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.     

Effect of cell‐based VEGF gene therapy on healing of a segmental bone defect

Fracture healing requires coordinated coupling between osteogenesis and angiogenesis in which vascular endothelial growth factor (VEGF) plays a key role. We hypothesized that targeted over‐expression of angiogenic and osteogenic factors within the fracture would promote bone healing by inducing development of new blood vessels and stimulating/affecting proliferation, survival, and activity of skeletal cells. Using a cell‐based method of gene transfer, without viral vector, 5.0 × 10⁶ fibroblasts transfected with VEGF were delivered to a 10‐mm bone defect in rabbit tibiae (Group 1) (n = 9); control groups were treated with fibroblasts (Group 2) (n = 7), or saline (Group 3) (n = 7) only. After 12 weeks, eight tibial fractures healed in Group 1, compared to four each in Groups 2 and 3. In Group 1, ossification was seen across the entire defect; in Groups 2 and 3, the defects were fibrous and sparsely ossified. Group 1 had more positively stained (CD31) vessels than Groups 2 and 3. MicroCT 3‐D showed complete bridging of the new bone for Group 1, but incomplete healing for Groups 2 and 3. MicroCT bone structural parameters showed significant differences between VEGF treatment and control groups (p < 0.05). These results indicate that the cell‐based VEGF gene therapy has significant angiogenic and osteogenic effects to enhance healing of a segmental defect in the long bone of rabbits. © 2008 Orthopaedic Research Society. Published by Wiley Periodicals, Inc. J Orthop Res 27:8–14, 2009     

冻干同种异体骨与冻干异种骨移植治疗骨缺损的比较实验研究

目的：对比观察冻干同种异体骨和冻干异种骨骨移植治疗骨缺损的效果。方法：48只中国大白兔一侧桡骨造成1cm骨缺损，随机分为同种异体骨组和异种骨组，每组24只，分别植入两种移植骨，术后4、8、12周分批取材，进行X线片和组织学检查，然后做对比分析。结果：术后4周异种骨组的X线片，Gary X线评分与同种异体骨组有统计学差异，而术后8周同种异体骨组组织学检查以及组织学评分与异种骨组有统计学差异，在其他时期两组比较无统计学差异。结论：冻干异种骨具有很好的成骨效果，可以作为修复骨缺损的移植材料。     

BMP induced inflammation: A comparison of rhBMP‐7 and rhBMP‐2

Concern has been raised because of reports of inflammatory swelling following the use of recombinant human bone morphogenetic protein‐2 (rhBMP‐2) and recombinant human bone morphogenetic protein‐7 (rhBMP‐7). The purpose of this study is to compare the inflammatory action of rhBMP‐7 with those of rhBMP‐2. ELISA assays (IL‐6, TNF‐α) were used to measure the cytokine response to different concentrations of rhBMP‐7 and ‐2. Recombinant human BMP‐7 was absorbed into absorbable collagen sponges and different amounts were implanted either subcutaneously (SC) or intramuscularly (IM) into the backs of rats. Using MRI and MIPAV software, we measured the degree of soft tissue edema at 3 h and at 2, 4, and 7 days postoperatively. After sacrificing rats on day 7 the inflammatory zone and mass were measured and the tissue examined histologically. Soft tissue edema after rhBMP‐7 and rhBMP‐2 implantation was dose‐dependent and peaked at 3 h for the subcutaneous implants and at 2 days for the intramuscular implants. RhBMP‐7 was associated with a significantly smaller soft tissue edema volume than was rhBMP‐2 only at the highest dose (20 µg/ml). Both rhBMP‐2 and rhBMP‐7 triggered dose‐dependent inflammatory reactions. Compared to rhBMP‐2, rhBMP‐7 is associated with somewhat smaller soft tissue edema volumes. Although rhBMP‐7 is associated with an inflammatory reaction leading to soft tissue edema, at high doses this response is significantly less than that seen with rhBMP‐2. Our animal model can be used to test materials that could ameliorate this reaction. © 2012 Orthopaedic Research Society. Published by Wiley Periodicals, Inc. J Orthop Res 30:1985–1994, 2012     

Repair of rat cranial bone defects with nHAC/PLLA and BMP‐2‐related peptide or rhBMP‐2

An ideal artificial substitute has good biocompatibility properties and is able to provide for rapid bone formation. Bone morphogenetic protein‐2 (BMP‐2) is considered as one of the most important growth factors for bone regeneration. In this study, a synthetic BMP‐2‐related peptide (designated P24) corresponding to residues of the knuckle epitope of BMP‐2 was introduced into a bioactive scaffold based on nano‐hydroxyapatite/collagen/poly(L ‐lactic acid) (nHAC/PLLA); its in vitro release kinetics was then measured. A 5 mm diameter cranial bone defect was created in the calvariae of 30 rats and randomly implanted with three groups of biomaterials: Group A (nHAC/PLLA alone); Group B (P24/nHAC/PLLA composite); and Group C (recombinant human BMP‐2 (rhBMP‐2)/nHAC/PLLA composite). The P24/nHAC/PLLA implants significantly stimulated bone growth similarly to the rhBMP‐2/nHAC/PLLA implants based on the radiographic and three‐dimensional CT evaluation and histological examination, thereby confirming the enhanced bone healing rate of these compounds compared with the stand‐alone nHAC/PLLA scaffold material. The osteoinductive ability of 3 mg P24 was similar to that of 1 µg rhBMP‐2. P24/nHAC/PLLA is a promising scaffold biomaterial for bone tissue regeneration. © 2011 Orthopaedic Research Society Published by Wiley Periodicals, Inc. J Orthop Res 29:1745–1752, 2011     

Characterization of a chronic infection in an internally-stabilized segmental defect in the rat femur.

The aim of this study was to characterize a new model of chronic osteomyelitis with clinically relevant features. A segmental defect of critical size was surgically created in the rat femur, stabilized with a polyacetyl plate and Kirschner wires, and contaminated with bacteria. The animals were allowed to recover while the contamination progressed to a chronic infection. At a later point in time, the defect was surgically débrided without removing the implant. Further treatments of interest, such as antibiotic therapy or application of an osteogenic agent, could be introduced at this time. To implement this model, an initial experiment was performed to determine the bacterial inoculum and time from contamination that would reliably result in an infected defect without causing excessive bone damage by the time débridement surgery was performed. The number of recovered bacteria, degree of radiographic bony lysis, and torsional stiffness of the defect fixation were measured in 192 rats as a function of 4 inocula of Staphylococcus aureus (10(3), 10(4), 10(5) or 10(6) CFUs) and 4 times from contamination (1, 2, 3 or 4 weeks). A 10(4) CFU inoculum over 2 weeks was found to consistently create an infection without severe lysis and loss of fixation stability. Based on these values, a second experiment was performed in 96 rats to characterize the débrided defect over time (2, 4, 8 and 12 weeks after débridement), with and without 4 weeks of the antibiotic ceftriaxone, in terms of the same outcome variables. Infection was persistent in all animals in spite of débridement and antibiotic therapy. Antibiotic therapy did not reduce the degree of bony lysis. Compared with animals not given antibiotic, bacterial counts significantly decreased during the period of antibiotic therapy, but then rebounded to significantly higher levels at 12 weeks. This model allows us to perform further studies on differing regimens of antibiotic therapy and their relationship to surgical débridement, and on the efficacy of osteogenic agents in the presence of infection.     

异种脱蛋白骨管移植修复大段骨缺损的力学评估

目的研究异种脱蛋白骨管移植修复大段骨缺损的生物力学变化。方法建立山羊双侧胫骨大段骨缺损模型,36只山羊(72只后肢)随机分为2组,实验组:异种脱蛋白骨管;对照组:异种脱蛋白骨粒;两组均在移植骨中添加了骨形态发生蛋白(BMP),并采用骨板、钢板双重固定修复骨缺损。术后5、10、15周对羊胫骨行影像学观察和生物力学的变化。结果影像学显示实验组在骨缺损修复及成骨方面较对照组高。生物力学测试结果表明,术后5、10、15周时实验组力学强度较对照组明显增高,差异有统计学意义(P<0.05);术后15周,实验组的生物力学强度与正常胫骨已无差异。结论应用异种脱蛋白骨管支撑复合BMP修复大段骨缺损,能够有效加强移植骨修复负重骨缺损区的力学结构。     

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.     

Augmentation of fracture healing by hydroxyapatite/collagen paste and bone morphogenetic protein‐2 evaluated using a rat femur osteotomy model

In fracture treatment, biological bone union generally depends on the bone's natural fracture healing capacity, even in surgically treated cases. Hydroxyapatite/collagen composite (HAp/Col) has high osteoconductivity and stimulates osteogenic progenitors. Furthermore, it has the potent capacity to adsorb bone morphogenetic proteins (BMPs). In this study, we prepared an injectable HAp/Col paste and evaluated its augmentation of bone union. Furthermore, the effect of HAp/Col paste combined with BMP‐2 was also evaluated. We used a rat femur osteotomy model with a defect size of 1 mm. Male Wistar rats were assigned to one of the following four groups; a control group without any implant, a HAp/Col implant group, a group that received an absorbable collagen sponge (ACS) implant impregnated with BMP‐2 (1 μg), and a group that received a HAp/Col implant impregnated with BMP‐2 implant. Micro‐CT analysis, three‐point bending tests, and histological evaluation were performed. Bone union was achieved in two of eight cases in the HAp/Col group, five of eight cases in the ACS + BMP‐2 group, and all cases in the HAp/Col + BMP‐2 group at 8 weeks post‐surgery. The control group did not achieve bone union. In addition, in the HAp/Col + BMP‐2 group, the biomechanical strength of the fused femurs was comparable to that of the contralateral intact femur; the ratio of the mechanical load at the breaking point of the osteotomy side relative to that of the contralateral side was 1.00 ± 0.151 (SD). These results indicate that HAp/Col paste with or without BMP‐2 augments bone union. © 2017 Orthopaedic Research Society. Published by Wiley Periodicals, Inc. J Orthop Res 36:129–137, 2018.     

Recombinant human bone morphogenetic protein‐type 2 (rhBMP‐2) enhances local bone formation in the lumbar spine of osteoporotic sheep

The failure of orthopedic implants in osteoporotic patients is attributed to the lack of sufficient bone stock and regenerative capacity but most treatments for osteoporosis fail to address this issue. rhBMP‐2 is known to promote bone formation under normal conditions but has not been used clinically in the osteoporotic condition. Osteoporosis was induced in 19 ewes using ovariectomy, low calcium diet, and steroid injection. After induction, the steroid was withdrawn and pellets containing inert carrier with rhBMP‐2 in either slow or fast‐release formulation were implanted into the lumbar vertebrae of each animal. After 2, 3, and 6 months the spines were harvested and assessed for changes in BMD and histomorphometric indices. BMD did not change after cessation of steroid treatment. After 2 months BV/TV increased in the vicinity of the pellets containing the fast‐release rhBMP‐2 and was sustained for the duration of the study. Focal voids surrounding all implants, particularly the slow‐release formulation, were observed initially but resolved with time. Increased BV/TV adjacent to rhBMP‐2 pellets suggests it could be used for localized treatment of osteoporosis. Refinement of the delivery system and supplementary treatments may be necessary to overcome the initial catabolic effects of rhBMP‐2. © 2013 Orthopaedic Research Society. Published by Wiley Periodicals, Inc. J Orthop Res 31:1390–1397, 2013.     

Estimation of defect volume in segmental defects of the tibia and femur

BACKGROUND: With the advent of modern limb salvage techniques, segmental bone loss in the lower extremity has become more common. METHODS: To aid preoperative planning when dealing with segmental bone loss in the femur and tibia, we performed a cadaveric study to estimate the volume of autogenous or allograft material required to fill defects located in various areas of the bones. RESULTS: The greatest volume was generally required in metaphyseal defects, with an average of 12 cc/cm in the distal femur and proximal tibia, 11 cc/cm in the proximal femur, and 6 cc/cm in the distal tibia. Diaphyseal defects were found to have the least variability with regard to the volume of graft material required for different specimens. Femoral diaphyseal defects required 7 cc/cm and tibial diaphyseal defects required 5 cc/cm. A slightly larger volume of allograft material was needed to fill all defects compared with autograft. CONCLUSION: This method allows one to estimate the amount of graft required for a defect of the femur and the tibia.     

A Comparative Study of Incorporation Rates between Non-xenograft and Bovine-based Structural Bone Graft in Foot and Ankle Surgery

Several types of structural bone grafts are available, each with different characteristics. Our previous study showed poor performance with the bovine-based xenograft in foot and ankle applications. In the present study, we compared the incorporation rates of non-xenografts, including allografts and autografts, with the bovine-based xenograft to determine whether the poor result was unique to the graft type and not institutional. The proportion of incorporated grafts at 12, 24, 36, and 48 weeks was compared between the nonxenograft and xenograft groups. Furthermore, Cox regression analysis was used to evaluate the factors associated with nonunion. A total of 61 patients (23 women and 38 men) with a median age of 24.0 years were enrolled. The factors associated with slower incorporation included side of operation (p = .033), tobacco use (p = .010), and graft type (p = .001). At 48 weeks, 5% of the nonxenografts and 58% of the xenografts were not incorporated. The median incorporation time for the non-xenograft and xenograft group was 16 and 57 weeks, respectively. We have concluded that it is not advisable to use a bovine-based bone xenograft in foot and ankle surgery.     

Enhanced effects of BMP‐binding peptide combined with recombinant human BMP‐2 on the healing of a rodent segmental femoral defect

BMP‐binding peptide (BBP) enhances the osteogenic activity of recombinant human bone morphogenetic protein‐2 (rhBMP‐2), but the mechanism underlying the enhancement remains unclear. We aimed to elucidate the potential enhanced efficacy of BBP using critical‐sized segmental femoral bone defects in rats. Seventy defects in seven groups of rats were filled with various amounts (0, 2, 5, and 10 µg) of rhBMP‐2 with or without 1000 µg BBP. Radiographs were obtained after 4 and 8 weeks. The animals were euthanized at 8 weeks, and femoral specimens were assessed manually, evaluated for bone volume using microcomputed tomography, and subjected to histological or biomechanical analysis. Although 10 µg rhBMP‐2 yielded consistent results in terms of bone healing and quality of bone repair across the segmental defect, lower doses of rhBMP‐2 failed to induce satisfactory bone healing. However, the combined administration of lower doses of rhBMP‐2 and BBP induced the formation of significantly large amounts of bone. Our results suggest that the combined administration of rhBMP‐2 and BBP facilitates bone healing and has potential clinical applications. © 2009 Orthopaedic Research Society. Published by Wiley Periodicals, Inc. J Orthop Res 28:258–264, 2010     

No effect of dynamic loading on bone graft healing in femoral segmental defect reconstructions in the goat

We studied if a static or dynamic mode of nail fixation influenced the healing of segmental defect reconstructions in long bones. Defects in the femur of goats were reconstructed using a cage filled with firmly impacted morsellised allograft mixed with a hydroxyapatite paste (Ostim). All reconstructions were stabilised with an intramedullary nail. In one group (n = 6) the intramedullary nail was statically locked, in the second group (n = 6) a dynamic mode of nail fixation was applied. We hypothesised that dynamisation of the nail would load, and by that stimulate the healing of the bone graft. Mechanical torsion strength of the reconstructions of the femur with the static and dynamic mode of nail fixation appeared to be 74.8 ± 17.5% and 73.0 ± 13.4%, respectively as compared with the contralateral femurs after 6 months. In all reconstructions, the grafts united radiographically and histologically to the host bone, and remodelled into a new vital bone structure. No large differences were found between newly formed bone areas inside and outside the mesh of the two groups. The area of callus outside the mesh in the dynamic mode of fixation group was smaller (p = 0.042), whilst the percentage of bone outside the mesh was larger (p = 0.049), as compared to the static mode of fixation group. The data suggest that healing of these defects with impacted morsellised graft in a cage is not significantly influenced by the mode of fixation of the nail in this model.     

Osteogenic and chondrogenic potential of biomembrane cells from the PMMA‐segmental defect rat model

A layer of cells (the “biomembrane”) has been identified in large segmental defects between bone and surgically placed methacrylate spacers or antibiotic‐impregnated cement beads. We hypothesize that this contains a pluripotent stem cell population with potential valuable applications in orthopedic tissue engineering. Objectives using biomembranes harvested from rat segmental defects were to: (1) Culture biomembrane cells in specialized media to direct progenitor cells along bone or cartilage cell differentiation lineages; (2) evaluate harvested biomembranes for mesenchymal stem cell markers, and (3) define relevant gene expression patterns in harvested biomembranes using microarray analysis. Culture in osteogenic media produced mineralized nodules; culture in chondrogenic media produced masses containing chondroitin sulfate/sulfated proteoglycans. Molecular analysis of biomembrane cells versus control periosteum showed significant upregulation of key genes functioning in mesenchymal stem cell differentiation, development, maintenance, and proliferation. Results identified significant upregulation of WNT receptor signaling pathway genes and significant upregulation of BMP signaling pathway genes. Findings confirm that the biomembrane has a pluripotent stem cell population. The ability to heal large bone defects is clinically challenging, and novel tissue engineering uses of the biomembrane hold great promise in treating non‐unions, open fractures with large bone loss and/or infections, and defects associated with tumor resection. © 2012 Orthopaedic Research Society. Published by Wiley Periodicals, Inc. J Orthop Res 30:1198–1212, 2012     

Union of a chronically infected internally stabilized segmental defect in the rat femur after debridement and application of rhBMP-2 and systemic antibiotic

OBJECTIVES: The goal of this study was to determine whether recombinant human bone morphogenetic protein-2 (rhBMP-2) would induce new bone formation in an internally stabilized segmental defect with a chronic bacterial infection in the rat femur and whether treatment with systemic antibiotic would enhance this effect. METHODS: A 6-mm unilateral femoral segmental defect was surgically created in 120 Sprague-Dawley rats, internally stabilized with a polyacetyl plate and 6 Kirschner wires, and contaminated with 10(4) colony-forming units of Staphylococcus aureus. After 2 weeks, all defects were surgically debrided and implanted with 0, 20, or 200 microg of rhBMP-2 in a type 1 bovine collagen sponge. Half of the animals in each treatment group received 4 weeks of systemic antibiotic, and half did not. Animals were euthanized at 4 or 12 weeks after debridement. Bone formation within and adjacent to the defect was assessed using microcomputed tomography, torsional failure testing and undecalcified histology. RESULTS: No substantial callus formed in the infected defects without rhBMP-2. Significantly more mineralized callus was induced with the higher dose of rhBMP-2 than with the lower dose (P = 0.001), with systemic antibiotic therapy than without (P < 0.001), and at 12 weeks after debridement compared with 4 weeks (P < 0.001). CONCLUSIONS: Recombinant human bone morphogenetic protein-2 maintained its osteoinductive capability in the presence of a chronic infection, and this property was enhanced by systemic antibiotic. This study presents an intervention that may potentially 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 infection.     

Differences of bone healing in metaphyseal defect fractures between osteoporotic and physiological bone in rats

Discrepancies in bone healing between osteoporotic and non-osteoporotic bone remain uncertain. The focus of the current work is to evaluate potential healing discrepancies in a metaphyseal defect model in rat femora. Female Sprague-Dawley rats were either ovariectomized (OVX, n = 14) and combined with a calcium-, phosphorus- and vitamin D3-, soy- and phytoestrogen-free diet or received SHAM operation with standard diet rat (SHAM, n = 14). Three months post-ovariectomy, DEXA measurement showed a reduction of bone mineral density reflecting an osteoporotic bone status in OVX rats. Rats then underwent a 3 mm wedge-shaped osteotomy at the distal metaphyseal area of the left femur stabilized with a T-shaped mini-plate and allowed to heal for 6 weeks. Biomechanical competence by means of a non-destructive three-point bending test showed significant lower flexural rigidity in the OVX rats at 3 mm lever span compared to SHAM animals (p = 0.048) but no differences at 10 mm lever span. Microcomputer tomography (μCT) showed bridging cortices and consolidation of the defect in both groups, however, no measurable differences were found in either total ossified tissue or vascular volume fraction. Furthermore, histology showed healing discrepancies that were characterized by cartilaginous remnant and more unmineralized tissue presence in the OVX rats compared to more mature consolidation appearance in the SHAM group. In summary, bone defect healing in metaphyseal bone slightly differs between osteoporotic and non-osteoporotic bone in the current 3 mm defect model in both 3 mm lever span biomechanical testing and histology.