and collagen was characterized by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE). Finally, the collagen cross-link residues hydroxylysylpyridinoline (HP) and lysylpyridinoline (LP) were quantified by HPLC. Within 4 weeks the cells became confluent on all of the studied biomaterials. All samples synthesized bone specific LP and collagen type I. However, in rhBMP-7-stimulated samples, the amount of HP and LP found was increased by 45% compared to non-stimulated samples. Cell proliferation and collagen synthesis was similar on the different biomaterials, but was consistently reduced in specimen not stimulated with rhBMP-7. In vitro stimulation of osteoblasts on Bio-Oss, Tutoplast, or PepGen p-15 with rhBMP-7 and subsequent transplantation of the constructs might lead to an enhanced osseointegration of the biomaterials in vivo.     

Computer-designed prostheses for orbitocranial reconstruction

Three-dimensional imaging is an adjunct to preoperative evaluation and surgical management in some patients with complex anatomic defects of various etiologies. Deformities defined by conventional computerized tomography can be viewed as accurate three-dimensional images calculated from the original scan. The images are viewed on a high-resolution video monitor and can be photographed for a permanent record. A computer-controlled milling device can use these data to fabricate prostheses. The prostheses aid reconstructive surgery through use as an alloplastic implant, as a template to fashion autogenous bone grafts, or as a model for tissue removal. We have utilized three-dimensional imaging in combination with computer-assisted prosthesis manufacture in six patients with complex orbitocranial deformities. Four patients have undergone reconstructive surgery with satisfactory results and no complications thus far. The use of computer-designed prostheses adds a new aspect to orbitocranial reconstructive surgery that facilitates increased accuracy in the correction of anatomic defects.     

旋转加压种植法在新型生物人工复合血管体外内皮化过程中的实验研究

目的：探讨在新型生物人工复合血管内腔面联合种植平滑肌细胞和内皮细胞的方法,比较研究旋转加压种植与普通灌注种植两种方法的内皮化效果,方法：先制备新型生物人工复合血管及获取培养鉴定平滑肌细胞和内皮细胞,再和旋转加压种植与普通灌注种植两种方法将平滑肌细胞和内皮细胞培养种植于新型复合血管内腔面,以光镜及扫描电镜等观察评价内皮化的效果。结果：旋转加压种植2小时末的复合血管腔内有大量内皮细胞,旋转加压种植9天后已形成完整的内皮细胞单层;普通灌注种植的复合血管内腔有内皮细胞附着,分布不均匀,未形成完整的内皮细胞层,结论：以旋转加压种植法在新型复合血管内腔面联合种植平滑肌细胞和内皮细胞效果满意,基本实现内皮化,可以满足复合血管内皮化的要求。     

A biomechanical comparison of vascularized and conventional autogenous bone grafts

Vascularized and conventional autogenous rib grafts were used to reconstruct 6-cm ulnar defects in the forelegs of the nine dogs. Each dog served as its own control. Biomechanical torsional testing of the grafted ulnas showed that vascularized grafts were 234 percent stronger than the conventional grafts. Bone toughness (energy absorbed) was 483 percent greater in the vascularized grafts, and elastic modulus and proportional limits were 263 and 246 percent greater, respectively. We conclude that vascularized bone grafts are significantly stronger than conventional autogenous bone grafts after 3 months of healing in the dog ulna model.     

Iliac crest bone graft harvesting techniques: a comparison

This study was undertaken to compare the morbidity of traditional iliac bone graft harvesting techniques for grafting alveolar clefts to minimally invasive techniques. Fifty-five age-matched patients, ages 6.5 to 16 years (mean, 11.2 years), 22 girls and 33 boys, were divided into three groups. The traditional bone window open harvesting technique served as the control group. Two different minimally invasive techniques, one that used a bone grinder and another that used a trephine, for bone harvesting were compared with the control. Both invasive techniques were statistically superior, p < 0.05, in terms of total time pain medication was necessary (mean of 12.0 hours for bone grinder, 17.6 hours for trephine, 26.0 hours for control), operative time for bone harvest (mean of 11 minutes for bone grinder and trephine, 20 minute for control), and mean incision length (2 cm for bone grinder and trephine, 5 cm for control). Patients exposed to the minimally invasive techniques had fewer complications, a trend toward earlier ambulation, and shorter hospital stays when compared with the bone grinder technique. The patients exposed to the bone grinder demonstrated earlier ambulation and fewer requirements for analgesia when compared with the trephine technique, although these results did not reach statistical significance. The trephine technique was useful when maxillary osteotomies were combined with alveolar bone grafting, because it provided structural bone grafts and cancellous bone. On the basis of these findings, the bone grinder is the preferred technique for harvesting alveolar bone grafts when no structural support is required. These authors no longer use the traditional bone window open harvesting technique.     

The comparative analysis of homologous fresh frozen bone and autogenous bone graft,associated or not with autogenous bone marrow,in rabbit calvaria: a clinical and histomorphometric study

The aim of this study was to evaluate the potential of fresh frozen homologous and autogenous grafts, associated or not with autogenous bone marrow, to form bone. Sixty titanium cylinders were used, and were fixed to the skulls of 30 rabbits. These cylinders were filled with (A) autogenous bone (AM) autogenous bone associated with the bone marrow (H) fresh frozen homologous bone (HM) fresh frozen homologous bone associated with the bone marrow (M) pure autogenous bone marrow and (C) blood clot. The animals were sacrificed after 02 and 03 months. After clinical evaluation, the samples were stained with hematoxylin, eosin and Mallory Trichrome dyes for optical microscopy analysis and histomorphometric analysis. Experimental groups that received mineralized materials (A, AM, H, HM) showed the best bone formation results, presenting no statistical difference between them (P > 0.05). Groups that did not receive mineralized materials (M and C) showed the worst results (P < 0.05), but the M group showed better results than the C group. Most of the autogenous and homologous bone particles were resorbed and there was a larger amount of residual particles in the homologous graft (H, HM) when compared with the autogenous graft (A, AM; P < 0.05). These findings suggest that fresh frozen homologous grafts produced similar amounts of new bone when compared with the autogenous grafts. However, the amount of residual bone particles was larger in the homogenous groups, which may indicate a slower remodeling process. The homologous fresh frozen bone seems to be a good osteoconductive material. The use of only autogenous bone marrow showed better results when compared to the bood clot. However, this research indicates that association with mineralized materials is required.     

Lyophilized allogeneic bone tissue as an antibiotic carrier

The rising number of primary joint replacements worldwide causes an increase of revision surgery of endoprostheses due bacterial infection. Revision surgery using non-cemented implants seems beneficial for the long-term outcome and the use of antibiotic-impregnated bone grafts might control the infection and give a good support for the implant. In this study we evaluated the release of antibiotics from fresh-frozen and lyophilized allogeneic bone grafts. Lyophilized bone chips and fresh frozen bone chips were mixed with gentamicin sulphate, gentamicin palmitate, vancomycin, calcium carbonate/calcium sulphate impregnated with gentamicin sulphate, and calcium carbonate/calcium sulphate bone substitute material impregnated with vancomycin. The efficacy of each preparation was measured by drug release tests and bacterial susceptibility using B. subtilis, S. aureus and methicillin-resistant Staphylococcus aureus. The release of gentamicin from lyophilized bone was similar to the release rate from fresh frozen bone during all the experimental time. That fact might be related to the similar porosity and microstructure of the bone chips. The release of gentamicin from lyophilized and fresh frozen bone was high in the first and second day, decreasing and keeping a low rate until the end of the second week. Depending on the surgical strategy either polymethylmethacrylate or allogeneic bone are able to deliver sufficient concentrations of gentamicin to achieve bacterial inhibition within two weeks after surgery. In case of uncemented revision of joint replacements allogeneic bone is able to deliver therapeutic doses of gentamicin and peak levels immediately after implantation during a fortnight. The use of lyophilized and fresh frozen bone allografts as antibiotic carriers is recommended for prophylaxis of bone infection.     

Nasal augmentation with split calvarial grafts in Orientals

This study reports on my experience with autogenous split calvarial grafts in nasal augmentation in 62 Orientals. In 78 percent of patients, the procedure was performed under local anesthesia in an outpatient setting. Total operating time for harvesting of split calvarial grafts ranged from 20 to 55 minutes, with a mean of 32 minutes. Patients ranged in age from 16 to 48 years, with a mean of 27 years. Follow-up was from 6 months to 8 years, with an average of 3.1 years. Intraoperative discomfort was uniformly low and well tolerated when local anesthesia was used. The complication rate was 8.0 percent, with three cases of minor seroma-hematoma formation at the bone-graft donor site. These were treated with aspiration. There were two recipient-site complications, with one case of complete bone resorption that occurred in a densely fibrotic nose with preexisting septal perforation and a case of overcorrection that was successfully rasped 1 year later. Because of their easy accessibility beneath the scalp, split calvarial grafts to the nose are useful in various types of nasal augmentation, and the technique is offered as a practical alternative to the use of alloplastic materials.     

Effect of isolation of periosteum and dura on the healing of rabbit calvarial inlay bone grafts

Little is understood about the role of the recipient site in the revascularization and incorporation of autogenous inlay bone grafts in the craniofacial skeleton. Clinical experience demonstrates that secondary complex cranial vault reconstruction performed with scarred avascular dura or poor soft-tissue coverage may undergo significant resorption, thus compromising the aesthetic outcome. This study was designed to determine the effect of isolating autogenous orthotopic inlay calvarial bone grafts from the surrounding dura and/or periosteum on graft revascularization, healing, and volume maintenance in the adult rabbit. Adult rabbits were randomized into four groups (n = 10 per group); in each rabbit, the authors created a circular, 15-mm in diameter, full-thickness cranial defect followed by reconstruction with an autogenous calvarial bone graft, which was replaced orthotopically and held with microplate fixation. Silicone sheeting (0.5 mm thickness) was used to isolate the dura (group II), the periosteum (group II), or both dura and periosteum (group IV) from the graft interface. No silicone was placed in group I. Animals were killed 10 weeks postoperatively, and calvaria were harvested to assess graft surface area, morphology, quantitative histology, fluorochrome staining, and revascularization. Grafts isolated from both the dura and periosteum exhibited significant decreases in total bone (cortical and trabecular) surface area, blood vessel count, and interface healing compared with nonisolated control grafts. Isolation of either the dura or periosteum significantly (p < 0.05) decreased blood vessel count but had no significant effect on interface healing. Isolation of the dura alone was associated with a significant (p < 0.05) decrease in graft cross-sectional surface area and dural cortical thickness compared with nonisolated control grafts, but this effect was not observed when the periosteum alone was isolated. Quantitative histology performed 10 weeks after surgery indicated that graft isolation was associated with increased marrow fibrosis and necrosis compared with nonisolated controls; it also demonstrated evidence of increased activity in bone remodeling (osteoblast and osteocyte count, new trabecular bone, and surface resorption). Triple fluorochrome staining suggested increased bone turnover in the nonisolated grafts compared with isolated grafts at 1 and 5 weeks postoperatively. This study demonstrates that isolating a rabbit calvarial inlay autogenous bone graft from the dura and/or periosteum results in significantly (p < 0.05) decreased revascularization, interface healing, and cross-sectional areas of amount of mature bone compared with nonisolated control grafts 10 weeks after surgery. At this time point, histologic examination demonstrates a paradoxical increase in bone remodeling in isolated bone grafts compared with controls. It is possible that the inhibition of revascularization results in a delayed onset of the remodeling phase of graft incorporation. However, in the model studied, it is not known whether the quantitative histologic and morphometric parameters measured in these isolated grafts exhibit a "catch-up" phenomenon at time points beyond 10 weeks after surgery. The results of this study emphasize the importance of a healthy recipient site in the healing and incorporation of calvarial bone grafts but stress the need for further investigation at later time points.     

Porous hydroxyapatite as an onlay bone-graft substitute for maxillofacial surgery

This paper chronicles 3 years of a continuing study comparing porous hydroxyapatite to autogenous bone grafts as onlays in maxillofacial surgery. Twenty-five patients, seen from June of 1984 to May of 1985, underwent onlay augmentation on various maxillary and mandibular locations. A total of 68 onlay augmentation sites comparing Interpore porous hydroxyapatite and autogenous bone were followed for 2 years or more. This long-term study compares these substances in radiologic longevity, histologic incorporation, clinical function, and aesthetic appearance.     

A new transplant bone for maxillary alveolar cleft

An innovative technique with distraction osteogenesis has been developed in our research group to explore autogenous bone transplantation into craniofacial bone defects. This technique was designed to investigate bone-marrow transplantion using a chondroid or fibula bone graft into simulated alveolar bone defects in mice in terms of the osteogenic process and activity. As an experimental model of maxillary alveolar bone cleft available for testing bone-inductive materials, a critical-size defect was formed in the pre-maxillary bone of male mice using a surgical trephine bur with a low-speed dental engine. Distraction osteogenesis was performed using an external fixation device. The osteotomy site was occupied by an external callus consisting of hyaline cartilage with a large quantity of chondroid bone. Moreover, bone remodelling with new bone formation was demonstrated 30 days after the transplantation. Bone adhesion was better in chondroid bone grafting than in fibula bone grafting. The present findings are the first to demonstrate the potential of chondroid bone transplantation as a new therapeutic system of bone grafting, suitable for bone substitutes in craniofacial bone defects.