Segmental Long Bone Defects Research Articles

Cancellous bone wrapping grafting for segmental defects of long bone

Objective To investigate the principles and effects of cancellous bone wrapping grafting for treatment of segmental defects of long bone. Methods From January 2008 to December 2015, 50 patients with segmental long bone defect were treated by various wrapping grafting with rich autogenous can-cellous bone. They were 31 males and 19 females, aged from 13 to 69 years (average, 34.6 years). The cancellous bone was wrapped by titanium mesh in 8 cases, by wire mesh in 10, by line binding in 13, and by induced membrane in 19. The bone defect was located at tibia in 22 cases, at radius in 10, at humerus in 8, at ulna in 7 and at femur in 3. The length of bone defect ranged from 3 to 9 cm, averaging 5.9 cm. Bone healing, complications and functionary recovery of adjacent joint were recorded. The bone defect healing and functionary recovery of adjacent joint were evaluated according to the Paley criteria. Results The inci-sions healed by the first intention in 48 cases and by the second in 2. All were followed up for 12 to 48 months (average, 19.1 months). All the bone defects healed by the first intention but one treated by induced membrane wrapping which was healed 15 months later by the secondary grafting due to nonunion at ends. The total clinical healing time ranged from 3 to 16 months (average, 6.1 months). The last follow-ups showed that all the affected limbs resumed weight-bearing activities. The healing of bone defects was graded as excellent in all but one in the group of induced membrane wrapping. Totally, the functionary recovery of adjacent joint was excellent in 18, good in 22, fair in 7 and poor in 3 cases (an excellent and good rate of 80.0%). Conclusions Cancellous bone wrapping grafting can avoid or significantly reduce loosening and absorption of cancellous bone graft after tradi-tional bone grafting. Although it is effective for treatment of large segmental bone defect, its methods should vary according to the specific conditions of the patient. Key words: Bones; Fractures, ununited; Free bone graft; Wrapping bone graft

In situ repair of bone and cartilage defects using 3D scanning and 3D printing

Three-dimensional (3D) printing is a rapidly emerging technology that promises to transform tissue engineering into a commercially successful biomedical industry. However, the use of robotic bioprinters alone is not sufficient for disease treatment. This study aimed to report the combined application of 3D scanning and 3D printing for treating bone and cartilage defects. Three different kinds of defect models were created to mimic three orthopedic diseases: large segmental defects of long bones, free-form fracture of femoral condyle, and International Cartilage Repair Society grade IV chondral lesion. Feasibility of in situ 3D bioprinting for these diseases was explored. The 3D digital models of samples with defects and corresponding healthy parts were obtained using high-resolution 3D scanning. The Boolean operation was used to achieve the shape of the defects, and then the target geometries were imported in a 3D bioprinter. Two kinds of photopolymerized hydrogels were synthesized as bioinks. Finally, the defects of bone and cartilage were restored perfectly in situ using 3D bioprinting. The results of this study suggested that 3D scanning and 3D bioprinting could provide another strategy for tissue engineering and regenerative medicine.

Repair of segmental radial defects in dogs using tailor-made titanium mesh cages with plates combined with calcium phosphate granules and basic fibroblast growth factor-binding ion complex gel.

Repair of large segmental defects of long bones are a tremendous challenge that calls for a novel approach to supporting immediate weight bearing and bone regeneration. This study investigated the functional and biological characteristics of a combination of a tailor-made titanium mesh cage with a plate (tTMCP) with tetrapod-shaped alpha tricalcium phosphate granules (TB) and basic fibroblast growth factor (bFGF)-binding ion complex gel (f-IC gel) to repair 20-mm segmental radial defects in dogs. The defects were created surgically in 18 adult beagle dogs and treated by implantation of tTMCPs with TB with (TB-gel group) or without (TB group) f-IC gel. Each tTMCP fitted the defect well, and all dogs could bear weight on the affected limb immediately after surgery. Dogs were euthanized 4, 8 and 24weeks after implantation. Histomorphometry showed greater infiltration of new vessels and higher bone union rate in the TB-gel group than in the TB group. The lamellar bone volume and mineral apposition rate did not differ significantly between the groups, indicating that neovascularization may be the primary effect of f-IC gel on bone regeneration. This combination method which is tTMCP combined with TB and f-IC gel, would be useful for the treatment of segmental long bone defects.

Optimal internal fixation of anatomically shaped synthetic bone grafts for massive segmental defects of long bones

BackgroundLarge segmental bone defects following tumor resection, high-energy civilian trauma, and military blast injuries present significant clinical challenges. Tissue engineering strategies using scaffolds are being considered as a treatment, but there is little research into optimal fixation of such scaffolds. MethodsTwelve fresh-frozen paired cadaveric legs were utilized to simulate a critical sized intercalary defect in the tibia. Poly-ε-caprolactone and hydroxyapatite composite scaffolds 5cm in length with a geometry representative of the mid-diaphysis of an adult human tibia were fabricated, inserted into a tibial mid-diaphyseal intercalary defect, and fixed with a 14-hole large fragment plate. Optimal screw fixation comparing non-locking and locking screws was tested in axial compression, bending, and torsion in a non-destructive manner. A cyclic torsional test to failure under torque control was then performed. FindingsBiomechanical testing showed no significant difference for bending or axial stiffness with non-locking vs. locking fixation. Torsional stiffness was significantly higher (P=0.002) with the scaffold present for both non-locking and locking compared to the scaffold absent. In testing to failure, angular rotation was greater for the non-locking compared to locking constructs at each torque level up to 40N-m (P<0.05). The locking constructs survived a significantly higher number of loading cycles before reaching clinical failure at 30 degrees of angular rotation (P<0.02). InterpretationThe presence of the scaffold increased the torsional stiffness of the construct. Locking fixation resulted in a stronger construct with increased cycles to failure compared to non-locking fixation.

Effects of Ovariectomy and Corticosteroid-Induced Osteoporosis on the Osteoinductivity of rhBMP-2 in a Segmental Long-Bone Defect Model.

This study used the segmental long-bone defect model to assess the effects of osteoporosis on the formation of new bones and the osteoinductivity of recombinant human bone morphogenetic protein-2 (rhBMP-2). Seventy-two female Sprague-Dawley rats were divided into two groups: an osteoporosis group with ovariectomies and dexamathasone intramuscular injections and a sham group. When they reached 22 weeks in age, each group was further divided into two groups and a 5-mm defect was made in both fibular mid-shafts of each rat. One fibula in each rat was picked randomly and was injected with 0.05 mL of hydrogel carrier; the opposite fibula was injected with the same carrier mixed with rhBMP-2 (10 μg). After rearing for a further 5 and 9 weeks, the ratios of the lengths of the newly formed bones in the fibular defects were determined using micro-CT and undecalcified histology. The sham rhBMP-2-injected group-in all of the 5- and 9-week-kept groups-showed a significantly higher bridging bone formation ratio than the other three groups. The osteoporosis rhBMP-2-injected group showed a significantly higher ratio than both the non-rhBMP-2-injected sham hydrogel and the osteoporosis hydrogel groups. The comparison of the micro-CT parameters of the newly formed bones showed that the sham rhBMP-2 group at both 5 and 9 weeks compared with the osteoporosis rhBMP-2 group had significantly higher percentage bone volumes, trabecular thicknesses, and trabecular numbers, in addition to significantly lower specific surfaces, trabecular pattern factors, and structural model indices. The histology results showed that the sham-rhBMP-2 group began forming bridging bones in the defect areas at 5 weeks, and at 9 weeks, trabeculae and marrow spaces were observed. However, the osteoporosis rhBMP-2 group exhibited a relatively minor level of new bone and trabecula formation. Consequently, the rhBMP-2 group showed significantly increased bone formation in the osteoporosis rat fibular defect model compared with the hydrogel group, whereas the new bone quantities, qualities, and remodeling in the osteoporosis rhBMP-2 group were less effective than those in the sham-rhBMP-2 group, signaling that ovariectomy and corticosteroid-induced osteoporosis significantly undermines rhBMP-2 osteoinductivity.

Treatment of Traumatic Segmental Long-Bone Defects: A Critical Analysis Review.

Long-bone defects are one of the most challenging problems associated with limb reconstruction following high-energy extremity trauma. Without reconstitution of structural integrity, amputation is the likely result. The current orthopaedic literature on segmental bone loss is limited to case reports, case series, and observational studies, making it difficult to establish sound conclusions on the basis of the levels of evidence provided. Ten-year data from a fracture registry indicated that 0.4% of all fractures that were treated at a Level-I orthopaedic trauma unit were associated with bone loss1. Data from the same registry also indicated that the most common site of bone loss was the tibia (68%), followed by the femur (22%), with the remaining fractures occurring evenly at different sites. An additional retrospective review of tibial bone defects …

Effect of VEGF-A165 addition on the integration of a cortical allograft in a tibial segmental defect in rabbits.

Long-bone segmental defects caused by infection, fracture, or tumour are a challenge for orthopaedic surgeons. Structural allografts are sometimes used in their treatment but their poor biological characteristics are a liability. The objective of this study was to determine whether the addition of recombinant vascular endothelial growth factor-A (VEGF) to a structural allograft improved its integration into a rabbit tibial segmental defect in a non-union model. Tibial segmental defects were filled with heat sterilized allogenic tubular tibiae sections and then stabilized with a screw plate. In the VEGF treatment group (n = 6 tibiae), 2 μg of VEGF added to a 50 μl matrigel solution was inserted into the allograft cavity. In the control group (n = 6 tibiae), only matrigel was added. After 12 weeks, macroscopic and microscopic analysis, radiographs, and computerized micro-tomography (micro-CT) were performed. If allograft consolidation was present, a torsional resistance analysis was performed. Addition of VEGF to the allograft decreased the rate of osteosynthesis failure compared with the control group (1/6 vs. 5/6, p = 0.08), increased trabecular continuity evaluated by micro-CT in the bone-allograft interphases (8/12 vs. 2/12, p = 0.036) and histological trabecular continuity (7/12 vs. 0/12, p = 0.0046). Full consolidation was observed in three tibiae of the VEGF group and one in the control group (differences not significant); however, torsional resistance showed no significant differences (n.s.). Addition of VEGF to a structural allograph inserted into a rabbit tibial segmental defect increased allograft integration rate. Further research in this direction might help clinicians in dealing with large bone defects.

Repair of segmental long-bone defects by stem cell concentrate augmented scaffolds: a clinical and positron emission tomography - computed tomography analysis

Treating segmental long-bone defects remains a major challenge. For defects >3 cm, segmental transport represents the gold standard, even though the method is time consuming and afflicted with several complications. The aim of this study was to evaluate healing of such defects after grafting an osteogenic scaffold previously seeded with stem cell concentrate. We evaluated five patients with segmental long-bone defects (3-14 cm) treated with bone marrow aspirate concentrates (BMAC) seeded onto a bovine xenogenous scaffold. The healing process was monitored by X-rays and positron emission tomography-computed tomography (PET-CT) three months after surgery. Centrifugation led to a concentration of leukocytes by factor 8.1 ± 7.5. Full weight bearing was achieved 11.3 ± 5.0 weeks after surgery. PET analysis showed an increased influx of fluoride by factor 8.3 ± 6.4 compared with the contralateral side (p < 0.01). Bone density in the cortical area was 75 ± 16 % of the contralateral side (p < 0.03). The patient with the largest defect sustained an implant failure in the distal femur and finally accomplished therapy by segmental transport. He also had the lowest uptake of fluoride of the patient collective (2.2-fold increase). Stem cell concentrates can be an alternative to segmental bone transport. Further studies are needed to compare this method with autologous bone grafting and segmental transport.

The Effect of Structural Design on Mechanical Properties and Cellular Response of Additive Manufactured Titanium Scaffolds

Restoration of segmental defects in long bones remains a challenging task in orthopedic surgery. Although autologous bone is still the ‘Gold Standard’ because of its high biocompatibility, it has nevertheless been associated with several disadvantages. Consequently, artificial materials, such as calcium phosphate and titanium, have been considered for the treatment of bone defects. In the present study, the mechanical properties of three different scaffold designs were investigated. The scaffolds were made of titanium alloy (Ti6Al4V), fabricated by means of an additive manufacturing process with defined pore geometry and porosities of approximately 70%. Two scaffolds exhibited rectangular struts, orientated in the direction of loading. The struts for the third scaffold were orientated diagonal to the load direction, and featured a circular cross-section. Material properties were calculated from stress-strain relationships under axial compression testing. In vitro cell testing was undertaken with human osteoblasts on scaffolds fabricated using the same manufacturing process. Although the scaffolds exhibited different strut geometry, the mechanical properties of ultimate compressive strength were similar (145–164 MPa) and in the range of human cortical bone. Test results for elastic modulus revealed values between 3.7 and 6.7 GPa. In vitro testing demonstrated proliferation and spreading of bone cells on the scaffold surface.

Finite element analysis on the biomechanical stability of open porous titanium scaffolds for large segmental bone defects under physiological load conditions

Repairing large segmental defects in long bones caused by fracture, tumour or infection is still a challenging problem in orthopaedic surgery. Artificial materials, i.e. titanium and its alloys performed well in clinical applications, are plenary available, and can be manufactured in a wide range of scaffold designs. Although the mechanical properties are determined, studies about the biomechanical behaviour under physiological loading conditions are rare. The goal of our numerical study was to determine the suitability of open-porous titanium scaffolds to act as bone scaffolds. Hence, the mechanical stability of fourteen different scaffold designs was characterized under both axial compression and biomechanical loading within a large segmental distal femoral defect of 30mm. This defect was stabilized with an osteosynthesis plate and physiological hip reaction forces as well as additional muscle forces were implemented to the femoral bone. Material properties of titanium scaffolds were evaluated from experimental testing. Scaffold porosity was varied between 64 and 80%. Furthermore, the amount of material was reduced up to 50%. Uniaxial compression testing revealed a structural modulus for the scaffolds between 3.5GPa and 19.1GPa depending on porosity and material consumption. The biomechanical testing showed defect gap alterations between 0.03mm and 0.22mm for the applied scaffolds and 0.09mm for the intact bone. Our results revealed that minimizing the amount of material of the inner core has a smaller influence than increasing the porosity when the scaffolds are loaded under biomechanical loading. Furthermore, an advanced scaffold design was found acting similar as the intact bone.

Technique de la membrane induite pour la reconstruction de pertes de substances osseuses après résection tumorale chez l’enfant : une étude préliminaire

Segmental long-bone defect due to tumor resection remains a challenge to treat. The induced membrane technique is a new alternative for biological reconstruction. During the first stage, a cement spacer is inserted after bone resection and stabilisation. The cement spacer is removed during a second stage procedure performed after chemotherapy, and corticocancellous bone autograft was placed in the biological induced chamber. The aim of this study was to assess preliminary results in eight children. This prospective study included six girls and two boys, with a mean age of 12.1 years (range 9.5 to 18) and treated for a mean of 15 cm defect (range 10 to 22 cms) due to resection of osteosarcoma ( n = 4), Ewing sarcoma ( n = 3) and low-grade sarcoma. All patients except one, were given pre- and postoperative chemotherapy. Surgery was performed for three patients with a distal femur tumor, two patients with a proximal tibial tumor and three patients who had proximal humerus, shaft of humerus and fibular tumors. Fixation was mainly performed with locking compression plate ( n = 4) and locked nail ( n = 2). The mean operating times for first and second step procedures were 4.8 and 4 hours respectively. The healing process was radiologically assessed. After a mean follow-up of 21,6 months (15 to 30), all patients were free of disease and seven had bony union. For the lower limb reconstructions, full weight bearing was possible after a mean of 116 days (range 90 to 150) following the second stage. Mean time to bone union was 4.8 months (1.5 to 10). The early MSTS score was 25.2/30 (range 20 to 30). Complications were: non-union ( n = 1), paradoxical graft resorption ( n = 1) requiring graft revision. This two-stage procedure reduces the operating time during the first stage and it also reduces early complications. Rapid bone union is objectively obtained despite major bone resection and the patients receiving chemotherapy. The induced membrane technique could be an excellent alternative for biological reconstruction after tumor resection in children.

Induced membrane technique for reconstruction after bone tumor resection in children: A preliminary study

Segmental long-bone defect due to tumor resection remains a challenge to treat. The induced membrane technique is a new alternative for biological reconstruction. During the first stage, a cement spacer is inserted after bone resection and stabilisation. The cement spacer is removed during a second stage procedure performed after chemotherapy, and cortico-cancellous bone autograft was placed in the biological induced chamber. The aim of this study was to assess preliminary results in eight children. This prospective study included six girls and two boys, with a mean age of 12.1 years (range 9.5 to 18) and treated for a mean 15 cm defect (range 10 to 22 cms) due to resection of osteosarcoma (n=4), Ewing sarcoma (n=3) and low grade sarcoma. All patients except one, were given pre- and postoperative chemotherapy. Surgery was performed for three patients with a distal femur tumor, two patients with a proximal tibial tumor and three patients who had proximal humerus, shaft of humerus and fibular tumors. Fixation was mainly performed with locking compression plate (n=4) and locked nail (n=2). The mean operating times for first and second step procedures were 4.8 and 4h respectively. The healing process was radiologically assessed. After a mean follow-up of 21.6 months (15 to 30), all patients were free of disease and seven had bony union. For the lower limb reconstructions, full weight bearing was possible after a mean of 116 days (range 90 to 150) following the second stage. Mean time to bone union was 4.8 months (1.5 to 10). The early Musculoskeletal Tumor Society (MSTS) score was 25.2/30 (range 20-30). Complications were: non-union (n=1), paradoxical graft resorption (n=1) requiring graft revision. This two stage procedure reduces the operating time during the first stage and it also reduces early complications. Rapid bone union is objectively obtained despite major bone resection and the patients receiving chemotherapy. The induced membrane technique could be an excellent alternative for biological reconstruction after tumor resection in children.

Oriented bioactive glass (13-93) scaffolds with controllable pore size by unidirectional freezing of camphene-based suspensions: Microstructure and mechanical response

Scaffolds of 13-93 bioactive glass (composition 6.0 Na 2O, 7.9 K 2O, 7.7 MgO, 22.1 CaO, 1.7 P 2O 5, 54.6 SiO 2 (mol.%)) containing oriented pores of controllable diameter were prepared by unidirectional freezing of camphene-based suspensions (10 vol.% particles) on a cold substrate (−196 °C or 3 °C). By varying the annealing time (0–72 h) to coarsen the camphene phase, constructs with the same porosity (86 ± 1%) but with controllable pore diameters (15–160 μm) were obtained after sublimation of the camphene. The pore diameters had a self-similar distribution that could be fitted by a diffusion-controlled coalescence model. Sintering (1 h at 690 °C) was accompanied by a decrease in porosity and pore diameter, the magnitude of which depended on the pore size of the green constructs, giving scaffolds with a porosity of 20–60% and average pore diameter of 6–120 μm. The compressive stress vs. deformation response of the sintered scaffolds in the orientation direction was linear, followed by failure. The compressive strength and elastic modulus in the orientation direction varied from 180 MPa and 25 GPa (porosity = 20%) to 16 MPa and 4 GPa (porosity = 60%), respectively, which were 2–3 times larger than the values in the direction perpendicular to the orientation. The potential use of these 13-93 bioactive glass scaffolds for the repair of large defects in load-bearing bones, such as segmental defects in long bones, is discussed.

Current management of long bone large segmental defects

Large segmental defects of long bones comprise a complex pathology resulting from a variety of aetiologies. Their prolonged, painful and uncertain treatment is usually beset with a range of consequences for the patient, varying from the psychological to the socioeconomic. Trauma, osteomyelitis, bone tumour resections or treatment of congenital deformities are main causes of bone deficiency. Their treatment has been thoroughly studied for the last 35 years and both vascularized bone grafting and distraction osteogenesis with the Ilizarov technique have emerged as gold standards. Novel techniques have arisen during the last 10 years, giving new perspectives to the management of this problem. Intramedullary lengthening devices, bioactive membranes, osteogenic proteins and tissue engineering are the new weapons in the armamentarium of orthopaedic surgeons. This study describes the aforementioned treatment techniques (classic and novel) and elaborates on their indications, advantages/disadvantages and complications. Algorithms for the assessment and treatment of critically size long-bone segmental defects are also proposed.

Repair of a segmental long bone defect in human by implantation of a novel multiple disc graft

Large segmental defects of the weight bearing long bones are very difficult to reconstruct. Current treatment options are afflicted with several limitations and disadvantages. We describe a novel approach to regenerate a segmental long bone defect in a patient using a multiple disc graft. Decellularized bovine trabecular bone discs were seeded with autologous bone marrow cells and cultured in a perfusion chamber for three weeks. Multiple cell-seeded discs were implanted to close a 72mm defect of the distal tibia in a 58-year-old woman, and fixed by an intramedullary nail. Bone formation was assessed non-invasively by plain radiographs and 18F-labeled sodium fluoride-based co-registration of positron emission- and computed tomography (PET/CT). Bone was actively formed around the grafted defect as early as six weeks after surgery. Because the tibia was sufficiently stabilized, the patient was able to freely walk with full weight bearing 6weeks after surgery. The uneventful two-year follow-up and the satisfaction of the patient demonstrated the success of the procedure. Therefore the use of multiple cell-seeded disc grafts can be considered as a treatment alternative for patients with segmental long bone defects.

Combination of anorganic bovine‐derived hydroxyapatite with binding peptide does not enhance bone healing in a critical‐size defect in a rabbit model

Anorganic bovine-derived hydroxyapatite (ABM) in combination with binding peptid (P-15) has demonstrated the capacity to improve the healing of periodontal defects. This study evaluated the benefit of ABM/P-15 to promote healing of cortical long bone defects in a rabbit model. A 5-mm segmental bone defect was created in the femur and fixed with a plate. There were two treatment groups: no implant (n = 12) and ABM/P-15 (n = 12). At 4, 8, and 12 weeks, healing of the defect was evaluated with radiographs and histomorphometric examination of the treated femora. After 4 weeks, radiographs showed bone formation without signs of complete consolidation in three of four animals in the control group and two of four ABM/P-15 treated animals. At the later course of the treatment, no radiologic difference was evident between the treatment groups. Histomorphometric evaluation revealed an area of 1.29 +/- 0.11 mm(2) and 0.97 +/- 0.21 mm(2) of newly produced bone in animals of the control group and ABM/P-15 group after 4 weeks. After 8 and 12 weeks, animals in the control group had an area of 2.44 +/- 0.62 mm(2) and 2.5 +/- 0.2 mm(2) of newly produced bone within the osteotomy gap compared to 1.6 +/- 0.65 mm(2) and 1.56 +/- 0.27 mm(2) in the ABM/P-15 group (p = 0.0004). An enhanced or accelerated ingrowth of bone, as reported in previous studies, was not observed. Our results imply that the ABM/P-15 is not a suitable graft for the treatment of critical-sized segmental defects in long bones.

New Approaches in the Treatment of Critical‐Size Segmental Defects in Long Bones

AbstractSummary: The treatment of large segmental diaphyseal bone deficiencies presents a formidable challenge. The standard treatment modalities such as cancellous bone grafting, cortical allografts, vascularized bone transfer, or distraction osteogenesis exhibit extremely high complication rates, and can culminate in limb amputation or major functional deficits. Recent efforts to develop new treatment modalities for segmental bone loss have resulted in designing new biodegradable polymeric and metallic mesh implants that can incorporate novel osteogenic, osteoinductive, and/or osteoconductive bone healing augmentation materials. These biologic implant composites are capable of further enhancing the efficacy of the treatment applied. This paper briefly reviews the limitations of the currently applied standard treatment modalities for segmental critical size bone defects, provides insight into the specific treatment challenges, and presents the animal and initial clinical results of new alterative treatment approaches that involve the application of cylindrical mesh implants consisting of biodegradable polylactide membranes or titanium cages as a means of potentiating the efficacy of bone graft.

Use of the Ilizarov method to correct lower limb deformities in children and adolescents.

The introduction to the West in the early 1980s of the Ilizarov circular external fixator and method resulted in rapid advances in limb lengthening, deformity correction, and segmental long-bone defect reconstruction. The mechanical features of and biologic response to using distraction osteogenesis with the circular external fixator are the unique aspects of Ilizarov's contribution. The most common indications for children and adolescents are limb lengthening and angular deformity correction. Surgical application and postoperative management of the device require diligent attention to detail by both patient and surgeon. Also required of the surgeon is a thorough appreciation of the basic principles of the apparatus, mechanical axial realignment, potential complications, and biologic response to stretching.

Gene transfer approaches to the healing of bone and cartilage.

Gene transfer approaches to the healing of bone and cartilage.

Influence of bone marrow on membrane-guided bone regeneration of segmental long-bone defects in rabbits.

Defects 10 mm long were created in long bone in the diaphysis of both radii of 18 rabbits (test and control side). On the test side, ingrowth of bone marrow into the defects was hindered or delayed by: plugging the opening of the cut bone ends with gutta-percha points (n = 7); plugging with Gelfoam (n = 6); or by removing the bone marrow by flushing with saline (n = 5). The defects on both test and control side were covered with an expanded polytetrafluoroethylene membrane, shaped as a tube. Healing was followed with radiographs for four to five months, after which the animals were killed and ground sections of the areas of the defects were prepared for histological examination. On the control side, nine of 18 animals had complete osseous bridging of the defect, and a small transverse non-mineralised zone remained in the centre of the healed defect in the other animals. This zone consisted of loose connective and cartilagenous tissue as well as connective tissue obviously derived from the outside of the membrane. By preventing or delaying the ingrowth of bone marrow we retarded the regeneration of mineralised bone, particularly in the gutta-percha and flushed bone marrow groups. The principle of guided tissue regeneration may be used to achieve regeneration of extensive long-bone defects. Any attempts to delay or prevent bone marrow ingrowth into the defects did retard regeneration of segmental long-bone defects.