经皮自体骨髓移植在骨缺损瘢痕组织内成骨作用的实验研究

目的 观察经皮自体骨髓移植在骨缺损瘢痕组织内的成骨作用。方法 选健康家兔１８只，建立双侧桡骨中段骨及骨膜缺损１ｃｍ模型，两骨断端髓腔用骨蜡封闭。６周后，实验侧（右）桡骨骨缺损区经皮注射自体红骨髓２ｍｌ，对照侧（左）桡骨骨缺损区注射自身外周血２ｍｌ。于注射前及注射后不同时间内分别行Ｘ线、组织学检查及骨缺损区瘢痕组织内钙磷含量的测定。结果 注射一１周，实验侧Ｘ线显示以骨缺损为中心形成一个密度增高的弧形     

墨汁灌注法观察细胞-陶瓷颅骨修复过程中血管生成与骨形成的关系

目的 通过双侧颈总动脉墨汁灌注法观察组织工程大鼠颅骨极量骨缺损修复过程中骨形成、血管生成与陶瓷降解之间的关系及血供来源和走行。方法 28只成年雄性SD大鼠随机分成实验组和对照组，在全麻及无菌条件下离断其左侧股，胫骨，将骨髓冲入α-MEM培养基中诱导培养大鼠的自体骨髓基质细胞。实验组将已分化的成骨细胞复合含孔磷钙陶瓷修复与所取细胞对应的大鼠颅骨极量骨缺损，对照组植入无成骨细胞的陶瓷。于术后第4、8、12、16、20、24及28周每线各处死2只大鼠，取材前行双侧颈总动脉墨汁灌注，制成脱钙切片和不脱钙磨片，光学显微镜下观察。结果 实验组大鼠颅骨缺损修复过程中，动脉血供主要来源于周围骨床，纵横直行并呈离心性分布，静脉回流蜿蜒迂曲呈向心性分布。术后16-28周，血管密集处大部分陶瓷已降解并被新骨所取代。对照组虽有大量血管杂乱地渗入陶瓷孔隙，也可见陶瓷为支架的组织工程骨修复过程中，陶瓷内部交通的孔隙有利于血管生成和新骨形成；血管生成与新骨形成及陶瓷降解关系密切。     

经皮自体骨髓移植在骨缺损瘢痕组织内成骨作用的实验研究与临床应用

目的观察经皮自体骨髓移植在骨缺损瘢痕组织内的成骨作用,探索治疗骨不连的新途径。方法选健康家兔１８只,建立双侧桡骨中段骨及骨膜缺损１ｃｍ模型,６周后,实验侧（右）骨缺损区经皮注射自体红骨髓２ｍｌ,对照侧（左）骨缺损区经皮注射自身外周血２ｍｌ。在不同时间内进行Ｘ线片、组织学检查及新生组织钙、磷含量测定。临床应用经皮自体骨髓移植治疗骨不连１５例,其中腕舟骨不连７例,肱骨不连３例,股骨不连２例,胫骨不连３例。骨不连时间为７～４８个月,平均１３个月。结果实验侧骨缺损区在骨髓注射后不同时间的Ｘ线片、组织学检查均显示新骨形成逐渐增多,钙、磷含量均高于对照侧。对照侧无成骨现象。临床应用的１５例中,１３例在５～９个月骨折愈合,２例未愈合。结论经皮自体骨髓移植在骨缺损瘢痕组织内有成骨作用,在临床上可作为治疗骨不连的方法,尤其适用于骨不连部位软组织条件差、不具备开放植骨的病例。     

The osteogenic potential of culture-expanded rat marrow mesenchymal cells assayed in vivo in calcium phosphate ceramic blocks

When whole marrow is introduced into porous calcium phosphate ceramic, bone forms on the walls of the pores. As an extension of earlier studies, bone marrow cells derived from the femora of inbred rats were introduced into tissue culture, and the adherent cells were cultivated, mitotically expanded, subcultured, harvested, placed in small cubes of porous calcium phosphate ceramic, and grafted into subcutaneous sites of syngeneic rats. Primary marrow-derived, cultured mesenchymal cells introduced into ceramic showed strong osteogenic potential, with bone forming in the pore regions of ceramic as early as two weeks after in vivo implantation; cartilage was observed infrequently in pores that appeared to be avascular. Osteogenesis could be observed after the 18th subculture (over 36 population doublings) when the cells were tested in ceramic at subcutaneous sites, whereas chondrogenesis was observed with only the first and second subcultured cells in the ceramic delivery vehicle. With increasing numbers of subcultures, the initiation of osteogenesis and the apparent rate of bone formation declined, and the course of osteogenesis was delayed. Cultured, marrow-derived mesenchymal cells, even after the 21st subculture (over 40 population doublings), exhibited a positive histochemical reaction for alkaline phosphatase. However, the in vivo osteogenic potential of these cells was not correlated with their alkaline phosphatase activity. The implantation of cell pellets or the injection of cell suspensions of fresh or cultured, adherent marrow cells never produced bone or cartilage in heterotopic sites. These data indicate that porous ceramic provides an excellent delivery vehicle for cells that are capable of osteogenic expression and suggest that the composite graft of marrow-derived mesenchymal cells and porous ceramic may be useful for repair of massive bone defects. It may be possible to culture marrow mesenchymal cells as a source for reparative cells for implantation back into autogeneic sites.     

VEGF producing bone marrow stromal cells (BMSC) enhance vascularization and resorption of a natural coral bone substitute

Bone graft substitutes often exhibit poor bone regeneration in large defects because of inadequate vascularization. Studies have shown that if blood supply is compromised, application of osteogenic factors alone could not induce successful healing. This study was to evaluate the effects of vascular endothelial growth factor, which combined with a coralline scaffold, on vascularization, scaffold resorption and osteogenesis in a rabbit radius critical size defect model. The scaffold was either coated with a control-plasmid DNA (group 1), coated with VEGF-plasmid DNA (group 2), loaded with mesenchymal stem cells (BMSC) transfected with control plasmid (group 3) or with both stem cells and the VEGF plasmid (group 4). X-rays were taken every 4 weeks up to week 16, when animals were euthanized. The volume of new bone was measured by mu-CT scans and blood vessels were counted after anti-CD31 staining of endothelial cells. The results from the solitary VEGF- and VEGF-transfected cells (groups 2 and 4) demonstrated significantly enhanced vascularization, osteogenesis and resorption of the carrier when compared to the control group. The highest degree of osteogenesis was found when the carrier was loaded with BMSC (group 3), whereas VEGF-transfected cells led to the highest vascularization and fastest resorption of the bone substitute. Additionally, VEGF-transfected BMSC led to a more homogenous vascularization of the defect. The results indicate that VEGF can be a helpful factor to improve healing in large bone defects, in which bone substitutes will otherwise not be vascularized and replaced by fresh bone.     

Direct percutaneous gene delivery to enhance healing of segmental bone defects

BACKGROUND: Healing of segmental bone defects can be induced experimentally with genetically modified osteoprogenitor cells, an ex vivo strategy that requires two operative interventions and substantial cost. Direct transfer of osteogenic genes offers an alternative, clinically expeditious, cost-effective approach. We evaluated its potential in a well-established, critical-size, rat femoral defect model. METHODS: A critical-size defect was created in the right femur of forty-eight skeletally mature Sprague-Dawley rats. After twenty-four hours, each defect received a single, intralesional, percutaneous injection of adenovirus carrying bone morphogenetic protein-2 (Ad.BMP-2) or luciferase cDNA (Ad.luc) or it remained untreated. Healing was monitored with weekly radiographs. At eight weeks, the rats were killed and the femora were evaluated with dual-energy x-ray absorptiometry, micro-computed tomography, histological analysis, histomorphometry, and torsional mechanical testing. RESULTS: Radiographically, 75% of the Ad.BMP-2-treated femora showed osseous union. Bone mineral content was similar between the Ad.BMP-2-treated femora (0.045 +/- 0.020 g) and the contralateral, intact femora (0.047 +/- 0.003 g). Histologically, 50% of the Ad.BMP-2-treated defects were bridged by lamellar, trabecular bone; the other 50% contained islands of cartilage. The control (Ad.luc-treated) defects were filled with fibrous tissue. Histomorphometry demonstrated a large difference in osteogenesis between the Ad.BMP-2 group (mean bone area, 3.25 +/- 0.67 mm(2)) and the controls (mean bone area, 0.65 +/- 0.67 mm(2)). By eight weeks, the Ad.BMP-2-treated femora had approximately one-fourth of the strength (mean, 0.07 +/- 0.04 Nm) and stiffness (mean, 0.5 +/- 0.4 Nm/rad) of the contralateral femora (0.3 +/- 0.08 Nm and 2.0 +/- 0.5 Nm/rad, respectively). CONCLUSIONS: A single, percutaneous, intralesional injection of Ad.BMP-2 induces healing of critical-size femoral bone defects in rats within eight weeks. At this time, the repair tissue is predominantly trabecular bone, has normal bone mineral content, and has gained mechanical strength.     

Autogeneic bone marrow and porous biphasic calcium phosphate ceramic for segmental bone defects in the canine ulna.

The purpose of this study was to evaluate a porous biphasic hydroxyapatite-calcium phosphate ceramic as a modifier and extender of an autogeneic marrow graft for filling a 2.5-cm segmental bony defect. Twenty adult mongrel dogs were surgically treated to create diaphyseal defects in the left ulnae. The defects were (1) filled with autogeneic bone marrow mixed with granular hydroxyapatite-tricalcium phosphate ceramic (granular ceramic); (2) grafted with a solid block of ceramic soaked in autogeneic bone marrow (block ceramic); (3) received no graft (no implant); or (4) were grafted with autogeneic bone marrow alone (bone marrow). All animals were followed clinically and roentgenographically for 24 weeks and then killed. Repair of diaphyseal defects with the block ceramic led to three solid unions and three fibrous unions; with the granular ceramic implants and marrow, the defects of five dogs formed solid unions, and one progressed to a fibrous union. Defects in all five dogs grafted with autogeneic bone marrow united. The three dogs with no implant formed nonunions. Histology showed normal marrow and only a light immune reaction. Complete bridging of the defect in the dogs treated with the granular ceramic occurred significantly earlier than bridging in the dogs grafted with bone marrow alone. Histomorphometry, performed on the block ceramic implants indicated active resorption of ceramic. Clinically, addition of ceramic to a marrow graft improved the handling characteristics of the graft material and accelerated healing according to roentgenographic evaluation.     

Treatment of nonunion by percutaneous injection of bone marrow and demineralized bone matrix. An experimental study in dogs

Successful treatment of nonunited fractures remains a major clinical challenge. Because bone marrow and demineralized bone matrix (DBM) are capable of stimulating osteogenesis, experiments were designed to test the effectiveness of bone marrow or DBM or both when injected percutaneously into a canine nonunion model. Six-millimeter segmental defects were created in the midtibial diaphysis of 24 adult mongrel dogs and held distracted by external fixation. For comparative purposes, a 0.5-mm osteotomy was created in five dogs. Five weeks later, the 6-mm defects were injected with either saline, autogeneic marrow, DBM powder, a composite of bone marrow and DBM, or treated by open grafting techniques with autogenic cancellous bone. Healing of the defect was evaluated roentgenographically, biomechanically (three-point bending), histologically, and biochemically 13 weeks postsurgery. Marrow and DBM stimulated defect healing. However, the combination of bone marrow with DBM produced a synergistic response in the defect, which was greater than the sum of either marrow or DBM alone. Healing in the composite-grafted dogs was comparable to those treated by standard cancellous bone grafting. These data suggest that percutaneous injection of bone marrow and DBM may be a potential alternative that offers numerous advantages over standard open grafting techniques in the treatment of fractures with nonunited defects.     

延时脱钙和骨基质几何形态对其成骨能力的影响

目的：研究延时脱钙和骨的几何形状对骨基质成骨能力的影响。方法：切取鼠股骨干分成5组。骨干24h组，骨干48h组（对照组），骨干48B组和骨干72h组，分别用0.6NHC1脱钙24，48或72h，或颗粒48B组的骨碾磨成125-800μm的颗粒，50只Wistar鼠作为受体，术后6周取标本测定重量，干燥后的重量，钙含量，碱性磷酸酶含量和组织学观察。结果：颗粒48H组的干燥重量高于控制组，二者有统计学差别。结论：骨延时脱钙不影响其成骨能力。125-800μm的颗粒状鼠骨基质成骨能力优于块状骨基质。     

钻孔复合人工骨体内植入成骨的实验研究

目的探索符合临床需要的复合人工骨移植材料。方法以磷酸三钙＋多孔羟基磷灰石 (TCP＋ HA)为载体 ,分为钻孔、未钻孔及空白对照三组。自成年新西兰兔股骨转子部取得骨髓基质细胞进行传代培养,所得骨髓基质细胞再与钙磷陶瓷载体复合培养制成复合人工骨。然后植入兔背部肌肉内,分别于手术后第 2、 4、 8、 12周取材。利用组织学和电子显微镜等方法观察细胞在载体内的生长、复合人工骨植入肌肉内的成骨等情况,以及载体钻孔对细胞长入和成骨的影响。结果电子显微镜观察显示,传代的骨髓基质细胞与载体共同培养生长良好,并可长入钻孔载体内部。大体标本可见,复合人工骨植入后,人工骨与周围肌肉连接,无包膜形成。组织学观察显示,术后第 2周组织和血管长入;术后第 4～ 8周有少量骨形成;术后第 12周板层骨形成。三组均无淋巴细胞浸润。与未钻孔者比较,钻孔人工骨的细胞、组织和血管向中心部位生长速度快,中心部位成骨多且更均匀。结论磷酸三钙＋多孔羟基磷灰石载体具有良好的生物相容性及骨传导性;骨髓基质细胞具有成骨能力;载体钻孔有利于增加新骨形成的速度和数量,使成骨更为均匀。     

Repair of bone defects with marrow cells and porous ceramic. Experiments in rats

We studied the role of bone-marrow reconstituted porous ceramics in enhancing healing of a 5-mm femoral diaphyseal defect fixed with a rigid polyethylene plate in rats. Osseous repair was evaluated by histologic scoring. When blocks of porous calcium phosphate ceramics alone were introduced into the defects, most cases showed fibrous tissue interposition at the host bone-ceramic junction 1 month after implantation, and only four of 12 defects developed osseous or osteochondral union at both the proximal and distal junctions 2 months after surgery. However, when the ceramic was combined with syngeneic viable marrow cells, new bone formation occurred in isolated pore regions of the ceramic at 1 month, and extensive bone formation was seen in most pore regions 2 months after implantation. Out of 12 implants, complete bone union was seen in eight, and one showed osseous or osteochondral union at both junctions 2 months after surgery. Our results indicated that composite grafts of porous calcium phosphate ceramics and marrow cells may be clinically applicable to enhance osteogenesis and osteoconduction.     

Tissue-engineered bone constructed in a bioreactor for repairing critical-sized bone defects in sheep

Purpose

Repair of bone defects, particularly critical-sized bone defects, is a considerable challenge in orthopaedics. Tissue-engineered bones provide an effective approach. However, previous studies mainly focused on the repair of bone defects in small animals. For better clinical application, repairing critical-sized bone defects in large animals must be studied. This study investigated the effect of a tissue-engineered bone for repairing critical-sized bone defect in sheep.

Methods

A tissue-engineered bone was constructed by culturing bone marrow mesenchymal-stem-cell-derived osteoblast cells seeded in a porous β-tricalcium phosphate ceramic (β-TCP) scaffold in a perfusion bioreactor. A critical-sized bone defect in sheep was repaired with the tissue-engineered bone. At the eighth and 16th week after the implantation of the tissue-engineered bone, X-ray examination and histological analysis were performed to evaluate the defect. The bone defect with only the β-TCP scaffold served as the control.

Result

X-ray showed that the bone defect was successfully repaired 16 weeks after implantation of the tissue-engineered bone; histological sections showed that a sufficient volume of new bones formed in β-TCP 16 weeks after implantation. Eight and 16 weeks after implantation, the volume of new bones that formed in the tissue-engineered bone group was more than that in the β-TCP scaffold group (P?Conclusion Tissue-engineered bone improved osteogenesis in vivo and enhanced the ability to repair critical-sized bone defects in large animals.     

Pulverized bone matrix as an injectable bone graft in rabbit radius defects

A segment of the rabbit radius diaphysis was excised, demineralized, and pulverized. The demineralized matrix particles were mixed with autologous bone marrow from the femoral canal and injected into the defect from which it had been excised. On the contralateral side, the demineralized bone was reimplanted without pulverization, but with bone marrow. The bone yield was measured by radiographic planimetry and Tc99m MDP scintimetry. The forearms of the rabbits were sectioned into transverse segments, including the middle of the radius defect. The ash weights and the Ca45 content of these segments were measured. After two weeks, the ash weight was greater on the pulverized than on the unpulverized side; but by four weeks, measurements showed no difference. In general, the injectable bone matrix preparation did not interfere with bone repair. In comparing the Tc99m and Ca45 methods, the latter provided high-precision data with respect to the biologic variation.     

活性煅烧骨的临床应用

把骨形态发生蛋白（ＢｏｎｅＭｏｒｐｈｏｇｅｎｅｔｉｃＰｒｏｔｅｉｎ，ＢＭＰ）按１∶２０的重量比例，与煅烧异种松质骨（ＴｒｕｅＢｏｎｅＣｅｒａｍｉｃ，ＴＢＣ）组成活性煅烧骨（ＢＭＰ／ＴＢＣ复合物），经动物实验揭示该复合体具有良好的填充修复骨缺损的能力，临床应用５３例病人，４８例随访８个月～３．５年，平均１８．６月，均取得满意疗效。作者认为自制煅烧骨是理想的载体。活性煅烧骨（ＢＭＰ／ＴＢＣ复合物）具有高效的诱导成骨能力，不引起免疫排斥反应，来源丰富，易于大量加工贮存，有广阔的临床应用前景。     

基因修饰的组织工程骨联合带血管蒂骨膜移植修复长段骨缺损的研究

目的评价骨形态发生蛋白2（bone morphogenetic protein2,BMP-2）基因修饰的组织工程骨联合带血管蒂骨膜移植修复长段骨缺损的效果。方法分离培养兔骨髓基质干细胞,经BMP-2基因转染后复合异种骨支架体外构建基因修饰的组织工程骨（gene modified tissue engineering bone,GMB）。建立兔双侧桡骨缺损（长2.5cm）模型,采用5种方法修复。A组：GMB＋带血管蒂骨膜移植;B组：GMB＋血管束植入;C组：GMB＋游离骨膜移植;D组：GMB;E组：单纯支架。于术后第4、8、12周行X线、组织学、生物力学测定和微血管墨汁灌注等观察血管形成及成骨情况。结果①A组血运建立快,第8周时即可修复骨缺损,其修复机制包括膜内成骨和软骨成骨两种机制;②B组血管束发出分支向移植骨内长入,但中心区成骨缓慢,第12周时骨缺损得到完全修复;③C组第4周时游离骨膜成活并发出微小血管,第8周时形成薄层外骨痂,第12周时骨缺损基本修复;④D组在BMP-2基因诱导下成骨速度和质量优于E组,可在第12周时使骨缺损部分修复,但中心区呈＂空心＂现象;而E组第12周时形成骨不连,缺损区内被纤维组织填充。结论带血管蒂骨膜与BMP-2基因修饰的组织工程骨联合移植,既提供了血运又提供了骨膜成骨细胞,同时具有良好的骨生成、骨诱导和骨引导作用,是治疗节段性骨缺损较为理想的方法。     

BMP2基因修饰犬脂肪源性基质细胞修复自体大段骨缺损

目的 评价BMP2基因修饰的犬脂肪源性基质细胞(ADSCs)与β-磷酸三钙(β-TCP)复合修复自体大段骨缺损的疗效.方法 从比格犬背部脂肪组织中提取基质细胞,转染腺病毒介导的人BMP2基因(Adv-hBMP2),通过ELISA和裸鼠体内异位成骨实验鉴定BMP2的表达及异位成骨活性;取比格犬11只,制作双侧尺骨2.5 cm骨缺损模型,缺损处旷置(4侧)或随机填充:单纯TCP(6侧),AD-SCs+TCP(6侧),Adv-hBMP2-ADSCs+TCP(6侧).所有动物16周后处死.结果 ELISA显示犬ADSCs被腺病毒转染后可高表达具有成骨活性的BMP2;裸鼠体内异位成骨实验证实所分泌的BMP2具有骨诱导活性.骨缺损修复的X线片显示,单纯TCP和ADSCs+TCP组至16周时,骨缺损均未愈合;BMP2基因修饰ADSCs+TCP组6例中2例愈合,4例部分愈合.显微摄片和组织学观察示:单纯TCP和AD-SCs+TCP组仅在骨缺损断端形成编织骨,缺损中央被纤维组织填充,而Adv-hBMP2-ADSCs+TCP组骨.缺损处皮质连续,新生骨组织主要为编织骨,部分改建形成板层骨.组织形态学分析示BMP2基因修饰ADSCs明显促进了新骨形成.结论 BMP2基因修饰ADSCs可以修复犬尺骨大段骨缺损.     

带血管蒂骨膜、骨瓣移植和血管束植入修复骨缺损的实验研究

目的 将松质骨移植到骨缺损处,再将带血管蒂骨膜,骨瓣移植和血管束植入,以探讨不同方法对骨缺损修复过程的影响。方法 制作兔桡骨骨缺损模型,移植松质骨后,分别用带血管蒂的骨膜,骨瓣移植和血管束植入,单纯松质骨移植作对照,术后４,８,１６周进行大体标本,Ｘ线,光镜及电镜观察。结果 骨膜移植组血运建立快,成骨面积大,骨缺损修复快,质量最好。骨瓣移植组是通过带血管蒂的骨瓣移植增加了血液供应,成骨较快且好;但     

Ilizarov treatment of tibial nonunions with bone loss

Twenty-five patients aged 19-62 years were treated for tibial nonunions (22 atrophic, three hypertrophic) with bone loss (1-23 cm, mean 6.2 cm) by the Ilizarov technique and fixator. Thirteen had chronic osteomyelitis, 19 had a limb-length discrepancy (2-11 cm), 12 had a bony defect (1-16 cm), and 13 had a deformity. Six had a bone defect with no shortening, 13 had shortening with no defect, and six had both a bone defect and shortening. Nonunion, bone defects, limb shortening, and deformity can all be addressed simultaneously with the Ilizarov apparatus. Bone defects were closed from within without bone grafts by the Ilizarov bone transport technique of sliding a bone fragment internally, producing distraction osteogenesis behind it until the defect is bridged (internal lengthening). Length was reestablished by distraction of a percutaneous corticotomy or through compression and subsequent distraction of the pseudarthrosis site (external lengthening). Distraction osteogenesis resulting from both processes obviated the need for a bone graft in every case. Deformity was corrected by means of hinges on the apparatus. Infection was treated by radical resection of the necrotic bone and internal lengthening to regenerate the excised bone. Union was achieved in all cases. The mean time to union was 13.6 months, but it was only 10.6 months if the time taken for unsuccessful compression-distraction of the nonunion is eliminated from the calculation. The bone results were excellent in 18 cases, good in five, and fair in two based on union in all cases, persistent infection in three, deformity in four, and limb shortening in one. The functional results were excellent in 16 cases, good in seven, fair in one, and poor in one based on return to work and daily activities in all cases, limp in four cases, equinus deformity in five cases, dystrophy in four cases, pain in four cases, and voluntary amputation for neurogenic pain in one case.     

Distraction osteogenesis in management of composite bone and soft tissue defects

A soft tissue defect is one of the most difficult problems that may accompany bone defects. Plastic surgery is often required. During distraction osteogenesis, not only the bone but also the soft tissues are lengthened, which may help in spontaneous closure of the soft tissue defects. This study examines 11 cases of composite bone and soft tissue defects which were managed by distraction osteogenesis. After debridement of the necrotic tissues, the soft tissue defects ranged from three by four centimetres to five by 14 centimetres and the bone defects ranged from four to 12 centimetres. All the soft tissue defects healed during the process of bone transport without the need for plastic surgery, except in one case. The complications were successfully managed during the course of treatment. Distraction osteogenesis is a good method for simultaneous treatment of composite bone and soft tissue defects.     

Injizierbare Trägersysteme für die Wachstumsfaktorapplikation zur minimal-invasiven Knochenheilungsstimulation

The characterization and cloning of growth factors for bone healing provide an enormous potential for minimally invasive treatment procedures for bone defects or fractures. However, the clinical application of injection vehicles for these growth factors must be made user-friendlier. In this study, two different injection vehicles were tested for their practicability and efficacy to enhance callus maturation during distraction osteogenesis. Calcium phosphate carriers showed a rather low user-friendliness and were less efficient in the animal model of distraction osteogenesis in sheep. Collagen carriers provided both a higher practicability for injection procedures and a higher efficacy.