Repair of mandible defect with tissue engineering bone in rabbits

BACKGROUND: The aim of the present study was to investigate the effect of tissue engineering bone composed of bone marrow-derived osteoblasts and demineralized bone in repairing mandible defect. METHODS: Bone marrow-derived osteoblasts of 20 rabbits were cultured and seeded into scaffold of allogeneic demineralized bone to construct tissue engineering bone graft in vitro, which was used to repair the 10 x 5-mm bone defect made in the same rabbit mandible edge. Implant of demineralized bone alone was as the control. Rabbits were killed according to the schedule: five after 2 weeks, five after 4 weeks, five after 8 weeks, five after 12 weeks, and the implants were harvested for gross, radiographic, and histological observation. RESULTS: New bone formation at the margin region of defect and osteogenesis at the centre were observed in the implant of tissue engineering bone, and the bone formation pattern included osteogenesis, osteoconduction, and osteoinduction. In the implant of demineralized bone alone, the major bone formation pattern was 'creeping substitute'. CONCLUSIONS: The tissue engineering bone graft constructed by autogenous bone marrow-derived osteoblasts and allogeneic demineralized bone was better than demineralized bone alone in bone formation capability, which might be an ideal graft for bone defect repair.     

The presence of different growth factors does not influence bone response to hydroxyapatite: preliminary results

Abstract Porous, non-resorbable, granular hydroxyapatite (pHA), either pure or mixed with fibrin, or soaked in solutions of the growth factors PDGF, TGF, IGF, or TNF, has been implanted in rabbits under the derma and periosteum of the skull, in contact with the calvarial bone. The degree of osteogenesis in the implant was evaluated by histological analysis and histomorphometry 20 and 60 days after the implant. The results showed that new bone formation occurred in all the implants, most evidently in those containing TGF, TNF or IGF. The histomorphometric results, however, showed no significant differences between the groups, perhaps because of technical artifacts and the small number of animals. On the other hand, the formation of new bone was due to osteoconduction rather than osteoinduction, as shown by the observation that osteogenesis decreased with the distance of pHA granules from the calvarium. The earliest evidence of osteogenesis was the presence of dense borders on the surface of pHA: some of them were vacuolated and contained glycoproteins, others resembled osteoid borders. The bone formed at 20 days was of woven type, whereas that present at 60 days was of both woven and lamellar type, suggesting that some degree of remodeling takes place as the implant ages.     

Use of the Wagner apparatus in the treatment of compound fractures with segmental bone loss

Treatment of non-union of long bone fractures with segmental bone loss is difficult problem. Fracture healing is commonly delayed due to the severe associated soft tissue injuries, deep wound infections and poor osteogenesis. The slow healing time often necessitates prolonged joint immobilization resulting in joint stiffness. In order to obtain satisfactory fracture union without adjacent joint contractures, the Wagner apparatus in conjunction with massive cancellous bone grafting has been employed at the Kobe University Hospital. Autogenous cancellous grafts provide a scaffolding for rapid vascular ingrowth and viable osteoblasts for osteogenesis. Graft incorporation and fracture union can be monitored by serial radiographs and radioisotope studies.     

Selection of bone grafts for revision total hip arthroplasty

The selection of bone grafts to reconstruct deficient bone for revision hip replacement requires an understanding of specific bone graft functions and the critical steps of the biologic incorporation of the graft into the host. Bone grafts provide functions of osteogenesis, either graft derived or by osteoinduction, osteoconduction, or both, and mechanical support. Autologous cancellous bone provides excellent osteogenesis and osteoconduction without structural support. Nonvascularized cortical autografts provide mechanical support and are somewhat osteogenic. Allogeneic cancellous bone is osteoconductive and minimally osteoinductive, whereas cortical allografts provide structural support, if not freeze-dried, and are somewhat osteoconductive. Allogeneic demineralization bone matrix is highly osteoinductive. The selection of the appropriate bone graft depends on the classification of the bone deficiency. Cavitary (contained) defects can be reconstructed with cancellous morselized autograft, frozen or freeze-dried allograft, or allogeneic demineralized bone matrix. Segmental defects require bulk corticocancellous and/or cortical autografts or allografts. The ultimate incorporation of the bone graft depends on the interaction of the graft and the host's mechanical and biologic environment, and host-bone graft contact and stability. Optimum bone graft selection will enhance the clinical outcomes in revision total hip arthroplasty.     

The healing of biologic and synthetic bone implants. An experimental study

The aim of the present study was to investigate the osteogenic properties of different types of cancellous bone grafts inserted in large osseous defects in dogs and to compare these with those of sintered hydroxyapatite implants. Fresh cancellous autografts were rapidly revascularized and invariably induced a complete healing of the defect. Frozen and fresh cancellous allografts were largely resorbed, the latter evoking a strong antigenic response in two of the five cases. Sintered hydroxyapatite granules were largely encapsulated in fibrous tissue, neither stimulating nor inhibiting osseous ingrowth. Degradation of the hydroxyapatite implant was not observed.     

Effects of low-dose parathyroid hormone on bone mass, turnover, and ectopic osteoinduction in a rat model for chronic alcohol abuse

Parathyroid hormone (PTH) is used clinically in osteoporotic patients to increase bone mass by enhancing bone formation. PTH therapy is not uniformly effective at all skeletal sites and "life-style" factors may modulate the skeletal response to PTH. Alcohol may represent one of these factors. Chronic alcohol abuse is associated with osteoporosis and impaired fracture healing. Therefore, the present study investigated the effects of alcohol on the bone anabolic response to a dose of PTH similar to a human therapeutic dose 1) during normal cancellous and cortical bone growth and turnover, and 2) in a model of demineralized allogeneic bone matrix (DABM)-induced osteoinduction. Three-month-old male Sprague Dawley rats were fed a Lieber-DeCarli liquid diet with 35% of the calories derived from ethanol. The controls were pair-fed an alcohol-free isocaloric diet containing maltose-dextran. Following adaptation to the liquid diets, the rats were implanted subcutaneously with DABM cylinders prepared from cortical bone of rats fed normal chow. The rats were subsequently treated daily with PTH (1 microg/kg/d sc, 5 d/week) or vehicle and measurements on bone and DABM implants performed 6 weeks later. Total bone mass was evaluated on the day of necropsy using DXA. Tibiae were processed for histomorphometry. Bone mass and architecture in tibial diaphysis and DABM implants were evaluated by muCT. PTH treatment increased whole body bone mineral content (BMC) and bone mineral density (BMD). The hormone also increased bone formation and bone area/tissue area in the proximal tibial metaphysis. In contrast, PTH treatment had no effect on periosteal bone formation and minimal effects on DABM-induced osteoinduction. Alcohol consumption decreased whole body BMC. Alcohol also decreased cancellous as well as cortical bone formation and bone mass in tibia and impaired DABM-mediated osteoinduction. There was no interaction between PTH treatment and alcohol consumption for any of the endpoints evaluated. Our results indicate that the bone anabolic response to a therapeutic dose of PTH in the rat is largely confined to cancellous bone. In contrast, alcohol consumption inhibits bone formation at all sites. Furthermore, alcohol inhibits osteoinduction and reduces periosteal and cancellous bone formation, irrespective of therapeutic PTH administration. Based on the animal model, our findings suggest that alcohol consumption could impair the beneficial effects of PTH therapy in osteoporosis.     

Osteointegration of hydroxyapatite-titanium implants coated with nonglycosylated recombinant human bone morphogenetic protein-2 (BMP-2) in aged sheep

Osteointegration of metal implants into aged organisms can be severely compromised due to reduced healing capacity of bone, lack of precursor cells for new bone formation, or osteoporosis. Here, we report on successful implant healing in a novel model of aged sheep in the presence of nonglycosylated bone morphogenetic protein 2 (BMP-2). Ewes of 8 to 12 years with significant radiologic and histologic signs of osteoporosis and adipocytic bone marrow received a cylindrical hydroxyapatite-titanium implant of 12 x 10 mm. BMP-2 has been produced as a bacterial recombinant fusion protein with maltose-binding protein and in vitro generation of mature BMP-2 by renaturation and proteolytic cleavage. A BMP-2 inhibition ELISA was developed to measure the in vitro release kinetics of bioactive human BMP-2 from immersed solid implant materials by using Escherichia coli expressed and biotinylated recombinant human BMP-2 receptor IA extracellular domain (ALK-3 ECD). The implants were placed laterally below both tibial plateaus, with the left leg implant carrying 380 microg BMP-2. Both implant types became integrated within the following 20 weeks. The control implant only integrated at the cortical bone, and little new bone formation was found within the pre-existing trabecular bone or the marrow cavity. Marrow fat tissue was partially replaced by unspecific connective tissue. In contrast, BMP-2-coated implants initiated significant new bone formation, initially in trabecular arrangements to be replaced by cortical-like bone after 20 weeks. The new bone was oriented towards the cylinder. Highly viable bone marrow appeared and filled the lacunar structures of the new bone. In mechanical tests, the BMP-2-coated implants displayed in average 50% higher stability. This animal model provided first evidence that application of nonglycosylated BMP-2 coated on solid implants may foster bone healing and regeneration even in aged-compromised individuals.     

The healing of biologic and synthetic bone implants

Summary The aim of the present study was to investigate the osteogenic properties of different types of cancellous bone grafts inserted in large osseous defects in dogs and to compare these with those of sintered hydroxyapatite implants. Fresh cancellous autografts were rapidly revascularized and invariably induced a complete healing of the defect. Frozen and fresh cancellous allografts were largely resorbed, the latter evoking a strong antigenic response in two of the five cases. Sintered hydroxyapatite granules were largely encapsulated in fibrous tissue, neither stimulating nor inhibiting osseous ingrowth. Degradation of the hydroxyapatite implant was not observed.     

The effect of proximally and fully porous-coated canine hip stem design on bone modeling

Porous coated canine femoral hip replacement implants were evaluated for biological fixation by bone ingrowth and the effect of the extent of porous coating on bone modeling. The Co-Cr alloy implants were either fully porous coated or coated only on the proximal 40% of the stem. Two implants of each type were studied 9, 16, and 36 months after surgery. Implant fixation and bone modeling were assessed radiographically throughout the implant periods and histologically after the test animals were killed. All 12 implants appeared stably fixed within the femur and were bone-ingrown in the porous region. Radiographic features such as proximal medial and anterior cortical thinning, proximal cancellous bone hypertrophy, and new endosteal bone formation near the stem tip were noted within the first postoperative year, with no appreciable change thereafter. The extent of proximal cortical thinning varied from virtually none to as much as 40%, being more prominent with the proximally coated implants at 16 months and with the fully coated implants at 36 months. Of consistent note was cancellous hypertrophy at the junction of porous and smooth implant surfaces with proximally coated implants and new endosteal bone formation and ingrowth at the stem tip of fully coated implants. These results indicate that the proximally porous-coated implant design causes increased proximal stress transfer, but this does not necessarily preclude proximal cortical resorption.     

碱性成纤维细胞生长因子增强人重组骨形态发生蛋白-2诱导成骨的调节机理

目的：对ｂＦＧＦ增强ｒｈＢＭＰ－２诱导成骨的调节机理进行探讨。方法：２１０只ＢＡＬＢ／ｃ小鼠随机分为３组,每组７０只,试验侧均位于右后肢。设立ｒｈＢＭＰ－２／牛松质骨载体、单纯牛松质骨载体为对照组,分别于术后１２ｈ～２１ｄ共１１个时间点取材,观察其诱导成骨过程。结果：ｒｈＢＭＰ－２／ｂＦＧＦ／聚乙烯吡咯啉酮／牛松质骨载体组在诱导间充质细胞增殖、分化,软骨细胞、新生骨形成方面均早于ｒｈＢＭＰ－２／牛松质骨载体组,成骨量优于ｒｈＢＭＰ－２／牛松质骨载体组,而单纯牛松质骨载体组在２１ｄ仅出现了少量增殖的间充质细胞。结论：ｒｈＢＭＰ－２和ｂＦＧＦ在诱导成骨调节中存在着协同作用。     

Relevance of bone graft viability in a goat transverse process model

Little is known about the mechanism by which autologous bone grafts are so successful. The relevance of viable osteogenic cells, which is a prominent difference between autologous bone graft and conventional alternatives, is especially controversial. With the emergence of bone tissue engineering, knowledge of the exact role of these cells has become crucial. The most obvious question to answer is whether viability of the graft has an effect on bone formation. In the current study, we investigated this effect of bone graft viability in a transverse process model that represents the initial bone formation in posterolateral spinal fusion. Eight goats received viable and devitalized autologous bone grafts in chambers mounted on the decorticated lumbar transverse processes. In addition, five goats received empty chambers. Histology and histomorphometry were performed after a 12‐week implantation, and the dynamics of bone formation was monitored by sequential fluorochrome labeling. An obvious qualitative effect of viability was demonstrated by the presence of early onset osteogenesis distant from the transverse process bone in the viable grafts only. Quantitative analysis indicated about 30% more bone in the viable grafts, however, this difference was not statistically significant. In the empty chambers, bone was found in comparable quantities. We conclude that there is a qualitative advantage of graft viability in terms of early graft‐derived osteogenesis. However, this advantage did not lead to significantly more bone formation in the viable bone grafts. © 2008 Orthopaedic Research Society. Published by Wiley Periodicals, Inc. J Orthop Res 27: 1055–1059, 2009     

The biology of bone grafts

The clinical outcome of bone grafting procedures depends on many factors, including type and fixation of the bone graft as well as the site and status of the host bed. Bone grafts serve one or both of two main functions, as a source of osteogenesis and as a mechanical support. Autografts, both cancellous and cortical, are implanted fresh, provide a source of osteoprogenitor cells, and are osteoinductive. The latter is a process whereby the transplanted tissue induces mesenchymal cells of the recipient to differentiate into osteoblastic cells. Cortical grafts, whether autogeneic or allogeneic, at least initially act as weight-bearing space fillers. All bone grafts are initially resorbed; cancellous grafts are completely replaced in time by creeping substitution, whereas cortical grafts remain an admixture of necrotic and viable bone for a prolonged period of time. The three-dimensional framework that supports invasion of the bone grafts by capillaries and osteoprogenitor cells, termed "osteoconduction", is another important function of both autografts and allografts. Because fresh allographs evoke both local and systemic immune responses that diminish or destroy the osteoinductive and conductive processes, freezing or freeze-drying of allografts is used clinically to improve incorporation. Graft incorporation is also influenced by the vascularity and composition of the host bed. Thus, the interaction of the host and the bone graft determines the success of these procedures, which ultimately is to provide a mechanically efficient support structure.     

Systemic intermittent parathyroid hormone treatment improves osseointegration of press-fit inserted implants in cancellous bone

Background and purpose Intermittent administration of parathyroid hormone (PTH) has an anabolic effect on bone, as confirmed in human osteoporosis studies, distraction osteogenesis, and fracture healing. PTH in rat models leads to improved fixation of implants in low-density bone or screw insertion transcortically. Material and methods We examined the effect of human PTH (1-34) on the cancellous osseointegration of unloaded implants inserted press-fit in intact bone of higher animal species. 20 dogs were randomized to treatment with human PTH (1-34), 5 μg/kg/day subcutaneously, or placebo for 4 weeks starting on the day after insertion of a cylindrical porous coated plasma-sprayed titanium alloy implant in the proximal metaphyseal cancellous bone of tibia. Osseointegration was evaluated by histomorphometry and fixation by push-out test to failure. Results Surface fraction of woven bone at the implant interface was statistically significantly higher in the PTH group by 1.4 fold with (median (interquartile range) 15% (13-18)) in the PTH group and 11% (7-13) in control. The fraction of lamellar bone was unaltered. No significant difference in bone or fibrous tissue was observed in the circumferential regions of 0-500, 500-1,000, and 1,000-2,000 μm around the implant. Mechanically, the implants treated with PTH showed no significant differences in total energy absorption, maximum shear stiffness, or maximum shear strength. Interpretation Intermittent treatment with PTH (1-34) improved histological osseointegration of a prosthesis inserted press-fit at surgery in cancellous bone, with no additional improvement of the initial mechanical fixation at this time point.     

The biology of bone graft repair

Cancellous and cortical autografts histologically have three differences: (1) cancellous grafts are revascularized more rapidly and completely than cortical grafts; (2) creeping substitution of cancellous bone initially involves an appositional bone formation phase, followed by a resorptive phase, whereas cortical grafts undergo a reverse creeping substitution process; (3) cancellous grafts tend to repair completely with time, whereas cortical grafts remain as admixtures of necrotic and viable bone. Physiologic skeletal metabolic factors influence the rate, amount, and completeness of bone repair and graft incorporation. The mechanical strengths of cancellous and cortical grafts are correlated with their respective repair processes: cancellous grafts tend to be strengthened first, whereas cortical grafts are weakened. Bone allografts are influenced by the same immunologic factors as other tissue grafts. Fresh bone allografts may be rejected by the host's immune system. The histoincompatibility antigens of bone allografts are presumably the proteins or glycoproteins on cell surfaces. The matrix proteins may or may not elicit graft rejection. The rejection of a bone allograft is considered to be a cellular rather than a humoral response, although the humoral component may play a part. The degree of the host response to an allograft may be related to the antigen concentration and total dose. The rejection of a bone allograft is histologically expressed by the disruption of vessels, an inflammatory process including lymphocytes, fibrous encapsulation, peripheral graft resorption, callus bridging, nonunions, and fatigue fractures.     

Comparative study of human cancellous bone remodeling to titanium and hydroxyapatite-coated implants

The human cancellous bone response was compared in weight-bearing porous hydroxyapatite (HA) and titanium-coated implants placed in the distal medial femoral condyles of consenting staged bilateral knee patients. The Institutional Review Board approved study quantified the amount of bone ingrowth, the mineral apposition rate, and the bone mineral content. Results showed that the osteoconductive HA coating increased the amount of bone ingrowth by 8% (P=.018). The HA coating did not effect the mineral apposition rate of the bone but had an 8% lower bone mineral content at the implant interface (P=.042). The influence of HA coatings on human cancellous bone appears highly focal along the coating surface. Gaps of 50–500 μm filled with fibrous connective tissue were observed along the porous-coated surfaces of both implant types suggesting that HA coatings still require precision placement adjacent to human cancellous bone.     

Quantitative comparisons of healing in cranial fresh autografts, frozen autografts and processed autografts, and allografts in canine skull defects

Adult dog skull defects larger than 17 mm do not spontaneously heal. A quest for a potentially viable, cosmetically, mechanically, and technically acceptable template for human cranial reconstruction prompted a comparison of processed autogeneic and allogeneic bone implants with a fresh autograft control in the dog. Quantitative reproducible observations demonstrated that fresh calvarial bone autografts were superior to the nonviable implants in volume percent defect filled, mm2 new cortical bone, mm2 new and old cortical bone, and cortical bone porosity. Frozen autografts achieved 75%, antigen-extracted, autolyzed, partially demineralized auto- and allografts, 50% of the overall efficiency of fresh autografts. Fresh cancellous bone added to allografts did not improve long-term repair. Remodeling of all grafts appeared consistent with osteoconductive invasion by peripheral host endosteal and diploic elements; host external periosteum and dura contributed less. Central osteoinductive recruitment of mesenchymal cells from muscle or dura seemed not to occur in the adult dog. Partially demineralized dog calvarial grafts were resorbed without acting as a template for new bone formation. Surface demineralization, antigen extraction, and autolytic digestion of autografts and allografts, with or without fresh iliac bone, did not improve calvarial bone regeneration in adult dogs.     

Evaluation of bovine-derived bone protein with a natural coral carrier as a bone-graft substitute in a canine segmental defect model

The efficacy of a bone-graft substitute (bovine-derived bone protein in a carrier of natural coral) in the healing of a segmental defect of a weight-bearing long bone was evaluated. Twenty dogs, divided into two groups, underwent bilateral radial osteotomies with creation of a 2.5 cm defect. On one side of each dog, the defect was filled with autogenous cancellous bone graft. Contralateral defects received, in a blinded randomized fashion, cylindrical implants consisting of natural coral (calcium carbonate) or calcium carbonate enhanced with a standard dose of bovine-derived bone protein (3.0 mg/implant; 0.68 mg bone protein/cm³). The limbs were stabilized with external fixators, and all animals underwent monthly radiographs. They were killed at 12 (group 1) or 24 (group 2) weeks, and regenerated bone was Studied by biomechanical testing and histology. Radiographic union developed in all 20 radii with autogenous cancellous bone grafts and in all 10 of the radii with the composite implants. None of the radii with implants of calcium carbonate alone showed radiographic evidence of union. This represented a statistically significant difference between implant types. In addition, calcium carbonate implants both with and without bone protein demonstrated radiographic evidence of near total resorption of the radiodense carrier by 12 weeks. This resorption facilitated radiographic evaluation of healing. Mean values for. biomechanical parameters of radii with the composite implants exceeded those for the contralateral controls at 12 and 24 weeks; the difference was statistically significant at 12 weeks. Histology revealed scant residual calcium carbonate carrier at either time in the defects with calcium carbonate implants; however, a moderate amount was present in defects with the composite implants. In these specimens, the residual carrier was completely surrounded by newly formed bone that may have insulated the calcium carbonate from further degradation. The present study used a carrier of granular calcium carbonate reconstituted with bovine type-I collagen to deliver an osteoinductive protein to the defect site. This carrier is of nonhuman origin (eliminating the risk of disease transmission or antigenicity) and resorbs rapidly. In this model, bovine-derived bone protein in a natural coral carrier performed consistently better than the gold standard autogenous cancellous bone graft in terms of the amount of bone formation and strength of the healed defect. This may have implications for removal of hardware or resumption of weight-bearing in certain clinical situations. These data also indicate that coralline calcium carbonate alone. represents a poor option as a bone-graft substitute in this critical-sized segmental defect model.     

Effect of Er: YAG laser holes on osteoinduction in demineralized rat calvarial allografts

Massive cortical autografts and allografts have been found to incorporate into host bone very slowly and thus are subject to complications such as fatigue fracture and infection. In order to understand and improve the process of osteogenesis in these types of bone grafts, a new experimental model was developed using bone discs from rat calvaria prepared by demineralization and drilling of 0.5 mm diameter holes with a pulsed, 2.94 μm wavelength Erbium: Yttrium-Aluminum-Garnet laser. Four types of bone discs were analyzed: untreated (Type I), demineralized (Type II), laser-ablated (Type III), and laser-ablated then demineralized (Type IV). The discs were transplanted into a subcutaneous site in adult Sprague-Dawley rats and followed for as long as 6 weeks. Histologic analysis of the discs at weekly intervals with use of hematoxylin and eosin staining confirmed the presence of new bone growth in Type-II and Type-IV discs. The amount of new bone growth in each disc was estimated by determining the mineral x-ray attenuation coefficient, which is proportional to mineral density, from digitized radiographs of the discs. The results showed that the processes of demineralization (p < 0.001) and laser ablation with demineralization (p < 0.05) were both significant in enhancing new bone growth in this model. This study demonstrated that osteoinduction can be fostered in cortical bone through the processes of demineralization and laser ablation. To the extent that laser ablation may allow maintenance of structural integrity while altering the surface geometry in such a way as to promote ingrowth of new bone, this experimental model represents an advance in understanding how osteogenesis in cortical bone grafts might be improved.     

Osteogenesis after bone and bone-marrow transplantation. I. Studies with combined myelo-osseous grafts in the guinea pig.

There has been conflicting evidence in the past regarding the contribution made to new bone formation by various components of a bone graft. This study in guinea pigs has compared cortical and cancellous bone allografts, both non-decalcified and hydrochloric acid decalcified, inserted into a muscular site for up to thirteen days, both alone and combined with autologous bone marrow. New bone formation was seen only if the implant contained fresh autologous marrow and it was not seen before the ninth day of implantation. There were no differences in the cell populations about cortical and cancellous grafts. Undecalcified bone evoked giant cell formation as well as new bone.