Promoting lumbar spinal fusion by adenovirus-mediated bone morphogenetic protein-4 gene therapy

Objective: To determine whether an adenoviral construct containing bone morphogenetic protein-4(BMP-4) gene can be used for lumbar spinal fusion. Methods: Twelve New Zealand white rabbits were randomly divided into two groups, 8 in the experimental group and 4 in the control group. Recombinant, replication-defective type 5 adenovirus with the cytomegalovirus (CMV) promoter and BMP-4 gene (Ad-BMP-4) was used. Another adenovirus constructed with the CMV promoter andβ-galactosidase gene (Ad-β-gal) was used as control. Using collagen sponge as a carrier, Ad-BMP-4 (2.9×108 pfu/ml) was directly implanted on the surface of L5-L6 lamina in the experimental group, while Ad-β-gal was implanted simultaneously in the control group. X-ray was obtained at 3, 6, and 12 weeks postoperatively to observe new bone formation. When new bone formation was identified, CT scans and three-dimensional reconstruction were obtained. After that, the animals were killed and underwent histological inspection. Results: In 12 weeks after operation, new bone formation and fusion were observed on CT scans in the experimental group, without the evidence of ectopic calcification in the canal. Negative results were found in the control group. Histological analysis demonstrated endochondral bone formation at the operative site and fusion at early stage was testified. Conclusions: In vivo gene therapy using Ad-BMP-4 for lumbar posterolateral spinal fusion is practicable and effective.     

Effect of regional gene therapy with bone morphogenetic protein-2-producing bone marrow cells on spinal fusion in rats

BACKGROUND: Bone morphogenetic proteins (BMPs) are now being used as bone-graft substitutes to enhance spinal fusion. However, the large doses of BMP required to induce a spinal fusion in humans suggests that the delivery of these proteins should be improved. We used ex vivo adenoviral gene transfer to create BMP-2-producing bone marrow cells, and these autologous cells were found to induce a posterolateral fusion of the spine in syngeneic rats. METHODS: Intertransverse spinal arthrodesis (L4 and L5) was attempted in ten groups of Lewis rats with 5 x 10 (6) BMP-2-producing rat bone marrow cells (Ad-BMP-2 cells), created through adenoviral gene transfer with guanidine hydrochloride-extracted demineralized bone matrix as a carrier (Group I); 5 x 10 (6) Ad-BMP-2 cells on a collagen sponge carrier (Group II); 10 micro g of recombinant BMP-2 (rhBMP-2) in a guanidine hydrochloride-extracted demineralized bone matrix carrier (Group III); 10 micro g of rhBMP-2 in a collagen sponge carrier (Group IV); autogenous iliac crest bone-grafting (Group V); 5 x 10 (6) beta-galactosidase-producing rat bone marrow cells, created through adenoviral gene transfer with guanidine hydrochloride-extracted demineralized bone matrix as a carrier (Group VI); decortication of the transverse processes alone (Group VII); 5 x 10 (6) uninfected rat bone marrow cells with a guanidine hydrochloride-extracted demineralized bone matrix carrier (Group VIII); guanidine hydrochloride-extracted demineralized bone matrix only (Group IX); or a collagen sponge alone (Group X). Each specimen underwent plain radiography, manual palpation, and histological analysis. RESULTS: All spines in Groups I and II (BMP-2-producing bone marrow cells) and all spines in Groups III and IV were fused at four weeks postoperatively. In contrast, none of the spines in the other groups had fused at a minimum of eight weeks after implantation. Histological analysis of the specimens revealed that the spines that had received BMP-2-producing bone marrow cells (Groups I and II) were filled with coarse trabecular bone postoperatively, whereas those that had received rhBMP-2 (Groups III and IV) were filled with thin, lace-like trabecular bone. All of the other spines, including those that had been treated with autogenous iliac crest bone-grafting (Group V), produced little or no new bone. CONCLUSION: BMP-2-producing bone marrow cells, created by adenoviral gene transfer, produce sufficient BMP to induce an intertransverse fusion in the rat spine model.     

Bone induction by BMP-2 transduced stem cells derived from human fat.

PURPOSE: We have isolated pluripotent mesenchymal progenitor cells in large numbers from liposuction aspirates (processed lipoaspirate cells or PLAs). This study examines the osteogenic potential of PLAs and bone marrow aspirate cells (BMAs), when exposed to either recombinant human bone morphogenetic protein (BMP)-2 (rh-BMP-2) or adenovirus containing BMP-2 cDNA (Ad-BMP-2). METHODS: Liposuction aspirates underwent proteolytic digestion to obtain PLAs. After exposure to exogenous rh-BMP-2 or Ad-BMP-2 for four or seven days, PLAs and BMAs were assessed by histochemistry, spectrophotometry and RT-PCR. Western blotting and ELISA confirmed BMP gene transduction. Results were compared to osteoblasts and cells in osteogenic media only. PLA-Ad-BMP-2 cells were seeded on matrices and implanted in the hind limbs of SCID mice. RESULTS: Analysis of quantified bone precursor assays including extracellular ALP histomorphometry, intracellular ALP spectrophotometry, and calcified extracellular matrix (von Kossa) histomorphometry revealed that PLAs treated with exogenous rh-BMP-2 or transduced with a BMP-2 containing adenovirus (PLA-Ad-BMP-2) produced more bone precursors than osteoblasts (p=0.001). PLAs treated with exogenous rh-BMP-2 or PLA-Ad-BMP-2 also produced more bone precursors than BMAs (p=0.001), except for day 7 ALP histomorphometry (p=0.343). ELISA confirmed successful BMP-2 production by both progenitor cell groups transduced with Ad-BMP-2. H&E sections from collagen I matrices seeded with PLA-Ad-BMP-2 cells confirmed bone formation at six weeks. CONCLUSIONS: Liposuction aspirates contain PLAs that can be transfected with the BMP-2 gene, with rapid induction into the osteoblast phenotype at a rate comparable to rh-BMP-2 and osteoblast groups. Transduced PLAs produce more bone precursors with faster onset of calcified extracellular matrix than transduced BMAs. PLAs may be an ideal source of mesenchyme-lineage stem cells for gene therapy and tissue engineering.     

基因修饰细胞和成骨蛋白在脊柱融合中作用比浇

目的 观察骨形态发生蛋白-2(BMP-2)修饰的骨髓间充质干细胞(BMSCs)诱导大鼠脊柱融合的能力,并与已广泛商品化使用的BMP-2蛋白比较.方法 分别将携带BMP-2和β-半乳糖苷酶的腺病毒转染BMSCs,在大鼠脊柱融合模型中建立5个实验组,每组8只,分别植入:Ⅰ组,5×106个BMP-2转染细胞;Ⅱ组,5×106个β-半乳糖苷酶转染细胞;Ⅲ组,10μgBMP-2蛋白;Ⅳ组,5×106个未转染细胞;Ⅴ组,单独的胶原海绵.8周后处死大鼠,进行各项观察.结果 手触力学评价显示Ⅰ、Ⅲ组全部达到骨性融合,而其他组均未融合.X线评分Ⅰ组(5.02)和Ⅲ组(4.21)也明显高于其他组.Micro-CT和组织学切片显示Ⅲ组的新骨骨量较少,骨小梁较细.结论 基因修饰的骨髓间充质干细胞能够很好地诱导脊柱融合,诱导效果要好于商品化的BMP-2蛋白.     

Thoracoscopic intradiscal spine fusion using a minimally invasive gene-therapy technique

BACKGROUND: Gene therapy has been utilized to achieve posterior intertransverse process fusion in rodents. To our knowledge, however, no one has previously reported on the use of this technique to achieve anterior spinal fusion in mammals. The purpose of the present study was to determine if a gene-therapy technique can be utilized to achieve anterior intradiscal fusion in pigs with use of minimally invasive techniques. METHODS: Mesenchymal stem cells were isolated from each of three pigs, expanded in culture, and transduced with an adenovirus carrying either the gene for bone morphogenetic protein-2 (Adv-BMP2) or the control gene, beta-galactosidase (Adv-betagal). In vitro, assays were performed to detect BMP-2 expression as well as protein markers of bone formation. In vivo, four thoracic disc spaces in each of three pigs were injected thoracoscopically with cells after 1 cm (3) of the disc had been removed. In each of the three pigs, two discs were injected with autologous mesenchymal stem cells transduced with Adv-BMP2, the third disc was injected with cells transduced with Adv-betagal (control 1), and the fourth disc served as the sham-operated control (control 2). The three animals were killed six weeks after the implantation. Computerized tomographic scanning was performed on two of the specimens, and histological examination was performed on all specimens. The computerized tomographic scans and histological examinations were then interpreted in a blinded fashion. RESULTS: In the in vitro study, a human BMP-2 protein band was detected in the medium of Adv-BMP2-transduced stem cells but not in that of the control cells. The Adv-BMP2-transduced stem cells were associated with a fivefold increase in alkaline phosphatase activity compared with the controls as well as with matrix mineralization and increased protein expression of type-I collagen, osteopontin, and bone sialoprotein. In the in vivo study, radiographic examination demonstrated anterior spinal fusion in all six disc spaces that had been treated with implantation of Adv-BMP2-transduced stem cells. In contrast, the six control disc spaces had little or no intervening bone. Histological examination demonstrated bridging bone from end plate to end plate in all six disc spaces that had been treated with implantation of Adv-BMP2-transduced stem cells. The six control disc spaces had no bridging bone. CONCLUSION: The Adv-BMP2-transduced mesenchymal stem cells produced BMP-2 protein. Further, the cells differentiated into osteoblasts and induced anterior spinal fusion in six of six disc spaces in this pig model. Although many technical and practical challenges remain, the results of the present study suggest that it may eventually be possible to use similar techniques to achieve anterior spinal fusion in humans.     

Human skeletal muscle cells in ex vivo gene therapy to deliver bone morphogenetic protein-2

We have examined whether primary human muscle-derived cells can be used in ex vivo gene therapy to deliver BMP-2 and to produce bone in vivo. Two in vitro experiments and one in vivo experiment were used to determine the osteocompetence and BMP-2 secretion capacity of cells isolated from human skeletal muscle. We isolated five different populations of primary muscle cells from human skeletal muscle in three patients. In the first in vitro experiment, production of alkaline phosphatase by the cells in response to stimulation by rhBMP-2 was measured and used as an indicator of cellular osteocompetence. In the second, secretion of BMP-2 was measured after the cell populations had been transduced by an adenovirus encoding for BMP-2. In the in vivo experiment, the cells were cotransduced with a retrovirus encoding for a nuclear localised beta-galactosidase gene and an adenovirus encoding for BMP-2. The cotransduced cells were then injected into the hind limbs of severe combined immune-deficient (SCID) mice and analysed radiographically and histologically. The nuclear localised beta-galactosidase gene allowed identification of the injected cells in histological specimens. In the first in vitro experiment, the five different cell populations all responded to in vitro stimulation of rhBMP-2 by producing higher levels of alkaline phosphatase when compared with non-stimulated cells. In the second, the five different cell populations were all successfully transduced by an adenovirus to express and secrete BMP-2. The cells secreted between 444 and 2551 ng of BMP-2 over three days. In the in vivo experiment, injection of the transduced cells into the hind-limb musculature of SCID mice resulted in the formation of ectopic bone at 1, 2, 3 and 4 weeks after injection. Retroviral labelling of the cell nuclei showed labelled human muscle-derived cells occupying locations of osteoblasts in the ectopic bone, further supporting their osteocompetence. Cells from human skeletal muscle, because of their availability to orthopaedic surgeons, their osteocompetence, and their ability to express BMP-2 after genetic engineering, are an attractive cell population for use in BMP-2 gene therapy approaches.     

Gene-mediated osteogenic differentiation of stem cells by bone morphogenetic proteins-2 or -6.

Bone marrow-derived mesenchymal stem cells (BMDMSC) hold promise for targeted osteogenic differentiation and can be augmented by delivery of genes encoding bone morphogenetic proteins (BMP). The feasibility of promoting osteogenic differentiation of BMDMSC was investigated using two BMP genes in monolayer and three-dimensional alginate culture systems. Cultured BMDMSC were transduced with E1-deleted adenoviral vectors containing either human BMP2 or BMP6 coding sequence under cytomegalovirus (CMV) promoter control [17:1 multiplicities of infection (moi)] and either sustained in monolayer or suspended in 1 mL 1.2% alginate beads for 22 days. Adenovirus (Ad)-BMP-2 and Ad-BMP-6 transduction resulted in abundant BMP-2 and BMP-6 mRNA and protein expression in monolayer culture and BMP-2 protein expression in alginate cultures. Ad-BMP-2 and Ad-BMP-6 transduced BMDMSC in monolayer had earlier and robust alkaline phosphatase-positive staining and mineralization and were sustained for a longer duration with better morphology scores than untransduced or Ad-beta-galactosidase-transduced cells. Ad-BMP-2- and, to a lesser degree, Ad-BMP-6-transduced BMDMSC suspended in alginate demonstrated greater mineralization than untransduced cells. Gene expression studies at day 2 confirmed an inflammatory response to the gene delivery process with upregulation of interleukin 8 and CXCL2. Upregulation of genes consistent with response to BMP exposure and osteogenic differentiation, specifically endochondral ossification and extracellular matrix proteins, occurred in BMP-transduced cells. These data support that transduction of BMDMSC with Ad-BMP-2 or Ad-BMP-6 can accelerate osteogenic differentiation and mineralization of stem cells in culture, including in three-dimensional culture. BMP-2-transduced stem cells suspended in alginate culture may be a practical carrier system to support bone formation in vivo. BMP-6 induced a less robust cellular response than BMP-2, particularly in alginate culture.     

骨形成蛋白2基因修复兔桡骨缺损的实验研究

目的观察携带人骨形成蛋白2（bone morphogenetic protein 2，BMP-2）基因的腺病毒载体（adenovirus carrying BMP-2 gene，Ad—BMP2），通过纤维蛋白凝胶与牛松质骨支架（bovine cancellous bone，BCB）复合，修复骨缺损的效果。方法将60只新西兰大耳白兔随机分为4组，每组15只。制成双侧桡骨中段1．5cm骨缺损模型，采用4种材料植入修复。A组：Ad-BMP-2＋BCB；B组：重组BMP-2＋BCB；C组：携带D-半乳糖酐酶基因的腺病毒对照载体（adenovirus carrying β—galgene，Ad—Lacz）＋BCB；D组：单纯BCB支架。修复术后各组于4、8和12周各处死动物5只取材，行X线片、组织学、生物力学和免疫组织化学染色检查。结果A、B两组骨缺损均得到了修复，但术后各时间点，A组在成骨活跃程度、新生骨量、力学强度及BMP-2表达等方面均明显优于B组；C、D两组均无新骨形成。结论BMP-2直接基冈治疗，操作简便、骨诱导能力强，是修复节段性骨缺损的有效方法。     

Influence of short-term adenoviral vector and prolonged lentiviral vector mediated bone morphogenetic protein-2 expression on the quality of bone repair in a rat femoral defect model

The objective of this study was to compare the efficacy of adenoviral and lentiviral regional gene therapy in a rat critical sized femoral defect model. The healing rates and quality of bone repair of femoral defects treated with syngeneic rat bone marrow cells (RBMCs) transduced with either lentiviral vector (Group I) or adenoviral vector (Group II) expressing bone morphogenetic protein-2 (BMP-2) gene were assessed. RBMCs transduced with the adenoviral vectors produced more than 3 times greater (p < 0.001) BMP-2 when compared to RBMCs transduced with lentiviral vectors in an in vitro evaluation. Serial bioluminescent imaging demonstrated short duration luciferase expression (less than 3 weeks) in defects treated with RBMCs co-transduced with two adenoviral vectors (Group IV; adenovirus expressing BMP-2 and luciferase [Ad-BMP-2 + Ad-Luc]). In contrast, the luciferase signal was present for 8 weeks in defects treated with RBMCs co-transduced with two lentiviral vectors (Group III; lentivirus expressing BMP-2 and luciferase gene [LV-BMP-2 + LV-Luc]). There were no significant differences with respect to the radiological healing rates (p = 0.12) in defects treated with lentiviral versus adenoviral mediated BMP-2 gene transfer. Biomechanical testing of healed Group I femoral specimens demonstrated significantly higher energy to failure (p < 0.05) when compared to Group II defects. Micro CT analysis revealed higher bone volume/tissue volume fraction (p = 0.04) in Group I defects when compared to Group II defects. In conclusion, prolonged BMP-2 expression associated with lentiviral mediated gene transfer demonstrated a trend towards superior quality of bone repair when compared to adenoviral mediated transfer of BMP-2. These results suggest that the bone repair associated with regional gene therapy is influenced not just by the amount of protein expression but also by duration of protein production. This observation needs validation in a more biologically challenging environment where differences in healing rates and quality of bone repair are more likely to be significantly different.     

腺病毒介导人骨形态蛋白-7基因感染大鼠髓腔细胞的实验研究

目的 :观察已构建好的腺病毒介导的骨形态蛋白 7基因感染大鼠髓腔内细胞的情况 ,为腺病毒介导BMP 7的进一步基因治疗提供可靠的实验基础。材料和方法 :从人骨肉瘤细胞株U 2OS中获得hBMP 7的cDNA片段 ,插入穿梭质粒后在大肠杆菌内与腺病毒实现同源重组 ,阳性克隆转染 2 93细胞 ,扩增纯化得到大量病毒后注入大鼠椎体髓腔中 ,用冲洗髓腔的方法和流式细胞仪分析来观察注射椎体和上位椎体髓腔内细胞感染腺病毒BMP 7的情况 ,并观察病毒感染的时限。结果 :经体外检测能高效表达活性BMF 7的构建好的AdBMP 7注入椎体髓腔后 ,髓腔冲洗液的荧光显微镜观察 ,术后 1 2d和 2 4d都有感染了带有荧光蛋白标记的髓腔内细胞 ,而且在注射椎体的上位椎体中也出现了感染病毒的细胞。结论 :腺病毒介导的BMP -7基因能够在体内感染髓腔细胞并分泌活性的BMP -7,可以成为基因治疗的有效手段。     

BMP-14 gene therapy increases tendon tensile strength in a rat model of Achilles tendon injury

BACKGROUND: Molecular and cellular-based enhancements of healing combined with conventional methods may yield better outcomes after the surgical management of tendon injury. We examined the histological and biomechanical effects of adenovirus-mediated transgene expression of bone morphogenetic protein-14 (BMP-14) on healing in a rat Achilles tendon laceration model. Specifically, we hypothesized that this delivery system for gene therapy would hasten the restoration of the normal histological appearance and tensile strength of a surgically repaired tendon. METHODS: The right Achilles tendon of ninety male Sprague-Dawley rats was transected, repaired, and immediately infected with adenovirus expressing either the gene for green fluorescent protein (AdGFP) or the gene for human BMP-14 and green fluorescent protein (AdBMP-14). A sham control group received no viral-mediated infection after repair. Animals from each of the three groups were killed at one, two, and three weeks after surgery. The retrieved tendons were inspected, examined under light and fluorescent microscopy, and tested to determine their tensile strength. RESULTS: Tendons transduced with BMP-14 exhibited less visible gapping, a greater number of neotenocytes at the site of healing, and 70% greater tensile strength than did either those transduced with GFP or the sham controls at two weeks after repair. Histological examination revealed no inflammatory response to the adenovirus in tendons transduced with BMP-14 or GFP. No ectopic bone or cartilage formed in the tendons transduced with BMP-14. CONCLUSIONS: Adenovirus-mediated gene therapy with BMP-14 expedites tendon-healing in this animal model. No adverse immunological response to the adenoviral vector was detected in the host tissue, and the local production of BMP-14 did not induce unwelcome bone or cartilage formation within the healing tendon. CLINICAL RELEVANCE: The results of this animal study suggest that gene therapy with BMPs may improve the capacity of injured musculoskeletal tissue to heal.     

骨形态发生蛋白2基因治疗在修复骨缺损中对血管化影响的实验研究

目的观察骨形态发生蛋白-2（BMP-2）基因治疗在修复骨缺损中对血管化的影响。方法分离培养兔骨髓基质干细胞（MSCs），经BMP-2腺病毒载体转染后复合异种骨支架移植修复1．5cm桡骨缺损。分为5组：①BMP-2基因转染细胞＋去抗原牛松质骨支架（BCB）；②未转染细胞＋重组BMP-2＋BCB；③对照基因转染细胞＋BCB；④未转染细胞＋BCB；⑤BCB。术后4、8、12周行微血管墨汁灌注、血管内皮生长因子（VEGF）免疫组化染色、组织学等检测。结果术后4周，基因治疗组骨间血管的密度在周边区域较高，通常每一个骨小梁孔隙中都有一支新形成的小血管；透射电镜见成骨细胞总是毗邻血管内皮细胞而存在，并随着微血管的增生而逐渐向骨细胞发展；间质细胞VEGF表达明显增强。结论BMP-2基因治疗可通过上调VEGF表达，间接诱导移植骨血管化，对骨不连、骨缺损的治疗具有重要意义。     

BMP-2基因转染的人骨髓基质干细胞复合PLA/PCL支架体外构建组织工程骨

目的　用人骨形态发生蛋白 2腺病毒表达载体 (Ad -BMP - 2 )转染的人骨髓基质干细胞 (hBMSC) ,复合PLA/PCL(聚乳酸 /聚己内酯 )生物降解支架体外构建组织工程骨。方法　用Ad -BMP - 2转染体外培养的成人BMSC ,免疫组化、原位杂交染色和蛋白印迹方法检测细胞BMP - 2的表达 ,并通过流式细胞仪和ALP活性检测分析其对细胞增殖、分化的影响。然后将转染后细胞接种到PLA/PCL支架上 ,扫描电镜观察细胞贴附、生长状况。结果　转染后 ,hBMP - 2基因在mRNA水平和蛋白水平均有表达 ;S期细胞比例和ALP活性明显增高。扫描电镜见转染细胞分布均匀 ,伸展良好。结论　Ad-BMP - 2可高效转染hBMSC ,且促进细胞增殖及成骨转化。转染后细胞在PLA/PCL上生长良好 ,BMP - 2基因治疗的组织工程骨构建成功     

骨形成蛋白2基因修饰的老年大鼠骨髓间充质干细胞成骨分化能力的研究

目的观察年龄因素对骨髓间充质干细胞(marrow mesenchymal stem cells, MSCs)成骨分化能力的影响;了解基因治疗对老年大鼠MSCs成骨分化能力的影响. 方法 1月龄(幼年组)、9月龄(成年组)及24月龄(老年组)雄性Wistar大鼠各6只,取MSCs经体外分离、培养及携带骨形成蛋白2(bone morphogenetic protein 2,BMP-2)基因的腺病毒载体(Ad-BMP-2)转染后,定量检测BMP-2、碱性磷酸酶(alkaline phosphate,ALP)表达,以及成骨细胞标志性蛋白:Ⅰ型胶原、骨涎蛋白(bone sialoprotein,BSP)和骨桥素(osteopontin, OPN)的表达.将转染的各组MSCs分别与磷酸三钙(tricalcium phosphate, TCP)复合后植入裸鼠体内,3周后取材,比较各组诱导异位成骨能力. 结果 ELISA检测表明BMP-2基因修饰的MSCs可以有效表达BMP-2,且表达量在各年龄组间差异无统计学意义(P>0.05);各组ALP于诱导后第9天达高峰,但组间差异均无统计学意义(P＞0.05);诱导后第7天,RT-PCR半定量检测示各组均有成骨细胞特征性蛋白,即:Ⅰ型胶原、OPN及BSP的明显表达,表达量在各组间差异无统计学意义(P>0.05);BMP-2基因转染的MSCs与TCP复合后可诱导裸鼠体内异位成骨,各组成骨量差异无统计学意义(P>0.05). 结论 BMP-2基因修饰的老年大鼠MSCs可以恢复成骨分化能力,基因治疗可能为老年性骨骼疾病提供一种新的治疗途径.     

Induction of Bone Formation Using a Recombinant Adenoviral Vector Carrying the Human BMP-2 Gene in a Rabbit Spinal Fusion Model

Bone marrow-derived mesenchymal stem cells are pluripotential cells that have the capacity to differentiate into an osteoprogenitor line. It has been demonstrated that BMP-2 can enhance this differentiation process. In an attempt to prolong the transforming effect of BMP-2, we used an adenoviral vector carrying the human BMP-2 gene to transduce marrow-derived mesenchymal stem cells of New Zealand white rabbits. Assays on tissue culture demonstrated that these cells indeed produced the BMP-2 protein. These transduced stem cells were then autologously reimplanted into the donor rabbits. The cells were placed in the intertransverse process area of five rabbits. In one out of the five rabbits, this resulted in the production of new bone which was demonstrable on both radiographic and histologic examination. We conclude that it is possible to successfully transduce mesenchymal stem cells with the gene for BMP-2 such that these cells will produce the BMP-2 protein in vitro. Further, the transduction results in transformation of these cells into an osteoprogenitor line capable of producing bone in vivo. These data suggest the feasibility of employing gene therapy using recombinant adenoviral vectors as a tool for enhancing spine fusion. Further work to improve the fidelity and longevity of the gene transfer is warranted. Received: 22 January 1998 / Accepted: 19 February 1998     

The effects of recombinant human bone morphogenetic protein-2, recombinant human bone morphogenetic protein-12, and adenoviral bone morphogenetic protein-12 on matrix synthesis in human annulus fibrosis and nucleus pulposus cells

BACKGROUND CONTEXT: Bone morphogenetic proteins (BMPs) are potential therapeutic factors for degenerative discs, and BMP-12 does not have the osteogenic potential of BMP-2, making it better suited for intradiscal injection. However, no reports have compared the actions of BMP-2 and -12 on human annulus fibrosus (AF) and nucleus pulposus (NP) cells nor evaluated adenoviral-mediated gene therapy in human AF cells. PURPOSE: To evaluate and compare the effects of recombinant human (rh) BMP-2, rhBMP-12, and adenoviral BMP-12 (Ad-BMP-12) on nucleus pulposus and annulus fibrosis cell matrix protein synthesis. STUDY DESIGN: In vitro study using rhBMP-2 and -12 and adenoviral BMP-12 with human intervertebral disc (IVD) cells. METHODS: Human NP and AF IVD cells were isolated, maintained in monolayer, and incubated with BMP-2 or -12 for 2 days. AF and NP cells were transduced with Ad-BMP-12, pellets formed, and incubated for 6 days. Growth factor-treated cells were labelled with either 35-S or 3H-proline to assay matrix protein synthesis. RESULTS: rhBMP-2 increased NP proteoglycan, collagen, and noncollagen protein synthesis to 355%, 388%, and 234% of control. RhBMP-12 increased the same NP matrix proteins' synthesis to 140%, 143%, and 160% of control. Effects on AF matrix protein synthesis were minimal. Ad-BMP-12 significantly increased matrix protein synthesis and DNA content of AF and NP cells in pellet culture. NP synthesis of all matrix proteins and AF synthesis of proteoglycans was increased when the data were normalized to pellet DNA. AF synthesis of noncollagen protein and collagen was not modulated by Ad-BMP-12 if the data are normalized to pellet DNA content. CONCLUSIONS: Both rhBMP-2 and -12 increase human NP cell matrix protein synthesis while having minimal effects on AF cells. However, Ad-BMP-12 did increase matrix protein synthesis in both NP and AF cells, making it a potential therapy for enhancing matrix production in the IVD. These responses plus the proliferative action of Ad-BMP-12 seen in the current studies, and the lack of an osteogenic action noted in other studies justifies future studies to determine if gene therapy with BMP-12 could provide protective and/or reparative actions in degenerating discs.     

Effect of bone morphogenetic protein-2-expressing muscle-derived cells on healing of critical-sized bone defects in mice.

BACKGROUND: Cells that express bone morphogenetic protein-2 (BMP-2) can now be prepared by transduction with adenovirus containing BMP-2 cDNA. Skeletal muscle tissue contains cells that differentiate into osteoblasts on stimulation with BMP-2. The objectives of this study were to prepare BMP-2-expressing muscle-derived cells by transduction of these cells with an adenovirus containing BMP-2 cDNA and to determine whether the BMP-2-expressing muscle-derived cells would elicit the healing of critical-sized bone defects in mice. METHODS: Primary cultures of muscle-derived cells from a normal male mouse were transduced with adenovirus encoding the recombinant human BMP-2 gene (adBMP-2). These cells (5 yen 10(5)) were implanted into a 5-mm-diameter critical-sized skull defect in female SCID (severe combined immunodeficiency strain) mice with use of a collagen sponge as a scaffold. Healing in the treatment and control groups was examined grossly and histologically at two and four weeks. Implanted cells were identified in vivo with use of the Y-chromosome-specific fluorescent in situ hybridization (FISH) technique, and their differentiation into osteogenic cells was demonstrated by osteocalcin immunohistochemistry. RESULTS: Skull defects treated with muscle cells that had been genetically engineered to express BMP-2 had >85% closure within two weeks and 95% to 100% closure within four weeks. Control groups in which the defect was not treated (group 1), treated with collagen only (group 2), or treated with collagen and muscle cells without adBMP-2 (group 3) showed at most 30% to 40% closure of the defect by four weeks, and the majority of the skull defects in those groups showed no healing. Analysis of injected cells in group 4, with the Y-chromosome-specific FISH technique showed that the majority of the transplanted cells were located on the surfaces of the newly formed bone, but a small fraction (approximately 5%) was identified within the osteocyte lacunae of the new bone. Implanted cells found in the new bone stained immunohistochemically for osteocalcin, indicating that they had differentiated in vivo into osteogenic cells. CONCLUSIONS: This study demonstrates that cells derived from muscle tissue that have been genetically engineered to express BMP-2 elicit the healing of critical-sized skull defects in mice. The cells derived from muscle tissue appear to enhance bone-healing by differentiating into osteoblasts in vivo. Clinical Relevance: Ex vivo gene therapy with muscle-derived cells that have been genetically engineered to express BMP-2 may be used to treat nonhealing bone defects. In addition, muscle-derived cells appear to include stem cells, which are easily obtained with muscle biopsy and could be used in gene therapy to deliver BMP-2.     

A light and electron microscopic study of ectopic tendon and ligament formation induced by bone morphogenetic protein-13 adenoviral gene therapy.

OBJECT: Bone morphogenetic proteins (BMPs) are involved in the growth and development of many tissues, but it is their role in skeletal development and their unique ability to induce ectopic and orthotopic osteogenesis that have attracted the greatest interest. Expression of the BMP-13 gene is predominantly localized to hypertrophic chondrocytes in regions of endochondral bone formation during development, as well as in mature articular cartilage in the adult. In addition, the application of BMP-13 on a collagen carrier induces neotendon/neoligament formation when delivered subcutaneously or intramuscularly in rodents. The aim of the present study was to determine the histological and ultrastructural changes that occur after the intramuscular injection of a first-generation BMP-13 adenoviral vector. METHODS: Athymic nude rats were injected with 3.75 x 10(10) plaque-forming units of adenovirus (Ad)-BMP-13 or Ad-beta-galactosidase in the thigh musculature, and the region was examined using light and electron microscopy at various time points between 2 days and 100 days postinjection. As early as 2 days after injection of Ad-BMP-13, progenitor cells were observed infiltrating between the transduced muscle fibers. These cells subsequently proliferated, differentiated, and secreted large amounts of collagenous extracellular matrix. By 100 days postinjection, the treated tissue displayed the histological and ultrastructural appearance of neotendon/neoligament, which was clearly demarcated from the surrounding muscle. Small foci of bone and fibrocartilage were also seen within the treated tissue. A short-term bromodeoxyuridine study also demonstrated rapid mesenchymal cell proliferation at the Ad-BMP-13 injection site as early as 48 hours postinjection. At all time points, the control AD-beta-gal injection sites were found to contain only normal muscle, without evidence of inflammation or mesenchymal cell proliferation. CONCLUSIONS: The results of this study indicate that in the future the use of the BMP-13 gene may have therapeutic utility for the healing of tendon and ligament tears and avulsion injuries.