同种异体骨髓间充质干细胞关节腔内成软骨分化的实验研究

目的检测同种异体间充质干细胞在不接受免疫抑制治疗的正常羊关节腔内分化形成软骨能力。方法将同种异体间充质干细胞复合β-磷酸三钙陶瓷植入正常羊关节腔内。在进行干细胞体内植入前进行淋巴细胞混合试验,确保供体与受体动物间存在免疫排斥反应。异体干细胞植入8周后,取出植入物进行组织学与免疫组化分析。将其结果与自体间充质干细胞复合β-磷酸三钙陶瓷关节腔内植入以及无细胞复合的单纯β-磷酸三钙陶瓷关节腔内植入相应检测结果进行对比分析。通过检测受体血清内产生的针对供体细胞的特异性抗体,评估系统性免疫反应。结果未检测到针对同种异体间充质干细胞移植所产生的特异性免疫排斥反应。在复合异体干细胞的植入物内没有发生明显炎性细胞浸润,并且没有检测到有针对异体细胞的抗体产生。组织学和免疫组化分析表明在复合异体及自体间充质干细胞的植入物内有新生软骨形成。在无细胞复合的植入物内未检测到软骨形成。结论受关节腔环境影响,同种异体间充质干细胞能够分化形成软骨组织。在被移植到不经免疫抑制治疗的同种异基因动物体内后,细胞陶瓷复合体不引起特异性免疫排斥反应。     

Repairing of goat Tibial Bone Defects with BMP-2 Gene–Modified Tissue-Engineered Bone

Bone defects larger than a critical size are major challenges in orthopedic medicine. We combined tissue-engineered bone and gene therapy to provide osteoprogenitor cells, osteoinductive factors, and osteoconductive carrier for ideal bone regeneration in critical-sized bone defects. Goat diaphyseal bone defects were repaired with tissue and genetically engineered bone implants, composed of biphasic calcined bone (BCB) and autologous bone marrow derived mesenchymal stem cells (BMSC) transduced with human bone morphogenetic protein-2 (hBMP-2). Twenty six goats with tibial bone defects were divided into groups receiving implants by using a combination of BCB and BMSCs with or without the hBMP-2 gene. In eight goats that were treated with BCB that contained hBMP-2 transduced BMSC, five had complete healing and three showed partial healing. Goats in other experimental groups had only slight or no healing. Furthermore, the area and biochemical strength of the callus in the bone defects were significantly better in animals treated with genetically engineered implants. We concluded that the combination of genetic and tissue engineering provides an innovative way for treating critical-sized bone defects.     

Bone regeneration by implantation of purified,culture-expanded human mesenchymal stem cells

Bone marrow contains a population of rare progenitor cells capable of differentiating into bone, cartilage, tendon, and other connective tissues. These cells, referred to as mesenchymal stem cells, can be purified and culture-expanded from animals and humans and have been shown to regenerate functional tissue when delivered to the site of musculoskeletal defects in experimental animals. To test the ability of purified human mesenchymal stem cells to heal a clinically significant bone defect, mesenchymal stem cells isolated from normal human bone marrow were culture-expanded, loaded onto a ceramic carrier, and implanted into critical-sized segmental defects in the femurs of adult athymic rats. For comparison, cell-free ceramics were implanted in the contralateral limb. The animals were elthanized at 4, 8, or 12 weeks, and healing bone defects were compared by high-resolution radiography, immunohistochemistry, quantitative histomorphometry, and biomechanical testing. In mesenchymal stem cell-loaded samples, radiographic and histologic evidence of new bone was apparent by 8 weeks and histomorphometry demonstrated increasing bone formation through 12 weeks. Biomechanical evaluation confirmed that femurs implanted with mesenchymal stem cell-loaded ceramics were significantly stronger than those that received cell-free ceramics. These studies demonstrate that human mesenchymal stem cells can regenerate bone in a clinically significant osseous defect and may therefore provide an alternative to autogenous bone grafts.     

Engineered allogeneic mesenchymal stem cells repair femoral segmental defect in rats.

Bone marrow derived mesenchymal stem cells (MSC) have been shown to be progenitor cells for mesenchymal tissues. These cells may also provide a potential therapy for bone repair. Our previous studies showed that MSC engineered with the gene for bone morphogenetic protein 2 (BMP-2), a growth factor for bone cells, were capable of differentiating into osteoblast lineage and inducing autologous bone formation in several animal models. Culturing individual MSC for autologous implantation, however, remains problematic. The number of human MSC with osteogenic potential decreases with age, and, in certain diseases, the patient's marrow may be damaged or the healthy cells reduced in number. In this study, we used rats with a femoral segmental defect to investigate whether allogeneic BMP-2 engineered MSC would facilitate bone healing. We show that BMP-2 engineered allogeneic MSC can repair critical bone defects to the same degree as rats treated with BMP-2 engineered autologous MSC, if the allogeneic group receives short-term treatment with immunosuppressant FK506. We also show that allogeneic gene transferred MSC are directly involved in bone repair, in addition to acting as gene deliverers.     

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.     

Scale‐up of MSC under hypoxic conditions for allogeneic transplantation and enhancing bony regeneration in a rabbit calvarial defect model

To realize the therapeutic potential of mesenchymal stem cells (MSCs), we aimed to develop a method for isolating and expanding New Zealand rabbit MSCs in a great scale. Rabbit MSCs expanded under hypoxic and normoxic conditions were compared in terms of replication capacity, differentiation potential, and the capacity for allogeneic transplantation in a calvarial defect model. The cells from all tested rabbits were expanded more rapidly when plated at low‐density under hypoxic conditions compared to under normoxic conditions. Moreover, cells expanded under hypoxic conditions increased in the potential of osteoblastic, adipocytic, and chondrocytic differentiation. More importantly, radiographic analysis and micro‐CT measurement of bone volume revealed the hypoxic cells when transplanted in the calvarial defects of another rabbit increased in the ability to repair bone defect compared to the normoxic cells. Six weeks after allogeneic transplantation of hypoxic MSCs, histological analysis revealed a callus spanned the length of the defect, and several bone tissues spotted in the implant. At 12 weeks, new bone had formed throughout the implant. Using BrdU labeling to track the transplanted cells, the hypoxic cells were more detected in the newly formed bone compared to the normoxic cells. For defects treated with allogeneic MSCs, no adverse host response could be detected at any time‐point. In conclusion, we have developed a robust method for isolation and expansion of rabbit MSCs by combining low‐density with hypoxic culture, which can be applied for the design of clinical trials in allogeneic transplantation of MSCs for bone healing. © 2012 Orthopaedic Research Society. Published by Wiley Periodicals, Inc. J Orthop Res 30:1213–1220, 2012     

构建组织工程骨修复兔颅骨极限缺损的实验研究

目的观察以胶原缓释重组人骨形成蛋白2(recom b inan t hum an bone m orphogenetic prote in 2,rhBM P-2)复合骨髓间充质干细胞(m arrow m esenchym a l stem ce lls,M SC s)及珊瑚构建的组织工程骨修复兔颅骨极限缺损的能力。方法新西兰大白兔40只,制备颅骨极限缺损,按植入的修复物不同随机分为5组,每组8只。Ⅰ组:自体髂骨,为阳性对照组;Ⅱ组:珊瑚,为阴性对照组;Ⅲ组:rhBM P-2+珊瑚;Ⅳ组:胶原+rhBM P-2+珊瑚;Ⅴ组:M SC s+胶原+rhBM P-2+珊瑚。将其分别植入兔颅骨极限缺损处,术后8、16周行大体观察、X线片、HE染色及M asson三色染色法观察比较骨缺损修复的情况。结果术后Ⅴ组材料与Ⅰ组修复颅骨极限缺损的效果相近,缺损区大体标本可见骨样组织充填,硬度与周边骨质相近,并与周边骨质形成明显骨融合;X线阻射程度高,16周时达80.45%±2.52%;组织学观察为板层状结构的新骨组织,空白孔隙区较少。Ⅳ组修复效果次之,Ⅲ组材料成骨能力较弱,Ⅱ组大部为半透明的纤维薄膜,缺损区界限清晰。结论胶原是rhBM P-2适宜的缓释载体,胶原及M SC s对促进复合支架材料修复骨缺损有重要意义。以M SC s+胶原+rhBM P-2+珊瑚构建的组织工程骨可成为一种良好的骨缺损修复材料。     

人脐血间充质干细胞修复颅骨缺损的实验研究

目的 应用人脐血间充质干细胞(umbilical cord blood derived mesenchymal stem cells,UCB-MSCs)复合脱钙骨材料构建组织工程化骨,修复裸大鼠颅骨标准缺损.方法 体外扩增培养、成骨诱导人UCB-MSCs,采用Alizarin Red染色和钙离子半定量的方法测定细胞成骨分化能力.将第2代细胞接种在脱钙骨支架材料上继续诱导培养,扫描电镜检测细胞在材料上的生长状况.制备裸大鼠双侧颅骨全层标准缺损(直径5 mm),一侧以细胞材料复合物修复作为实验侧(n=8);另一侧以单纯脱钙骨材料修复作为对照侧(n=8).术后6、12周取材,分别通过大体形态观察、显微CT(Micro-CT)、组织学方法检测颅骨缺损的修复效果.结果 UCB-MSCs体外能够诱导分化为成骨细胞,且在脱钙骨支架材料上生长良好.Micro-CT检测显示术后6周实验侧有部分新生骨组织形成,12周时骨缺损修复率达(78.19±6.45)%,脱钙骨材料降解完全;对照侧6周时无明显新骨生成,12周时材料完全降解,骨缺损未修复.组织学检测显示12周时实验侧有较多成熟骨生成,为骨性愈合;对照侧骨缺损为纤维愈合.结论 成骨诱导的人UCB.MSCs复合脱钙骨材料构建的组织工程化骨可修复裸大鼠颅骨全层标准缺损,有望成为新的组织工程骨种子细胞来源.     

异体DBM复合rhBMP-2修复兔桡骨缺损的实验研究

目的探讨异体脱钙骨基质（demineralized bonematrix，DBM）复合重组人骨形态发生蛋白2（reconstruction humn bonemorphology protein-2，rhBMP-2）修复节段性骨缺损的能力。方法48只新西兰大白兔采用桡骨15mm节段性骨缺损模型，随机分为3组，A组植入异体DBM与rhBMP-2复合材料，B组植入异体DBM，C组为空白对照组。术后4周、8周、12周、16周．进行放射学和组织学检查。结果A组：术后4周宿主结缔组织长入植骨材料内的骨小梁间，并有岛状新生软骨、骨组织形成；术后8周，新生软骨及骨形成并融合成片；术后12周，新骨改建成熟，但仍能见到植骨材料；术后16周，管状骨结构形成，髓腔再通。B组：术后4周，植骨材料周围有软骨形成；术后8周，大量软骨形成；术后12周，大片状骨形成；术后16周，有髓腔形成。C组：各时问点仅见有纤维结缔组织，只在两端有新骨形成。X片示A组成骨量大，新骨改建、成熟迅速，术后16周全部达骨性愈合。B组成骨量少，仅2例达骨性愈合。C组未见骨性愈合。结论异体DBM复合rhBMP-2材料通过骨诱导和骨传导两种方式修复骨缺损，是一种较理想、具有高效成骨活性的植骨材料。     

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

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

不同应力环境对兔骨髓间充质干细胞修复关节软骨缺损的影响

目的探讨不同应力环境对骨髓间充质干细胞(MSCs)修复关节软骨缺损的影响. 方法将日本大耳白兔15只制成髌骨外侧脱位动物模型,平均分成3组,每组5只:即单纯载体脱位组(对照组)、移植物正常应力组及移植物脱位组.对兔MSCs进行分离、培养,以兔MSCs为种子细胞构建自体组织工程移植物修复关节软骨缺损.6周后处死动物,观察修复组织的成分和结构. 结果术后6周,移植物正常应力组修复组织浅层为软骨组织,甲苯胺蓝染色接近正常关节软骨;深层为软骨下骨,与正常关节软骨结构相似.移植物脱位组为骨组织所修复,缺损周围的正常关节软骨变薄,软骨下血管侵入正常关节软骨内,遗留在股骨髁滑车槽内的移植物在滑车槽正常关节软骨表面形成新生类透明软骨组织.单纯载体脱位组为纤维组织修复. 结论 MSCs修复关节软骨缺损,只有在正常应力状态下修复效果最佳;提示维持负重关节正常的应力刺激,对组织工程软骨修复组织的形成和维持必不可少.     

Osteochondral progenitor cells in acute and chronic canine nonunions.

This study examined the ability of cells isolated from early healing segmental defects and from tissue from chronic nonunions to support bone and cartilage formation in vivo and their response to transforming growth factor-beta1 in vitro. Ostectomies (3 mm) were created in the radial diaphysis of four dogs. The dogs were splinted 3-5 days postoperatively and then allowed to bear full weight. At 7 days, tissue in the defect was removed and any periosteum was discarded; cells in the defect tissue were released by enzymatic digestion. The dogs were splinted again and allowed to bear full weight for 12 weeks. Radiographs confirmed a persistent nonunion in each dog. Defect tissue was again removed, any periosteum was discarded, and cells were isolated. Cells were also obtained from the defect tissue by nonenzymatic means with use of explant cultures. One-half of the tissue and one-half of any preconfluent, first-passage cultures were shipped to Cleveland by overnight carrier. At second passage, cells were loaded into ceramic cubes and implanted into immunocompromised mice for 3 or 6 weeks. Harvested cubes were examined histologically for cartilage and bone with use of a semiquantitative scoring system. Confluent fourth-passage cultures of 7 and 84-day defect tissue cells were cultured with 0.03-0.88 ng/ml transforming growth factor-beta1 for 24 hours, and [3H]thymidine incorporation and alkaline phosphatase specific activity were determined. Donor-dependent differences were noted in the rate at which defect cells achieved confluence; in general, cells from 7-day tissue divided most rapidly. Seven-day defect cells formed less bone and at a slower rate than was seen in the ceramic cubes containing samples from day 84. Cells derived enzymatically behaved similarly to those from explant cultures. Ceramic cubes contained fibrous connective tissue, cartilage, bone, and fat, indicating that multipotent cells were present. Stimulation of [3H]thymidine incorporation in response to transforming growth factor-beta1 was donor dependent and variable; only two of six separate isolates of cells exposed to it had measurable alkaline phosphatase activity (which was relatively low), and none of the cultures exhibited an increase in response to transforming growth factor-beta1 for 24 hours. This indicates that mesenchymal progenitor cells are present in the healing defect tissue at 7 and 84 days and that the relative proportion of osteochondroprogenitor cells is greater at the later time. The response to transforming growth factor-beta1 is typical of multipotent mesenchymal cells but not of committed chondrocytes or osteoblasts, indicating that these committed and differentiated cells are not present in early stages of healing and suggesting that their differentiation is inhibited in chronic nonunion.     

Massive bone reconstruction with heat‐treated bone graft loaded autologous bone marrow‐derived stromal cells and β‐tricalcium phosphate composites in canine models

Bone marrow‐derived stromal cells (BMSCs) contain mesenchymal stem cells that are capable of forming various mesenchymal tissues. We hypothesized that BMSCs and β‐tricalcium phosphate (β‐TCP) composites would promote the remodeling of large‐sized autologous devitalized bone grafts; therefore, the aim of this study was to evaluate the effects of the composites on the remodeling of autologous devitalized bone grafts. Autologous BMSCs cultured in culture medium containing dexamethasone (10^?7 M) were loaded into porous β‐TCP granules under low‐pressure. Theses BMSC/TCP composites were put into the bone marrow cavity of autologous heat‐treated bone (femoral diaphysis, 65‐mm long, 100°C, 30 min) and put back to the harvest site. In the contralateral side, β‐TCP without BMSC were used in the same manner as the opposite side as the control. Treatment with the BMSC/TCP composites resulted in a significant increase in thickness, bone mineral density, and matured bone volume of the cortical bone at the center of the graft compared to the control. Histological analysis showed matured regenerated bone in the BMSC loaded group. These results indicate that BMSC/TCP composites facilitated bone regeneration and maturation at the graft site of large‐sized devitalized bone. This method could potentially be applied for clinical use in the reconstruction of large bone defects such as those associated with bone tumors. © 2013 Orthopaedic Research Society Published by Wiley Periodicals, Inc. J Orthop Res 31:1308–1316, 2013

     

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.     

Modified Masquelet technique using allogeneic umbilical cord-derived mesenchymal stem cells for infected non-union femoral shaft fracture with a 12 cm bone defect: A case report

IntroductionNon-union due to large bone loss often causes significant long-term morbidity. We incorporate the use of allogeneic umbilical cord-derived mesenchymal stem cells (UC-MSCs) as part of the diamond concept of regenerative medicine in a case of infected non-union fracture.Presentation of caseWe reported a 54-year-old female patient presenting with pain on the right thigh. She was previously diagnosed with a closed fracture of the right femoral shaft and underwent four surgeries before finally being referred to Dr. Cipto Mangunkusumo General Hospital with infected non-union of the right femoral shaft. The patient was treated with a combination of UC-MSCs, bone morphogenetic protein-2 (BMP-2), Hydroxyapatite (HA), and mechanical stabilization using Masquelet Technique. The combination of allogeneic MSCs, BMP2, HA, and Masquelet Technique was successful in creating new bone with no apparent side effects.DiscussionBone loss might be caused by external factors (true defects), or structural loss of the existing bone. The combination of allogeneic UC-MSCs, BMP-2, HA and an induced membrane technique pioneered by Masquelet allowed for faster regeneration process and more optimal bone healing. This paper aims to assess and compare the result of such procedures with the previous four surgeries done to the patient, which did not yield satisfactory results.ConclusionThe application of allogeneic UC-MSC, BMP-2, HA and Masquelet technique as proposed in the diamond concept is a viable method in treating critical-sized bone defect and provides an effective way to overcome non-union caused by large defect.     

Use of mesenchymal stem cells in a collagen matrix for achilles tendon repair

This investigation tested the hypothesis that delivering mesenchymal stem cell-seeded implants to a tendon gap model results in significantly improved repair biomechanics. Cultured, autologous, marrowderived mesenchymal stem cells were suspended in a collagen gel delivery vehicle; the cell-gel composite was subsequently contracted onto a pretensioned suture. The resulting tissue prosthesis was then implanted into a 1-cm-long gap defect in the rabbit Achilles tendon. Identical procedures were performed on the contralateral tendon, but only the suture material was implanted. The tendon-implant constructs were evaluated 4, 8, and 12 weeks later by biomechanical and histological criteria. Significantly greater load-related structural and material properties were seen at all time intervals in the mesenchymal stem cell-treated tendons than in the contralateral, treated control repairs (p < 0.05), which contained suture alone with natural cell recruitment. The values were typically twice those for the control tissues at each time interval. Load-related material properties for the treated tissues also increased significantly over time (p < 0.05). The treated tissues had a significantly larger cross-sectional area (p < 0.05), and their collagen fibers appeared to be better aligned than those in the matched controls. The results indicate that delivering mesenchymal stem cell-contracted, organized collagen implants to large tendon defects can significantly improve the biomechanics, structure, and probably the function of the tendon after injury.     

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.     

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.     

Healing course of primate ulna segmental defects treated with osteogenic protein-1.

Twelve African green monkeys were implanted with recombinant human osteogenic protein-1 (rhOP-1) placed on a bovine bone-derived Type I collagen carrier to characterize healing in an ulna segmental bone defect model at 1, 3, 12, and 20 weeks postoperative. Defect healing was evaluated by plain film radiography, computed tomography (CT), magnetic resonance imaging (MRI), bone mineral density (BMD), and histologic analysis. Radiographically, new bone formation was observed as early as 3 weeks postoperative. By 6 weeks, new bone was visible in five of six defects. Increased quantity and mineralization of the new bone were apparent by 12 weeks. Reformation of the medullary cavity with appearance of marrow elements was demonstrated by CT and MRI at 20 weeks. BMD studies revealed a significant increase in the presence of bone with time. Histology at 1 week demonstrated that the implant material was well contained in the defect, and a proliferation of cells occurred at the defect borders. At 3 weeks cell proliferation continued and cell phenotype differentiation was recognized. By 12 weeks substantially less residual carrier was found in the defects, and calcifying tissues with plump chondrocytes, osteoblasts, and immature woven bone were observed. Areas of lamellar and woven bone were identified at 12 weeks, with advanced remodeling and revascularization observed at 20 weeks. The use of osteoinductive implants may provide an alternative to autologous and allogeneic bone tissue in the therapeutic approach to bone defects and promotion of fusion by eliminating the donor site morbidity associated with autogenous bone and the decreased efficacy and potential for disease transmission associated with allogeneic bone.     

Ivan Petrovich Pavlov

Twelve African green monkeys were implanted with recombinant human osteogenic protein-1 (rhOP-1) placed on a bovine bone-derived Type I collagen carrier to characterize healing in an ulna segmental bone defect model at 1, 3, 12, and 20 weeks postoperative. Defect healing was evaluated by plain film radiography, computed tomography (CT), magnetic resonance imaging (MRI), bone mineral density (BMD), and histologic analysis. Radiographically, new bone formation was observed as early as 3 weeks postoperative. By 6 weeks, new bone was visible in five of six defects. Increased quantity and mineralization of the new bone were apparent by 12 weeks. Reformation of the medullary cavity with appearance of marrow elements was demonstrated by CT and MRI at 20 weeks. BMD studies revealed a significant increase in the presence of bone with time. Histology at 1 week demonstrated that the implant material was well contained in the defect, and a proliferation of cells occurred at the defect borders. At 3 weeks cell proliferation continued and cell phenotype differentiation was recognized. By 12 weeks substantially less residual carrier was found in the defects, and calcifying tissues with plump chondrocytes, osteoblasts, and immature woven bone were observed. Areas of lamellar and woven bone were identified at 12 weeks, with advanced remodeling and revascularization observed at 20 weeks. The use of osteoinductive implants may provide an alternative to autologous and allogeneic bone tissue in the therapeutic approach to bone defects and promotion of fusion by eliminating the donor site morbidity associated with autogenous bone and the decreased efficacy and potential for disease transmission associated with allogeneic bone.