Bone morphogenetic protein-2b stimulation of growth and osteogenic phenotypes in rat osteoblast-like cells: comparison with TGF-beta 1.

The biologic effects of recombinant human bone morphogenetic protein-2b (BMP-2b = BMP-4) were studied and compared with transforming growth factor-beta 1 (TGF-beta 1) in fetal rat osteoblast-like (ROB) cells. Similar to the effects of TGF-beta 1, BMP-2b stimulated DNA and collagen synthesis as well as protein accumulation. Unlike TGF-beta 1, which inhibited alkaline phosphatase activity, BMP-2b enhanced enzyme activity eight-to ninefold over the control level. The present study demonstrates direct actions of BMP-2b on bone-associated cells to stimulate osteogenic phenotypes in vitro and provides a cellular mechanism for the induction of bone formation by BMP-2b in vivo.     

Bone marrow and recombinant human bone morphogenetic protein-2 in osseous repair

Bone marrow stem cells and recombinant human bone morphogenetic protein-2 each has the capacity to repair osseous defects. Recombinant human bone morphogenetic proteins require the presence of progenitor cells to function. It is hypothesized that a composite graft of recombinant human bone morphogenetic protein-2 and marrow would be synergistic and could result in superior grafting to autogenous bone graft. Syngeneic Lewis rats with a 5-mm critical sized femoral defect were grafted with recombinant human bone morphogenetic protein-2 and marrow, recombinant human bone morphogenetic protein-2, marrow, syngeneic cancellous bone graft, or carrier alone (control). Serial radiographs (3, 6, 9, 12 weeks) and torque testing (12 weeks) were performed. Bone formation and union were determined. The recombinant human bone morphogenetic protein-2 and marrow composite grafts achieved 100% union at 6 weeks. Recombinant human bone morphogenetic protein alone achieved 80% union by week 12. Both groups yielded a higher union rate and superior mechanical properties than did either syngeneic bone graft (38%) or marrow (47%) alone. The superior performance of recombinant human bone morphogenetic protein-2 combined with bone marrow in comparison with each component alone strongly supports a biologic synergism. This experimentation shows the clinical importance of establishing operative site proximity for the osteoinductive factors and responding progenitor cells.     

Local bone formation by injection of recombinant human bone morphogenetic protein-2 contained in polymer carriers

The regenerating potential of human bone is limited. The repair of large bone defects often associated with bone tumor resections is not observed, and nonunion or delayed union of bone is a serious problem for fracture treatment. In these cases, autogeneic or allogeneic bone grafting has been routinely indicated, but these approaches require invasive surgical procedures. An alternative approach described in this paper involves the injection of bone morphogenetic proteins (BMPs) in a polymeric delivery system. We demonstrate that synthetic biodegradable polymers, poly-D,L-lactic acid-polyethylene glycol (PLA-PEG) block copolymers, which exhibit an exquisite temperature-dependent liquid-semisolid transition, work well as an injectable delivery system for recombinant human (rh) BMP-2. The thermosensitive property of the PLA-PEG/rhBMP-2 composite is permissive to percutaneous injection when heated. The fluidity of this composite decreases as it cools down to body temperature and the resultant semisolid form provides a scaffold for bone formation through the gradual local release of the rhBMP-2. This new type of injectable osteoinductive material will enable a less invasive approach to surgeries involving the restoration or repair of bone tissues.     

基因治疗对兔下颌骨牵引过程中骨形成蛋白-2表达的影响

目的 探索电穿孔介导的基因治疗对兔下颌骨牵引成骨过程中骨形成蛋白-2(bone morphogenetic protein-2,BMP2)表达的影响.方法 新西兰大白兔双侧下颌骨截骨,术后3d开始牵引,连续牵引7d后,将实验动物分为A、B、C、D、E5组,A、B、C组分别在牵引区注射2 μg(0.1 μg/μl)重组质粒pIRES-hVEGF165-hBMP2、pIRES-hBMP2、pIRES-hVEGF165.D组与E组分别注射相同剂量的空质粒(pIRES)和生理盐水(NS).各组分别于固定期第7、14、28天处死动物,取材行免疫组织化学检测BMP2的表达情况,并利用病理图像分析系统进行分析.结果 BMP2主要在肉芽组织中的炎细胞及新生幼稚骨小梁表面的细胞组织中表达.固定7d时表达最强烈,各时点基因治疗组明显强于对照组.结论 电脉冲介导的基因治疗能使BMP2在牵引区的表达增强和表达时限延长并促进细胞的分裂增殖与分化,促进牵引区细胞基质的形成和新骨生成.     

Bone formation in the presence of platelet-rich plasma vs. bone morphogenetic protein-7

Growth factors contained in platelet-rich plasma (PRP) have recently been proposed to enhance maturation of bone grafts and, in combination with anorganic bovine bone, to support repair in the treatment of small bone defects in maxillofacial surgery. Bone morphogenetic proteins (BMP) carried in a matrix may be able to replace the autologous bone graft in the treatment of critical size defects. However, no studies have compared the bone stimulating capacity of PRP and BMP. Likewise there is no data comparing the effects of PRP in either an autologous bone graft or in anorganic bovine bone. We augmented the mandible of Wistar rats (n = 28) on both sides with either anorganic bovine bone (Bio-Oss) or autologous rib bone. On the test side we applied either 20 microl of autologous PRP or 10 microl of rhBMP-7 (4 groups, n = 7). In addition, bone induction was evaluated in an extraskeletal site (n = 14). A polychrome sequential labeling was performed. The animals were sacrificed by intra-vital perfusion on day 50. Undecalcified ground sections were evaluated by microradiography, digitized histomorphometry and under fluorescent light. The qualitative analysis of fluorochrome labels suggested that PRP and rhBMP-7 accelerated bone growth. However, histomorphometric analysis revealed no significant differences in the area of newly mineralized bone under either the influence of PRP or rhBMP-7 on autologous bone graft. Likewise, the addition of PRP to anorganic bovine bone showed no statistical difference to the control group. The strongest bone stimulating effect was seen for the combination of rhBMP-7 with anorganic bovine bone (p = 0.028). In the extraskeletal model, newly formed bone was evident in the presence of rhBMP-7, but not of PRP. In conclusion, according to the histomorphometry, the addition of platelet-rich plasma failed to enhance bone formation on anorganic bovine bone and on autologous bone grafts.     

RhBMP-2诱导犬气管移植体软骨再生的实验研究

目的 探讨重组人骨形态发生蛋白-2（rhBMP-2）植入犬气管移植体内诱导软骨再生效果。方法 12只犬随机等分两组，分别将rhBMP-2／胶原（collagen）或胶原植入犬气管移植段，进行自体移植，通过HE染色、Masson’s染色、免疫组化检测软骨再生。18只犬随机等分为6组，给予不同浓度的rhBMP-2，利用HPIAS-1000高清晰度彩色病理图像分析仪测定各组新生软骨的面积，确定最佳诱导浓度。12只犬随机等分成2组，分别进行自体、异体移植并植入rhBMP-2，测定各组移植段气管管腔及新生软骨面积。结果 rhBMP-2／胶原植入气管移植段后，可以在植入区及固有软骨的软骨膜处诱导软骨再生，新生软骨面积与对照组差异有统计学意义（P〈0．01）。rhBMP-2剂量与新生软骨面积呈剂量依赖关系，当rhBMP-2浓度为5mg/ml时，其诱导的新生软骨面积最多。在自体移植与异体移植实验中，植入rhBMP-2后，移植段气管管腔的狭窄情况均较对照组得到改善。结论 rhBMP-2可促进气管移植体软骨再生，其诱导作用不仅可以促进植入区的软骨再生，而且通过软骨细胞间的接触促进了软骨膜处的软骨再生。可见rhBMP-2可以促进移植段气管的软骨再生，克服气管移植段的软化狭窄。     

Bone morphogenetic protein-2 compared to autologous iliac crest bone graft in the treatment of long bone nonunion

This retrospective study investigated the effect of recombinant human bone morphogenetic protein-2 (rhBMP-2) mixed with cancellous allograft on fracture healing compared to iliac crest autograft in the treatment of long bone nonunion. Eighty-nine patients with 93 established long bone nonunions treated between January 2002 and June 2004 at a single academic Level I trauma center were evaluated. Patients with clinical and radiographic evidence of failed fracture union underwent nonunion debridement, revision of fixation, and implantation at the nonunion site of either rhBMP-2 or the standard treatment autologous iliac crest bone graft. Union rate, operative time, estimated intraoperative blood loss, hospital length of stay, and postoperative infections were recorded. Nineteen nonunions received rhBMP-2 on a specialized carrier matrix (an absorbable collagen sponge) mixed with cancellous allograft, and 74 nonunions were treated with autologous iliac crest bone graft. There was no statistical difference in the rate of healing between treatment groups (68.4% vs 85.1%, respectively; P=.09). Incidence of postoperative infection was 16.2% after autologous iliac crest bone graft and 5.3% after rhBMP-2/absorbable collagen sponge (P=.22). Iliac crest autograft was associated with longer operative procedures (257.9±93.0 vs 168.9±86.5 minutes; P=.0007) and greater intraoperative blood loss (554.6±447.8 vs 331.6±357.2 mL; P=.01). These outcomes suggest that rhBMP-2 may provide a suitable alternative to autologous iliac bone graft, with the possible advantages of shorter operative time and reduced intraoperative blood loss, and may be considered as part of the orthopedic surgeon's treatment options.     

A novel nano-hydroxyapatite/synthetic polymer/bone morphogenetic protein-2 composite for efficient bone regeneration

BACKGROUNDEfficient bone regeneration using recombinant human bone morphogenetic protein-2 (BMP-2) is needed to reduce side effects caused by high-dose BMP-2 use. The composite material of polylactic acid–polyethene glycol (PLA-PEG) for sustained release and an osteogenic nano-hydroxyapatite (nHAp) can contribute to efficient bone regeneration by BMP-2.STUDY DESIGNAn experimental in vitro and in vivo study.PURPOSEThe objective of this study is to investigate the effectiveness of a novel composite material of PLA-PEG and nHAp as a carrier for BMP-2.METHODSThe release kinetics of BMP-2 from the composites was investigated by ELISA. Thirty-six male Sprague–Dawley rats underwent posterolateral spinal fusion on L4–L5 with three different doses of BMP-2 (0 µg [control], 3 µg [low dose], and 10 µg [high dose]). Weekly µCT results and histology and a manual palpation test at 8 weeks postoperatively were used for assessment of the spinal fusion.RESULTSELISA demonstrated the sustained release of BMP-2 until day 21. µCT and manual palpation test demonstrated a solid fusion in 91.6% (11/12) of specimens in both the low- and high-dose groups. N mice in the control group attained bony fusion (0%, 0/9). nHAp was resorbed between 2 and 4 weeks postoperatively, and regenerated fusion mass at 8 weeks postoperatively consisted of only newly formed bone.CONCLUSIONSThe nHAp/PLA-PEG composite enabled efficient bone regeneration with low-dose BMP-2. The sustained release of BMP-2 by PLA-PEG and the osteogenic and biodegradable scaffold of nHAp might contribute to efficient bone regeneration.CLINICAL SIGNIFICANCEThis novel composite material has potential in clinical applications (spinal fusion, large bone defect and non-union) by enabling efficient bone formation by BMP-2.     

Adenovirus-mediated direct gene therapy with bone morphogenetic protein-2 produces bone

The need to improve bone healing permeates the discipline of orthopedic surgery. Bone morphogenetic proteins (BMPs) are capable of inducing ectopic and orthotopic bone formation. However, the ideal approach with which to deliver BMPs remains unknown. Gene therapy to deliver BMPs offers several theoretical advantages over implantation of a recombinant BMP protein, including persistent BMP delivery and eliminating the need for a foreign body carrier. A replication defective adenoviral vector was constructed to carry the rhBMP-2 gene (AdBMP-2). The direct in vivo gene therapy approach was applied in both immunodeficient and immunocompetent animals to produce intramuscular bone as early as 2 weeks following injection. Radiographic and histologic analysis revealed radiodense bone containing mature bone marrow elements. Adenovirus-mediated delivery of a marker gene (β-galactosidase) into control animals produced no bone but indicated the cells transduced with the AdBMP-2 vector. Furthermore, comparisons between immunodeficient and immunocompetent animals illustrated the magnitude and significance of the immune response. Gene therapy to deliver BMP-2 has innumerable potential clinical applications from bone defect healing to joint replacement prosthesis stabilization. This study is the first to establish the feasibility of in vivo gene therapy to deliver active BMP-2 and produce bone.     

Bone marrow-derived heparan sulfate potentiates the osteogenic activity of bone morphogenetic protein-2 (BMP-2)

Lowering the efficacious dose of bone morphogenetic protein-2 (BMP-2) for the repair of critical-sized bone defects is highly desirable, as supra-physiological amounts of BMP-2 have an increased risk of side effects and a greater economic burden for the healthcare system. To address this need, we explored the use of heparan sulfate (HS), a structural analog of heparin, to enhance BMP-2 activity. We demonstrate that HS isolated from a bone marrow stromal cell line (HS-5) and heparin each enhances BMP-2-induced osteogenesis in C2C12 myoblasts through increased ALP activity and osteocalcin mRNA expression. Commercially available HS variants from porcine kidney and bovine lung do not generate effects as great as HS5. Heparin and HS5 influence BMP-2 activity by (i) prolonging BMP-2 half-life, (ii) reducing interactions between BMP-2 with its antagonist noggin, and (iii) modulating BMP2 distribution on the cell surface. Importantly, long-term supplementation of HS5 but not heparin greatly enhances BMP-2-induced bone formation in vitro and in vivo. These results show that bone marrow-derived HS effectively supports bone formation, and suggest its applicability in bone repair by selectively facilitating the delivery and bioavailability of BMP-2.     

Bone morphogenetic protein-7 reduces cold ischemic injury in rat kidney

Background

Deceased donor kidneys have a high incidence of delayed graft function attributable to ischemia–reperfusion injury. Although preservation solution and cold storage reduce cold ischemic injury, the depletion of cellular energy and oxidative signalling leads to cellular damage. Because bone morphogenetic protein-7 has renoprotective effect, we have hypothesized that recombinant human bone morphogenetic protein-7 (rh BMP-7) will better preserve kidney tissue exposed to prolonged cold ischemia in comparison with University of Wisconsin (UW) solution. We evaluated how the duration of cold ischemia influences the cold ischemic injury.

Methods

Levels of lipid peroxidation, protein carbonyl content, as well as superoxide dismutase (SOD) and glutathione peroxidase (GSH-Px) activities were determined in the kidneys by spectrophotometry after 6, 12, and 24 hours of cold ischemia.

Results

Time-dependent increases in the levels of lipid peroxidation and protein carbonyl content at all time points were significantly lower in rhBMP-7–perfused kidneys versus the UW-treated group. SOD activity after 6 hours, as well as GSH-Px activity after 24 hours of cold ischemia was significantly higher in the kidney tissue perfused by rhBMP-7 than in UW group.

Conclusion

rhBMP-7 significantly decreases cellular damage in rat kidney versus UW preservation solution and this is attributed to lowering of cold ischemia injury and maintaining prolonged tissue antioxidant activity.     

Repair of bone allograft fracture using bone morphogenetic protein-2

Long-term clinical data have shown that reconstruction using bone allografts provide adequate function after extensive tumor surgery. Complications such as nonunion of allograft-host interface, infection, and allograft fracture often require major revision surgeries. Allograft fractures usually do not induce the same repair process that is seen in normal fracture healing. The authors did an experimental study to test whether bone morphogenetic protein-2 can induce and achieve osseous repair in an allograft osteotomy model. Recombinant human bone morphogenetic protein-2 was applied at femoral intercalary allograft osteotomy sites in 20 rats. Forty additional rats served as controls (carrier alone and sham). Specimens in all groups were examined histologically and radiographically at 4 and 8 weeks. Specimens in the control groups showed only fibrosis by 8 weeks. In contrast, none of 10 specimens in the experimental group showed radiographic union at 8 weeks. New bone formation and integration with underlying allografts were seen in the experimental group as early as 4 weeks. These data suggest that fracture repair in the allograft bone can be triggered by a biologic regulator that is expressed during normal fracture healing.     

Osteolysis in transforaminal lumbar interbody fusion with bone morphogenetic protein-2

STUDY DESIGN.: Retrospective radiographic review. OBJECTIVE.: To determine the incidence of osteolysis, graft subsidence, and cage migration after recombinant human bone morphogenetic protein-2 (rhBMP-2) use with transforaminal lumbar interbody fusion (TLIF). SUMMARY OF BACKGROUND DATA.: Osteolysis after TLIF is a recently described phenomenon associated with rhBMP-2 use. Although this is typically a self-limiting condition, complications such as graft subsidence and cage migration have been described. The incidence of this is not well defined and most studies use plain radiographs for diagnosis rather than more advanced imaging. This study serves to quantify the risk of osteolysis and its associated graft complications with routine use of computed tomography. METHODS.: A total of 58 patients who underwent primary TLIF from a single surgeon between 2004 and 2007 underwent routine postoperative computed tomographic scan. Seventy-seven levels of fusion were evaluated for osteolysis. All patients received the same dose of rhBMP-2 of 5 mg per level. Imaging was performed immediately postoperative and again at an average of 4.3 months postoperative (range = 2.4-9.0 months). These images were evaluated for the presence of osteolysis, graft subsidence, and cage migration. These changes were then graded according to their severity. RESULTS.: Osteolysis was found in 16 of the 58 (27.6%) patients and 19 of the 77 (24.7%) levels treated. No significant difference was found between single and two-level fusions. The degree of osteolysis ranged from 3 to 20 mm with an average of 12.5 mm. The osteolysis was characterized as severe (>1 cm) in 12 of the 19 levels. Of the patients with osteolysis, 31.6% demonstrated graft subsidence all of which occurred with severe osteolytic defects. Migration of the intervertebral cage was found in 8.8% of patients. CONCLUSION.: rhBMP-2 use with TLIF is associated with a significant risk of postoperative osteolysis. Patients who demonstrated postoperative osteolysis were associated with significant risk of subsidence or migration of the intervertebral cage. The clinical implications of these changes are not currently known.     

Lumbar spinal fusion using recombinant human bone morphogenetic protein in the canine. A comparison of three dosages and two carriers.

STUDY DESIGN: A randomized, prospective and controlled animal study. OBJECTIVE: To evaluate lumbar spinal fusion using recombinant human bone morphogenetic protein 2 in a canine model. SUMMARY OF BACKGROUND DATA: Spinal fusion using autogenous bone grafting is associated with donor site morbidity and a nonunion rate of 5% to 35%. The use of recombinant human bone morphogenetic protein 2 as a bone graft substitute would eliminate donor site morbidity and perhaps augment the rate of successful fusion. METHODS: Mature beagles underwent bilateral paraspinal exposure at L4-L5, followed by transverse process decortication and randomization into one of six groups using differing doses of recombinant human bone morphogenetic protein 2 implanted using either a Type I collagen carrier or a polylactic acid carrier. Two control groups were used: one group without recombinant human bone morphogenetic protein 2 and another group using autogenous rib graft alone. RESULTS: Groups treated with recombinant human bone morphogenetic protein 2 demonstrated complete fusion in all animals. Animals treated with collagen carrier alone (no recombinant human bone morphogenetic protein 2) demonstrated complete absence of fusion. Successful fusion occurred in one of three canines in the autogenous bone graft group. Fusion masses in the recombinant human bone morphogenetic protein 2 treatment groups were significantly larger in size at 3 months than in the autogenous bone graft group. The collagen carrier was more biocompatible and biodegradable because residual polylactic acid carrier was seen with adjacent multinucleated giant cells. There was no evidence of spinal canal or nerve root encroachment in the recombinant human bone morphogenetic protein 2 treatment groups. CONCLUSIONS: The use of recombinant human bone morphogenetic protein 2 implanted using a Type I collagen carrier resulted in 100% fusion without adverse effects.     

引导骨再生技术对血管化组织工程骨修复兔股骨缺损过程中骨形态发生蛋白-2成骨及表达的影响

目的 探讨引导骨再生(GBR)技术对血管化组织工程骨修复兔股骨缺损过程中局部骨形态发生蛋白-2(BMP-2)的成骨量及表达的影响,以明确GBR技术在血管化组织工程骨应用中的作用. 方法 将兔自体骨髓基质干细胞经诱导后与β-磷酸三钙材料复合,植入制备的兔股骨缺损处并在材料侧槽中植入股动静脉束,其中实验组9例,血管化组织工程骨用可吸收性GBR屏障膜包裹;对照组9例,单纯植入血管化组织工程骨,分别于术后4、8、12周通过形态学检测新生骨量,ELISA法检测骨缺损局部BMP-2的表达量. 结果 随着时间进展各组成骨量逐渐增加(实验组4、8、12周时新生骨的相对面积比分别为7.31%±0.55%,35.23%±3.07%,76.09%±3.71%,对照组4、8、12周时新生骨的相对面积比分别为17.26%±1.17%,54.50%±4.26%,82.57%±4.11%,差异均有统计学意义(P<0.05);且同一时间点实验组成骨量低于对照组,差异有统计学意义(P<0.05);术后4、8、12周时实验组骨缺损局部BMP-2 OD值分别为0.334±0.012,0.245±0.008,0.172±0.009,对照组骨缺损局部BMP-2 OD值分别为0.389±0.008,0.289±0.008,0.189±0.009;术后4周时两组骨缺损内BMP-2表达量均达峰值,此后即开始出现不同程度的下降;术后4、8、12周时骨缺损局部BMP-2表达量实验组均低于对照组,差异均有统计学意义(P<0.05). 结论 GBR屏障膜会降低血管化组织工程骨修复兔股骨缺损局部的成骨量,并减少骨缺损过程中局部BMP-2的表达量.     

Clinical evaluation of recombinant human bone morphogenetic protein-2

Recombinant human bone morphogenetic protein-2 is an osteoinductive protein that plays a pivotal role in bone growth and regeneration. Several hundred studies were conducted in the past 7 years in numerous animal models to establish unequivocally the efficacy, safety, mechanism of action, pharmacokinetics, and surgical handling properties of recombinant human bone morphogenetic protein-2, building a solid foundation for clinical development programs. Pilot clinical trials have shown the feasibility and safety of recombinant human bone morphogenetic protein-2 treatment, and defined the effective dose for its use in open long bone fractures and for augmentation or preservation of the alveolar bone in the dental ridge. Prospective observational clinical studies helped define clinical efficacy end points, identify significant variables, and estimate appropriate population sample size for pivotal clinical trials. Pivotal clinical trials of recombinant human bone morphogenetic protein-2 are underway in patients with open tibial shaft fractures and in patients with a deficiency of the alveolar ridge.