Recombinant human osteogenic protein-1 induces bone formation in a chronically infected, internally stabilized segmental defect in the rat femur

BACKGROUND: Recombinant human osteogenic protein-1 (rhOP-1), combined with a collagen carrier, has been shown to induce new-bone formation in a variety of animal models. The purpose of the present investigation was to test the hypotheses that rhOP-1 would accelerate bone formation in an internally stabilized, chronically infected, critical-size defect in the rat femur and that this effect would be enhanced by the administration of systemic antibiotic. METHODS: A 6-mm segmental defect was created surgically, stabilized with a polyacetyl plate and six Kirschner wires, and contaminated with 10(4) colony-forming units of Staphylococcus aureus in one femur in each of 168 Sprague-Dawley rats. After two weeks, these infected defects were débrided surgically and were assigned to one of six treatment groups. The defects in the thirty animals in the first group received lyophilized collagen carrier mixed with 200 microg of rhOP-1 dissolved in buffer, the defects in the thirty animals in the second group received carrier with 20 microg of rhOP-1 in buffer, and the defects in the twenty-four control animals in the third group received carrier mixed with buffer without rhOP-1. The last three groups were treated identically to the first three groups, except that the animals also received the antibiotic ceftriaxone for twenty-eight days after débridement. The animals were killed at two, four, eight, or twelve weeks after débridement. Newly mineralized callus within the defect, and adjacent to and bridging the outside of the defect, was assessed with use of quantitative high-resolution radiography, microcomputed tomography, torsional failure testing, and histological analysis of undecalcified sections. RESULTS: Bacterial cultures confirmed the presence of a chronic infection during the study period in all animals. At the later time-points, significantly more newly mineralized callus was present within and adjacent to the débrided defects that had been treated with 200 microg of rhOP-1, whereas minimal amounts of callus were present within and adjacent to the defects that had been treated without rhOP-1 and with 20 microg of rhOP-1. At eight and twelve weeks after débridement, there was significantly more newly mineralized callus in the group that had been treated with 200 microg of rhOP-1 with antibiotic than in the group that had been treated with 200 microg of rhOP-1 without antibiotic (p < 0.05). At twelve weeks, the values for torque, energy to failure, and linear stiffness for femora that had been treated with 200 microg of rhOP-1 with antibiotic were not significantly different from the values for intact, contralateral control femora, whereas the values for femora that had been treated with 200 microg of rhOP-1 without antibiotic remained significantly lower than those for the intact, contralateral controls (p < 0.05). CONCLUSIONS: Recombinant human osteogenic protein-1 maintained its osteoinductive capability in the presence of chronic infection, and this property was enhanced by antibiotic therapy. No substantial callus formed in the infected defects without a sufficiently high dose of rhOP-1. CLINICAL RELEVANCE: The treatment of an infection at the site of a fracture often necessitates removal of internal fixation. However, internal fixation is needed for fracture stability. This study presents an intervention that may accelerate fracture-healing in the presence of infection and colonized hardware, thereby permitting earlier removal of the hardware and more timely and effective treatment of the infection.     

The effect of osteogenic protein-1 dosing regimen on ectopic bone formation

Osteogenic protein-1 (also known as bone morphogenetic protein-7) is a member of the bone morphogenetic protein family. Several of the bone morphogenetic proteins, including osteogenic protein-1, have been shown to form bone at an ectopic site. However, optimal dosing regimens for the use of osteogenic protein-1 and other bone morphogenetic proteins in large bone defects have not been determined conclusively. We investigated the influence of osteogenic protein-1 (at doses of 0.1, 1, and 10 microg) with dosing regimens of single bolus, continuous infusion (alone), or bolus and continuous infusion in the rat ectopic subcutaneous bone formation assay (alkaline phosphatase units per microgram protein) in the presence of demineralized bone matrix. The biochemical results showed that the single bolus dosing regimen was significantly more effective than the continuous infusion alone and as effective as the bolus and continuous infusion regimens. The quality of the bone histologically confirmed the biochemical results. These data show that osteogenic protein-1 given as a single bolus is more effective in forming bone at an ectopic site than a continuous infusion of this protein.     

Effects of osteogenic medium on healing of the experimental critical bone defect in a rabbit model

Today, finding an ideal biomaterial to treat the large bone defects, delayed unions and non-unions remains a challenge for orthopedic surgeons and researchers. Several studies have been carried out on the subject of bone regeneration, each having its own advantages. At the same time, a variety of disadvantages still remain. The present study has been designed in vivo to evaluate the effects of osteogenic medium on healing of experimental critical bone defect in a rabbit model. Twenty New Zealand albino rabbits, 12 months old, of both sexes, weighing 2.0 ± 0.5 kg were used in this study. An approximately 10 mm segmental defect was created in the mid portion of each radius as a critical size bone defect. In the osteogenic medium group (n = 5) 1 ml osteogenic medium, in the maintenance medium group (n = 5) 1 ml maintenance medium, and in the normal saline group (n = 5) 1 ml normal saline were injected in the defected area while the defects of the rabbits of the control group (n = 5) were left empty. Radiological evaluation was done on the 1st day and then at the 2nd, 4th, 6th and 8th weeks post injury. Biomechanical and histopathological evaluations were done 8 weeks post injury. The radiological, histological and biomechanical findings of the present study indicated a superior bone healing capability in the osteogenic and maintenance medium groups, by the end of 8 weeks post-surgery, in comparison to the normal saline and control groups. In conclusion, this study demonstrated that the osteogenic medium and maintenance medium could promote bone regeneration in long bone defects better than the control group in rabbit model.     

The effect of recombinant human osteogenic protein-1 on growth plate repair in a sheep model

Injuries to the growth plate in children can result in bone bridge formation, which ultimately lead to limb length and angular deformities. The histological and molecular changes associated with growth plate repair following the Langenski?ld procedure, a surgical technique used to remove impeding bone bridges, in conjunction with administration of recombinant human osteogenic protein-1 (rhOP-1) were examined using a sheep model. Following treatment with rhOP-1 there was an increase in the height of the growth plate immediately adjacent to the defect compared to untreated animals. The expression of type I collagen, osteopontin and decorin were observed in the growth plate adjacent to the defect in the untreated animals at day 56, but this response was accelerated in the rhOP-1 treated animals, with these molecules seen as early as day 7. Therefore, treatment with rhOP-1 initiated a complex response that was both chondrogenic and osteogenic in nature.     

A comparison of two biomaterial carriers for osteogenic protein-1 (BMP-7) in an ovine critical defect model

Critical size defects in ovine tibiae, stabilised with intramedullary interlocking nails, were used to assess whether the addition of carboxymethylcellulose to the standard osteogenic protein-1 (OP-1/BMP-7) implant would affect the implant's efficacy for bone regeneration. The biomaterial carriers were a 'putty' carrier of carboxymethylcellulose and bovine-derived type-I collagen (OPP) or the standard with collagen alone (OPC). These two treatments were also compared to "ungrafted" negative controls. Efficacy of regeneration was determined using radiological, biomechanical and histological evaluations after four months of healing. The defects, filled with OPP and OPC, demonstrated radiodense material spanning the defect after one month of healing, with radiographic evidence of recorticalisation and remodelling by two months. The OPP and OPC treatment groups had equivalent structural and material properties that were significantly greater than those in the ungrafted controls. The structural properties of the OPP- and OPC-treated limbs were equivalent to those of the contralateral untreated limb (p > 0.05), yet material properties were inferior (p < 0.05). Histopathology revealed no residual inflammatory response to the biomaterial carriers or OP-1. The OPP- and OPC-treated animals had 60% to 85% lamellar bone within the defect, and less than 25% of the regenerate was composed of fibrous tissue. The defects in the untreated control animals contained less than 40% lamellar bone and more than 60% was fibrous tissue, creating full cortical thickness defects. In our studies carboxymethylcellulose did not adversely affect the capacity of the standard OP-1 implant for regenerating bone.     

The effect of recombinant human osteogenic protein-1 (bone morphogenetic protein-7) impregnation on allografts in a canine intercalary bone defect.

The utility of cortical allografts in repairing large bone defects is limited by their slow and incomplete incorporation into host bone. In order to determine the effects of recombinant human osteogenic protein-1 (rhOP-1) impregnation on allograft incorporation, we used a canine intercalary bone defect model. Bilateral resection of a 4 cm segment of the femoral diaphysis and reconstruction with structural bone allografts were performed. In one limb, the allograft was soaked in solution with rhOP-1 for 1 h before implantation. In the other limb, the allograft was soaked in the same solution without rhOP-1. Dynamic load-bearing, radiographic analysis, biomechanical testing, and histomorphometric analysis were conducted. Radiographic analysis showed significantly larger periosteal callus area in the rhOP-1 treated group at week 2. The rhOP-1 significantly increased allograft bone porosity and significantly increased the number of active osteons in the allografts. There were no significant differences between the rhOP-1 treated and non-treated allografts in load bearing and biomechanical analyses. These findings indicate that rhOP- I increases intercalary allograft remodeling without deleterious effects in mechanical and functional strength.     

Dual delivery of an angiogenic and an osteogenic growth factor for bone regeneration in a critical size defect model

This study investigated the effects of dual delivery of vascular endothelial growth factor (VEGF) and bone morphogenetic protein-2 (BMP-2) for bone regeneration in a rat cranial critical size defect. Four groups of scaffolds were generated with VEGF (12 microg), BMP-2 (2 mug), both VEGF (12 microg) and BMP-2 (2 microg), or no growth factor released from gelatin microparticles incorporated within the scaffold pores. These scaffolds were implanted within an 8 mm rat cranial critical size defect (n=8-9 for each group). At 4 and 12 weeks, implants were retrieved and evaluated by microcomputed tomography (microCT) and histological scoring analysis. Additionally, 4 week animals were perfused with a radiopaque material to visualize and quantify blood vessel formation. Histological analysis revealed that for all groups at 4 weeks, a majority of the porous scaffold volume was filled with vascularized fibrous tissue; however, bone formation appeared most abundant in the dual release group at this time. At 12 weeks, both dual release and BMP-2 groups showed large amounts of bone formation within the scaffold pores and along the outer surfaces of the scaffold; osteoid secretion and mineralization were apparent, and new bone was often in close or direct contact with the scaffold interface. MicroCT results showed no significant difference among groups for blood vessel formation at 4 weeks (<4% blood vessel volume); however, the dual release group showed significantly higher bone formation (16.1+/-9.2% bone volume) than other groups at this time. At 12 weeks, dual release and BMP-2 groups exhibited significantly higher bone formation (39.7+/-14.1% and 37.4+/-18.8% bone volume, respectively) than either the VEGF group or blank scaffolds (6.3+/-4.8% and 7.8+/-7.1% bone volume, respectively). This work indicates a synergistic effect of the dual delivery of VEGF and BMP-2 on bone formation at 4 weeks and suggests an interplay between these growth factors for early bone regeneration. For the doses investigated, the results show that the addition of VEGF does not affect the amount of bone formation achieved by BMP-2 at 12 weeks; however, they also indicate that delivery of both growth factors may enhance bone bridging and union of the critical size defect compared to delivery of BMP-2 alone.     

Strut-autografting with and without osteogenic protein-1 : a preliminary study of a canine femoral head defect model.

BACKGROUND: Osteonecrosis of the femoral head frequently leads to collapse of the articular cartilage and to disabling osteoarthritis, which ultimately may necessitate joint arthroplasty. One treatment method that has had moderate success is the so-called trapdoor approach, which involves excavation of diseased (necrotic) bone followed by bone-grafting. Augmentation of this procedure with various growth and differentiation factors may improve the outcome. We developed a canine model that mimics the clinical situation with trapdoor bone-grafting. The objective of this study was to evaluate the effect of the addition of osteogenic protein-1 on healing following the trapdoor procedure with strut-autografting. METHODS: Thirty-four skeletally mature dogs were used in the experiment. After capsulotomy, a trapdoor was created in the anterolateral surface of the femoral head and a 2-cm-diameter subchondral area of bone was removed. In the phase-I experiments, seven dogs had no treatment of the defect (Group I) and nine dogs were treated with strut-grafting (Group II). In phase II, the procedure was modified by collapsing the trapdoor into the created defect intraoperatively in eighteen dogs, which were divided into three equal groups: six untreated defects were left collapsed (Group III), six were treated with bone graft (Group IV), and six were treated with bone graft augmented with osteogenic protein-1 (Group V). RESULTS: Three of the seven femoral heads in Group I (untreated defect) and one of the nine heads in Group II (grafting without collapsing of the trapdoor) had evidence of cartilage collapse. Inspection of sagittal slices and radiographs revealed an unfilled residual defect in all Group-I heads, whereas all Group-II heads were well healed. The mean normalized stiffness value was significantly larger in Group II than it was in Group I. On visual inspection, depression was noted in all of the femoral heads in Group III (untreated defect; trapdoor left collapsed). In both Group IV and Group V (grafting without and with osteogenic protein-1), the trapdoor cartilage appeared to be essentially normal. Groups IV and V had more radiographic healing than did Group III. The defects in Group V (grafting with osteogenic protein-1) healed faster radiographically than did those in Group IV (grafting without osteogenic protein-1). CONCLUSIONS: Moderate-to-excellent healing was seen both radiographically and biomechanically by four months in the groups treated with grafting, with and without osteogenic protein-1, whereas untreated defects did not heal. Clinical Relevance: Symptomatic osteonecrosis of the femoral head is a clinical challenge. The animal model in the current study is a useful tool for the evaluation of methods to treat osteonecrosis of the femoral head. Studies investigating additional time-periods between implantation of osteogenic protein-1 and assessment of results as well as different doses of osteogenic protein-1 are warranted.     

Bone formation following OP-1 implantation is improved by addition of autogenous bone marrow cells in a canine femur defect model.

Osteogenic Protein-1 (OP-1, BMP-7) acts locally on connective tissue progenitors (CTPs) to induce bone formation. The response to OP-1 and similar agents is potentially limited by the number of local CTPs. This study tested the hypothesis that supplementing local CTPs using autogenous bone marrow will enhance bone formation at an OP-1 implant. Four 1.0-cm diameter unicortical cylindrical defects in the left proximal femur were grafted in each of seven dogs. Radial ingrowth of new bone formation was assessed at 4 weeks using micro CT. The OP-1 (3.5 mg rhOP-1 in 1 g bovine collagen I matrix) was implanted in each site combined with either clotted blood or aspirated bone marrow (BM). Bone formation was increased in the group augmented with transplanted marrow. These data suggest that increasing the local population of cells and CTPs using aspirated bone marrow can enhance the performance of OP-1, but may not eliminate the effects of site variation on the response to OP-1 and similar agents. The canine multiple femoral defect model defined in this study is well suited to quantitatively evaluate strategies for augmenting bone repair using local cell targeting and cell transplantation strategies.     

Osteogenic protein-1 induced bone formation in an infected segmental defect in the rat femur.

The goal of this study was to use a segmental defect model in the rat femur to determine if osteogenic protein-1 (OP-1) is capable of inducing bone formation in the presence of bacterial contamination. A 6 mm segmental defect was surgically created and stabilized with a polyacetyl plate and Kirschner wires in one femur in each of 126 Sprague-Dawley rats. The animals were divided into eight groups in which the defect was either left untreated, or subjected to various combinations of OP-1 (11 or 50 microg), lyophilized bovine type I collagen (carrier for the OP-1), and 10(5) colony-forming units of Staphylococcus aureus. The animals were euthanized at either 2, 4, or 9 weeks. Quantitative radiographic and histologic analyses were performed on the harvested tissue. The initial contamination progressed to infection in all animals receiving bacteria, as determined by qualitative bacteriology. There was very little, if any, bone formation in the untreated defects, and in the contaminated defects with or without collagen carrier. Bone formation was significantly greater in contaminated defects with either dose of OP-1, compared with contaminated defects without OP-1. The 50 microg dose of OP-1 induced significantly more bone formation than the 11 microg dose, both with and without bacteria. This investigation has demonstrated that OP-1 maintains its osteoinductive capability in a contaminated segmental defect. OP-1 may potentially be used in the clinical management of contaminated fractures.     

Wnt3a联合骨形态发生蛋白2对骨髓间充质干细胞增殖及成骨分化的影响

目的 观察经典Wnt/β-catenin以及骨形态发生蛋白(BMP)信号通路对大鼠骨髓来源的间充质干细胞(BMSCs)体外增殖以及向成骨方向分化的影响.方法 应用密度梯度离心联合贴壁筛选法分离培养骨髓间充质干细胞并分4组:对照组、Wnt组、BMP组、Wnt3a和BMP-2联合组,在不同时间点用细胞计数试剂盒-8(CCK-8)法检测细胞增殖活性,碱性磷酸酶(ALP)活性定量测定、Yon Kossa染色观察细胞向成骨分化和基质矿化程度.逆转录-聚合酶链反应(RT-PCR)检测成骨特异性标志物表达.结果 培养第7天,CCK-8测吸光度值联合组为0.845,Wnt组为0.738,明显高于对照组0.409(P＜0.05).培养第6天和第12天,ALP活性吸光度值联合组为63.8和144.3,BMP-2组为40.8和104.1,均明显高于对照组7.3和18.9(P ＜0.05).培养14 d后,联合组骨钙素mRNA表达量高于对照组,而Runx2及Osterix表达量与其他组比较增高不显著.培养3周后,Yon Kossa染色显示联合组钙结节数量及大小均高于对照组.结论 Wnt3a因子和骨形态发生蛋白-2联合诱导能有效地促进骨髓间充质干细胞增殖及向成骨方向分化.     

Evaluation of osteogenic cell differentiation in response to bone morphogenetic protein or demineralized bone matrix in a critical sized defect model using GFP reporter mice

We evaluated the osteoprogenitor response to rhBMP‐2 and DBM in a transgenic mouse critical sized defect. The mice expressed Col3.6GFPtopaz (a pre‐osteoblastic marker), Col2.3GFPemerald (an osteoblastic marker) and α‐smooth muscle actin (α‐SMA‐Cherry, a pericyte/myofibroblast marker). We assessed defect healing at various time points using radiographs, frozen, and conventional histologic analyses. GFP signal in regions of interest corresponding to the areas of new bone formation was quantified using a novel computer assisted algorithm. All defects treated with rhBMP‐2 healed. In contrast, the majority of the defects in the DBM (27/30) and control (28/30) groups did not heal. Quantitation of pre‐osteoblasts demonstrated a maximal response (% GFP+ cells/TV) in the Col3.6GFPtopaz mice at day 7 (7.2% ± 6.0, p < 0.05 compared to days 14, 21, 28, and 56). The maximal response of the Col2.3GFP cells was seen at days 14 (8.04% ± 5.0) and 21 (8.31% ± 4.32), p < 0.05. In contrast, DBM and control groups showed a limited osteogenic response at all time points. In conclusion, we demonstrated that the BMP and DBM induce vastly different osteogenic responses which should influence their clinical application as bone graft substitutes. © 2014 Orthopaedic Research Society. Published by Wiley Periodicals, Inc. J Orthop Res 32:1120–1128, 2014.     

Treatment of a congenital pseudarthrosis of the tibia by osteogenic protein-1 (bone morphogenetic protein-7): a case report

Congenital pseudarthrosis of the tibia remains one of the most difficult orthopaedic problems to treat. Before the use of a recombinant bone morphogenetic protein (bone morphogenetic protein-7; osteogenic protein-1) the patient with congenital pseudarthrosis of the tibia in this report had had 12 unsuccessful surgeries. Five months after radical resection of sclerotic tibial segments, Ilizarov fixation and administration of osteogenic protein-1 osteogenic device, the congenital pseudarthrosis of the tibia healed; at 45 months the tibia increased in size and the patient was fully weight bearing.     

Healing of segmental bone defects with granular porous hydroxyapatite augmented with recombinant human osteogenic protein-1 or autologous bone marrow.

Hydroxyapatite is a synthetic bone graft, which is used for the treatment of bone defects and nonunions. However, it is a rather inert material with no or little intrinsic osteoinductive activity. Recombinant human osteogenic protein-1 (rhOP-1) is a very potent biological agent, that enhances osteogenesis during bone repair. Bone marrow contains mesenchymal stem cells, which are capable of new bone formation. Biosynthetic bone grafts were created by the addition of rhOP-1 or bone marrow to granular porous hydroxyapatite. The performance of these grafts was tested in a sheep model and compared to the results of autograft, which is clinically the standard treatment of bone defects and nonunions. A 3 cm segmental bone defect was made in the tibia and fixed with an interlocking intramedullary nail. There were five treatment groups: no implant (n=6), autograft (n=8), hydroxyapatite alone (n=8), hydroxyapatite loaded with rhOP-1 (n=8), and hydroxyapatite loaded with autologous bone marrow (n=8). At 12 weeks, healing of the defect was evaluated with radiographs, a torsional test to failure, and histological examination of longitudinal sections through the defect. Torsional strength and stiffness of the healing tibiae were about two to three times higher for autograft and hydroxyapatite plus rhOP-1 or bone marrow compared to hydroxyapatite alone and empty defects. The mean values of both combination groups were comparable to those of autograft. There were more unions in defects with hydroxyapatite plus rhOP-1 than in defects with hydroxyapatite alone. Although the differences were not significant, histological examination revealed that there was more often bony bridging of the defect in both combination groups and the autograft group than in the group with hydroxyapatite alone. Healing of bone defects, treated with porous hydroxyapatite, can be enhanced by the addition of rhOP-1 or autologous bone marrow. The results of these composite biosynthetic grafts are equivalent to those of autograft.     

脂多糖对小鼠胫骨骨髓去除后早期骨形成的影响

目的 利用小鼠胫骨骨髓去除模型（bone marrow ablation, BMX）,观察脂多糖（lipopolysaccharide,LPS）对BMX后早期骨形成的影响,并初步探讨其机制。方法 建立小鼠胫骨BMX模型后,分为对照组（PBS处理）和LPS处理组,每组6只。BMX后1周,通过组织病理切片染色和组织形态计量观察胫骨骨髓腔中骨形成情况。通过免疫组化检测BMX后4 d骨髓腔中增殖细胞核抗原（PCNA）表达情况,BMX后1周β-catenin和Runx2表达情况。采用RT-PCR检测BMX后1周新生骨组织中成骨分化相关基因和Wnt/β-catenin信号通路下游靶基因表达水平。结果 BMX后1周,LPS处理组胫骨骨髓腔中新生骨组织较对照组减少,LPS处理组新生骨组织BV/TV较对照组显著减少;BMX后4 d,LPS处理组PCNA阳性细胞较对照组明显减少;BMX后1周,LPS处理组新生骨组织中Runx2阳性细胞和β-catenin阳性细胞较对照组明显减少;BMX后1周,LPS处理组新生骨组织中成骨分化相关基因和Wnt/β-catenin信号通路下游靶基因表达水平较对照组明显降低。结论 LPS可以抑制间充质细胞增殖,抑制成骨分化,导致BMX后早期骨形成减少,Wnt/β-catenin信号通路可能参与了LPS抑制BMX后早期骨形成的过程。     

急性骨短缩-延长加骨髓移植治疗胫骨骨折并骨缺损

随着高能量损伤的增多，胫骨开放性骨折并骨缺损病例逐年增多，由于胫骨解剖的特殊性，其治疗颇为棘手。2000年3月-2004年3月，应用半环槽外固定架采用急性骨短缩-延长加骨髓移植方法治疗胫骨骨折并骨缺损14例，取得了较满意的效果，现报告如下。