自体骨髓基质干细胞复合珊瑚修复犬下颌骨节段缺损的初步研究

目的应用自体骨髓基质干细胞(bonemarrowstromalcells,BMSCs)复合珊瑚构建组织工程化骨,修复犬下颌骨节段性缺损。方法体外扩增培养、成骨诱导犬BMSCs。将第二代细胞复合珊瑚后修复犬自体右侧3cm的下颌骨节段缺损(n=6);以单纯珊瑚植入缺损处为对照(n=6),术后12、32周分别通过影像学,大体形态观察,组织学和生物力学的方法检测骨缺损的修复效果。结果成骨诱导的BMSCs在珊瑚支架上生长良好。X线片显示12周时实验组骨痂较多,对照组材料明显吸收;32周时CT、X线片和大体观察显示术后实验组骨愈合良好,对照组为骨不连;骨密度检测示实验组显著高于对照组(P<0.05);组织学示实验组有较多成熟骨呈骨性愈合,对照组为纤维性愈合;生物力学测试实验组与正常下颌骨力学强度差异无统计学意义(P>0.05)。结论自体成骨诱导BMSCs复合珊瑚形成的组织工程化骨可修复犬下颌骨节段缺损。     

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。     

Coral grafting supplemented with bone marrow.

Limited success in regenerating large bone defects has been achieved by bridging them with osteoconductive materials. These substitutes lack the osteogenic and osteoinductive properties of bone autograft. A direct approach would be to stimulate osteogenesis in these biomaterials by the addition of fresh bone-marrow cells (BMC). We therefore created osteoperiosteal gaps 2 cm wide in the ulna of adult rabbits and either bridged them with coral alone (CC), coral supplemented with BMC, or left them empty. Coral was chosen as a scaffold because of its good biocompatibility and resorbability. In osteoperiosteal gaps bridged with coral only, the coral was invaded chiefly by fibrous tissue. It was insufficient to produce union after two months. In defects filled with coral and BMC an increase in osteogenesis was observed and the bone surface area was significantly higher compared with defects filled with coral alone. Bony union occurred in six out of six defects filled with coral and BMC after two months. An increase in the resorption of coral was also observed, suggesting that resorbing cells or their progenitors were present in bone marrow and survived the grafting procedure. Our findings have shown that supplementation of coral with BMC increased both the resorption of material and osteogenesis in defects of a clinical significance.     

自体脂肪干细胞复合珊瑚修复犬颅骨标准缺损的初步研究

目的 应用自体脂肪干细胞(adipose-derived stem cells,ADSCs)复合珊瑚构建组织工程化骨,修复犬颅骨标准缺损.方法 体外扩增培养、成骨诱导Beagle犬ADSCs,将第2代细胞接种在珊瑚支架上共同培养.制造实验犬双侧颅骨全层标准缺损(20 mm×20 mm),一侧以细胞材料复合物修复作为实验组(n=7),另一侧以单纯珊瑚材料修复作为对照组(n=7).术后24周分别通过影像学、大体形态观察、生物力学检测、组织学方法检测颅骨缺损的修复效果.结果 成骨诱导的犬ADSCs体外呈现成骨特性,在珊瑚支架上生长良好.3D-CT重建显示术后12周实验组有新生骨痂形成,对照组材料大部分降解;24周时实验组为骨性愈合,对照组为骨不连.24周时实验组缺损修复百分比为(84.19±6.45)%,显著高于对照组的(25.04 ±18.82)%(P<0.01).大体观察见实验组由新生骨痂修复缺损,对照组缺损边缘可见少量骨痂形成,主要为软组织充填;24周生物力学检测修复组织能耐受的最大压力载荷,实验组为(73.45±17.26)N,为犬顶骨最大压力负荷(104.27±22.71)N的70%,两者比较差异有统计学意义(P<0.01),对照组为软组织无法完成上述检测.HE染色见实验组有较多成熟骨呈骨性愈合,对照组为纤维性愈合.结论 自体成骨诱导的ADSCs复合珊瑚形成的组织工程化骨可修复犬颅骨标准缺损.     

Healing of an ulnar defect using a proprietary TCP bone graft substitute, JAX, in association with autologous osteogenic cells and growth factors

Currently, available synthetic bone substitutes have adequate osteoconductive properties but have little or no osteoinductivity. Recent research has focused on using osteogenic growth factors or cells to provide this. JAX is a beta tricalcium phosphate bone graft substitute that has a novel shape and interlocking design. This study investigated delivery methods and the use of autologous cell therapy to enhance healing of a bone defect using JAX as a scaffold. Bone marrow was harvested from 24 New Zealand White rabbits. The mononuclear cell fraction was isolated and culture expanded. Bilateral 1.5 cm defects in the ulna were filled with: Group 1: JAX alone, Group 2: JAX plus 1x10(7) autologous BMSCs injected at the time of surgery, Group 3: JAX plus 8x10(6) autologous BMSCs cultured on granules for 14 days prior to surgery, Group 4: JAX plus fresh bone marrow (BMA), Group 5: cortical autograft, Group 6: JAX plus 2.5 microg VEGF. Radiographs demonstrated that there was more new bone in the BMA and VEGF groups compared to JAX alone. Groups containing autologous BMSCs were only slightly better than JAX alone in the amount of bone in the defect but did improve bridging of the osteotomy. Histomorphometry identified a significant increase in bone volume in the BMA group compared to JAX alone. BMA and VEGF enhanced healing of bone defects whereas expanded BMSCs provided little advantage over scaffold alone. There was no difference between delivery methods of autologous BMSCs. These observations suggest that the provision of osteogenic cells alone is insufficient to enhance bone healing and that additional factors are required to initiate this process in vivo.     

Native multipotential stromal cell colonization and graft expander potential of a bovine natural bone scaffold

Graft expanders are bone scaffolds used, in combination with autografts, to fill large bone defects in trauma surgery. This study investigates the graft expander potential of a natural bone substitute Orthoss® by studying its ability to support attachment, growth and osteogenic differentiation of neighboring multipotential stromal cells (MSCs). Material consisting of bone marrow (BM) aspirate and reamer‐irrigator‐aspirator (RIA)‐harvested autograft bone was co‐cultured with commercially available Orthoss® granules. Native MSCs attached to Orthoss® were expanded and phenotypically characterized. MSCs egress from neighboring cancelous bone was assessed in 3D Matrigel co‐cultures. MSC differentiation was evaluated using scanning electron microscopy and measuring alkaline phosphatase (ALP) activity per cell. CD45⁺ hematopoietic lineage cells and highly proliferative CD90⁺CD73⁺CD105⁺ MSCs preferentially colonized Orthoss® granules, over RIA bone chips. MSC colonization was followed by their intrinsic osteogenic differentiation, assessed as mineral deposition and gradual rise in ALP activity, even in the absence of osteogenic stimuli. When in contact with mixed cell populations and RIA chips, Orthoss® granules support the attachment, growth and osteogenic differentiation of neighboring MSCs. Therefore, natural bone substitutes similar to Orthoss® can be used as void fillers and graft expanders for repairing large bone defects in conjunction with autologous BM aspirates and autografts. © 2013 Orthopaedic Research Society Published by Wiley Periodicals, Inc. J Orthop Res 31:1950–1958, 2013     

以自体诱导和无诱导的骨髓基质干细胞复合珊瑚修复犬下颌骨节段缺损的比较研究

目的分别采用诱导和无诱导的自体骨髓基质干细胞（Bone marrow stromal cells，BMSCs）复合珊瑚构建组织工程化骨，修复犬下颌骨节段性缺损，比较修复效果。方法体外扩增、成骨诱导或无诱导培养犬BMSCs，分别将第2代细胞复合珊瑚后修复犬自体右侧3cm的下颌骨节段缺损（诱导组n=6，无诱导组n=6）。术后32周，分别通过Micro-CT、大体形态观察和组织学方法检测骨缺损的修复效果。结果32周时，Micro-CT检测示诱导组骨容积率和密度均显著高于对照组（P〈0.05）；大体观察示诱导组骨愈合良好，无诱导组中的3条犬为骨不连；组织学检测诱导组有较多成熟骨形成，缺损部分均呈骨性愈合。无诱导组中的3只犬有新骨形成，但形态不完整，另3只犬的缺损部分呈纤维性愈合。结论成骨诱导的自体BMSCs复合珊瑚形成的组织工程化骨修复犬下颌骨节段缺损效果优于无诱导组。     

Low‐dose BMP‐2 and MSC dual delivery onto coral scaffold for critical‐size bone defect regeneration in sheep

Tissue‐engineered constructs (TECs) combining resorbable calcium‐based scaffolds and mesenchymal stem cells (MSCs) have the capability to regenerate large bone defects. Inconsistent results have, however, been observed, with a lack of osteoinductivity as a possible cause of failure. This study aimed to evaluate the impact of the addition of low‐dose bone morphogenetic protein‐2 (BMP‐2) to MSC‐coral‐TECs on the healing of clinically relevant segmental bone defects in sheep. Coral granules were either seeded with autologous MSCs (bone marrow‐derived) or loaded with BMP‐2. A 25‐mm‐long metatarsal bone defect was created and stabilized with a plate in 18 sheep. Defects were filled with one of the following TECs: (i) BMP (n = 5); (ii) MSC (n = 7); or (iii) MSC‐BMP (n = 6). Radiographic follow‐up was performed until animal sacrifice at 4 months. Bone formation and scaffold resorption were assessed by micro‐CT and histological analysis. Bone union with nearly complete scaffold resorption was observed in 1/5, 2/7, and 3/6 animals, when BMP‐, MSC‐, and MSC‐BMP‐TECs were implanted, respectively. The amount of newly formed bone was not statistically different between groups: 1074 mm³ [970–2478 mm³], 1155 mm³ [970–2595 mm³], and 2343 mm³ [931–3276 mm³] for BMP‐, MSC‐, and MSC‐BMP‐TECs, respectively. Increased scaffold resorption rate using BMP‐TECs was the only potential side effect observed. In conclusion, although the dual delivery of MSCs and BMP‐2 onto a coral scaffold further increased bone formation and bone union when compared to single treatment, results were non‐significant. Only 50% of the defects healed, demonstrating the need for further refinement of this strategy before clinical use. © 2017 Orthopaedic Research Society. Published by Wiley Periodicals, Inc. J Orthop Res 35:2637–2645, 2017.

     

Apatite-coated silk fibroin scaffolds to healing mandibular border defects in canines

Tissue engineering has become a new approach for repairing bony defects. Highly porous osteoconductive scaffolds perform the important role for the success of bone regeneration. By biomimetic strategy, apatite-coated porous biomaterial based on silk fibroin scaffolds (SS) might provide an enhanced osteogenic environment for bone-related outcomes. To assess the effects of apatite-coated silk fibroin (mSS) biomaterials for bone healing as a tissue engineered bony scaffold, we explored a tissue engineered bony graft using mSS seeded with osteogenically induced autologous bone marrow stromal cells (bMSCs) to repair inferior mandibular border defects in a canine model. The results were compared with those treated with bMSCs/SS constructs, mSS alone, SS alone, autologous mandibular grafts and untreated blank defects. According to radiographic and histological examination, new bone formation was observed from 4 weeks post-operation, and the defect site was completely repaired after 12 months for the bMSCs/mSS group. In the bMSCs/SS group, new bone formation was observed with more residual silk scaffold remaining at the center of the defect compared with the bMSCs/mSS group. The engineered bone with bMSCs/mSS achieved satisfactory bone mineral densities (BMD) at 12 months post-operation close to those of normal mandible (p > 0.05). The quantities of newly formed bone area for the bMSCs/mSS group was higher than the bMSCs/SS group (p < 0.01), but no significant differences were found when compared with the autograft group (p > 0.05). In contrast, bony defects remained in the center with undegraded silk fibroin scaffold and fibrous connective tissue, and new bone only formed at the periphery in the groups treated with mSS or SS alone. The results suggested that apatite-coated silk fibroin scaffolds combined with bMSCs could be successfully used to repair mandibular critical size border defects and the premineralization of these porous silk fibroin protein scaffolds provided an increased osteoconductive environment for bMSCs to regenerate sufficient new bone tissue.     

Strategies for improving the efficacy of bioengineered bone constructs: a perspective

Bioengineered bone scaffolds are intended for use in large bone defects. Successful bone constructs should stimulate and support both the onset and the continuance of bone ingrowth. In an attempt to improve their performance and to compete with the one of autologous bone grafts, a growing symbiosis at the biological and material level is required. Recent advances have been made to further exploit the osteogenic potential of MSCs in scaffold development. Current research encompasses new strategies for reducing cell death after implantation and the manufacturing of tailored, instructive scaffolds.     

"双相"组织工程软骨修复兔关节骨软骨缺损

目的探讨“双相”异体骨基质明胶（bonematrixgelatin,BMG）作为组织工程软骨载体,与同体骨髓间充质干细胞（marrowmesenchymalstemcells,MSCs）结合,构建组织工程软骨修复兔关节骨软骨缺损的效果。方法4月龄新西兰兔32只,雌雄不限,体重2～3kg。①体外实验：取5只新西兰兔,处死后取髂骨和四肢骨,制备一侧松质骨,一侧皮质骨的“双相”异体BMG载体,扫描电镜观察。另取新西兰兔18只,抽取骨髓,分离MSCs并诱导成软骨分化;将诱导而来的软骨前体细胞与“双相”BMG载体复合构建组织工程软骨,分别于1、3和5周取材行Masson、PAS染色和扫描电镜观察。②体内实验：将抽取骨髓的18只及余下的9只新西兰兔制成双侧股骨内髁骨软骨缺损模型,将前期制备的组织工程软骨同体植入18只兔的右股骨内髁骨软骨缺损（A组）,左侧缺损移植异体BMG（B组）,其余9只双侧软骨缺损未予处理作为空白对照（C组）,分别于术后1、3和6个月取材,行大体、组织学和Ⅱ型胶原mRNA原位杂交观察,改良Wakitani法评分,比较各组修复效果差异。结果①体外实验：“双相”BMG松质骨面孔隙大小100-800μm,细胞于其中增生,形成富含细胞的软骨层;皮质骨面孔隙大小10～40pm,细胞层状覆盖于其表面,可作为起支撑作用的软骨下骨。②体内实验：A组术后1个月即可重建关节骨软骨缺损;修复软骨在观察期内逐渐变薄,但在6个月内始终保持关节面及软骨下骨结构完整。B、C组未能修复缺损,缺损周边软骨磨损加剧。改良Wakitani评分显示A组在3个时间点的各项评分结果,除6个月软骨厚度外,其它指标均优于B、C组,且差异有统计学意义（P〈0．01）。Ⅱ型胶原mRNA原位杂交显示,A组缺损区修复组织中细胞阳性染色率明显高于B、C组,且差异有统计学意义（P〈0．01）。结论“双相”异体BMG可作为组织工程软骨载体材料,其结合自体MSCs诱导的软骨前体细胞制备的组织工程软骨,可修复兔关节软骨和软骨下骨。     

Mesenchymal stem cells used for rabbit tendon repair can form ectopic bone and express alkaline phosphatase activity in constructs.

Mesenchymal stem cells (MSCs) have been used to repair connective tissue defects in several animal models. Compared to "natural healing" controls (no added cells), MSC-collagen gel constructs in rabbit tendon defects significantly improve repair biomechanics. However, ectopic bone forms in 28% of MSC-treated rabbit tendons. To understand the source of bone formation, three studies were performed. In the first study, the hypothesis was tested that MSCs delivered during surgery contribute to bone formation in the in vivo repair site. Adjacent histological sections in the MSC-treated repair tissue were examined for pre-labeled MSCs and for cells showing positive alkaline phosphatase (ALP) activity. Both cells were observed in serial sections in regions of ectopic bone. Contralateral "natural healing" tendons lacked both markers. In the other two studies, the effects of osteogenic supplements and construct geometry (monolayer vs. 3-D) on ALP activity were studied to test three hypotheses: that rabbit MSCs increase ALP activity over time in monolayer culture conditions; that adding osteogenic inducing supplements to the culture medium increases cellular protein in monolayer culture; and that rabbit MSCs increase ALP activity both in monolayer and in 3-D constructs, with and without media supplements. Culture in monolayer under similar conditions to in vivo (as in the first study) did not increase ALP at 2 or 4 weeks. Medium designed to increase osteogenic activity significantly increased cell numbers (cellular protein increased by 260%) but did not affect ALP activity either in monolayer or 3-D constructs (p>0.12). However, MSCs in 3-D constructs exhibited higher ALP activity than cells in monolayer, both in the presence (p<0.045) and absence of supplement (p<0.005). These results suggest that in vitro conditions may critically influence cell differentiation and protein expression. Mechanisms responsible for these effects are currently under investigation.     

自体骨髓基质细胞复合支架材料修复兔尺骨干节段骨缺损的实验研究

目的探讨组织工程骨的体内血管化情况和修复大节段骨缺损的能力。方法采用骨髓穿刺、密度梯度离心的方法获取兔骨髓基质细胞，经体外诱导分化，接种到聚磷酸钙纤维／L-聚乳酸／胶原(CPPF／PLLA／Collagen)支架材料上，植入自体尺骨中下段1．5cm长的骨膜骨缺损区，对照组为缺损区单纯植入CPPF／PLLA／Collagen，术后观察12周。结果术后4周缺损区为新生骨样组织充填，血循环丰富，12周时形态结构已接近正常骨组织；随修复时间延长，修复组织的力学性能逐渐增强。结论骨髓基质细胞与CPPF/PLLA／Collagen复合物具有较强的成骨能力，有望成为修复骨缺损的治疗方法。     

组织工程化骨修复下颌骨缺损(附3例报告)

目的探讨以人自体骨髓基质干细胞(hBMSC)作为种子细胞的组织工程化骨在治疗肿瘤术后下颌骨缺损的可行性。方法回顾性分析2005年1月至2005年12月收治3例组织工程骨修复颌面部肿瘤术后下颌骨缺损的临床资料。抽取人骼前上棘骨髓,分离出hBMSC,经体外诱导和扩增,将hBMSC与部分脱钙骨(30%～40%)复合,于体外培养1个月后,手术植入骨缺损区。分别于术后2周、3个月、2年进行PET-CT检查随访。结果 PET-CT示,术后3个月能形成组织工程化骨,并修复了骨组织缺损,临床治疗效果稳定。结论以自体hBMSC为种子细胞,利用组织工程技术可在人体内形成稳定的组织工程化骨组织,并能修复下颌骨缺损。     

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.     

Engineering Vascularized Bone Graft With Osteogenic and Angiogenic Lineage Differentiated Bone Marrow Mesenchymal Stem Cells

Tissue‐engineered bone provides a promising method for the rehabilitation of acquired bone defects and congenital deformities. However, generating a vascular supply to the engineered graft remains a major challenge. We report a novel strategy to engineer vascularized bone grafts with osteogenic and angiogenic lineage differentiated marrow mesenchymal stem cells (MSCs). MSCs were expanded to form an osteogenic cell sheet using a continuous culture method and a scraping technique under osteogenic culture conditions. Another portion of MSCs was directed to differentiate into highly proliferative endothelial progenitor cells (EPCs), which were then seeded onto the cell sheets. Cell sheet–EPC complexes were implanted subcutaneously in nude mice. Cell sheets without EPCs were also implanted as a control. The mice were sacrificed, and the samples were harvested for evaluation consisting of micro‐CT scanning, histological analysis and scanning electronic microscopy 4 and 8 weeks after implantation. The results showed that cell sheets were composed of viable cells and extracellular matrix and showed apparent mineralization. The obtained EPCs could express the specific antigen marker of CD31 and form capillary‐like structures in vitro. The osteogenic cell sheet–EPC complexes yielded well‐vascularized bone grafts 4 and 8 weeks after implantation. Both bone density and vascular density were significantly higher in the cell sheet–EPC complex group than in the control group. The results demonstrated that the introduction of EPCs could not only generate a vascular network but also increase bone formation for cell sheet‐based bone engineering. These findings suggest that the strategy of engineering bone grafts with osteogenic and angiogenic lineage differentiated MSCs has great potential for clinical applications to repair large bone defects.     

Tissue engineering‐based cartilage repair with mesenchymal stem cells in a porcine model

This in vivo pilot study explored the use of mesenchymal stem cell (MSC) containing tissue engineering constructs in repair of osteochondral defects. Osteochondral defects were created in the medial condyles of both knees of 16 miniature pigs. One joint received a cell/collagen tissue engineering construct with or without pretreatment with transforming growth factor β (TGF‐β) and the other joint from the same pig received no treatment or the gel scaffold only. Six months after surgery, in knees with no treatment, all defects showed contracted craters; in those treated with the gel scaffold alone, six showed a smooth gross surface, one a hypertrophic surface, and one a contracted crater; in those with undifferentiated MSCs, five defects had smooth, fully repaired surfaces or partially repaired surfaces, and one defect poor repair; in those with TGF‐β‐induced differentiated MSCs, seven defects had smooth, fully repaired surfaces or partially repaired surfaces, and three defects showed poor repair. In Pineda score grading, the group with undifferentiated MSC, but not the group with TGF‐β‐induced differentiated MSCs, had significantly lower subchondral, cell morphology, and total scores than the groups with no or gel‐only treatment. The compressive stiffness was larger in cartilage without surgical treatment than the treated area within each group. In conclusion, this preliminary pilot study suggests that using undifferentiated MSCs might be a better approach than using TGF‐β‐induced differentiated MSCs for in vivo tissue engineered treatment of osteochondral defects. © 2011 Orthopaedic Research Society Published by Wiley Periodicals, Inc. J Orthop Res 29:1874–1880, 2011     

Chest wall repair with engineered fetal bone grafts: an efficacy analysis in an autologous leporine model

Purpose

We sought to compare the efficacy of engineered fetal bone grafts with acellular constructs in an autologous model of chest wall repair.

Methods

Rabbits (n = 10) with a full-thickness sternal defect were equally divided in 2 groups based on how the defect was repaired, namely, either with an autologous bone construct engineered with amniotic mesenchymal stem cells on a nanofibrous scaffold or a size-matched identical scaffold with no cells. Animals were killed at comparable time-points 18 to 20 weeks postimplantation for multiple analyses.

Results

Gross evidence of nonunion confirmed by micro-computed tomography scanning was present in 3 (60%) of 5 of the acellular implants but in no engineered grafts. Histology confirmed the presence of bone in both types of repair, albeit seemingly less robust in the acellular grafts. Mineral density in vivo was significantly higher in engineered grafts than in acellular ones, with more variability among the latter. There was no difference in alkaline phosphatase activity between the groups.

Conclusions

Chest wall repair with an autologous osseous graft engineered with amniotic mesenchymal stem cells leads to improved and more consistent outcomes in the midterm when compared with an equivalent acellular prosthetic repair in a leporine model. Amniotic fluid-derived engineered bone may become a practical alternative for perinatal chest wall reconstruction.     

Repair of large segmental bone defects using bone marrow stromal cells with demineralized bone matrix

Objective: The aim of the present study was to evaluate the effect of tissue‐engineered constructs on repair of large segmental bone defects in goats. Methods: Allogenic demineralized bone matrix (aDBM) was seeded with autologous marrow stromal cells (aMSC) for seven days to construct DBM–MSC grafts prior to implantation. 24 goats were randomly divided into three groups (eight in each). In each group, 3 cm diaphyseal femoral defects were created unilaterally, and subsequently filled with the DBM‐MSC grafts, DBM alone and an untreated control, respectively. Radiological analysis and biomechanical evaluation were performed at 12 and 24 weeks after operation. Results: Obvious increases in radiological scoring and biomechanical strength were found in the DBM‐MSC group when compared to the DBM group. X‐ray examination showed excellent bone healing in the DBM‐MSC group, whereas only partial bone repair was seen in the DBM group, and no healing in untreated controls. Histologically, a tendency to bone regeneration and remodeling was far more obvious for the DBM‐MSC group than the DBM only and untreated controls. Conclusion: Our results strongly suggest that transplantation of bone MSC within a DBM could have advantages for the bone repair of large segmental defects.     

Effects of age on the repair ability of mesenchymal stem cells in rabbit tendon.

Successful tissue engineered repair in the aging adult requires an abundant source of autologous, multipotent mesenchymal stem cells (MSCs). Although the number of bone marrow-derived MSCs declines dramatically with aging, their effectiveness in repair with increasing age has not been studied. We tested the hypothesis that MSCs harvested from geriatric rabbits would not repair patellar tendon defects as well as MSCs harvested from younger adult rabbits. In a novel within-subjects experiment, autologous MSCs were isolated from 1-year old rabbits, culture expanded, and cryogenically preserved. After housing the rabbits for 3 years, MSCs were re-harvested from the 4-year old rabbits and expanded. Five hundred thousand thawed and fresh MSCs were each separately mixed with type I collagen gel (333.3 x 10(3) cells/mg collagen) 24 h before surgery, and the resulting constructs implanted in bilateral full-length central third tendon defects. Twelve weeks post-surgery, the bone-tendon repair-bone units were failed in tension. Intra-animal (paired) comparisons between repair tissue treated with 1-year old MSCs and repair tissue treated with 4-year old MSCs resulted in no significant differences (alpha=0.05) in material properties including maximum stress (10.8 MPa vs. 9.9 MPa; p=0.762), modulus (139.8 MPa vs. 146.2 MPa; p=0.914), and strain energy density (0.52 N mm/mm(3) vs. 0.53 N mm/mm(3); p=0.966). Despite an age-related trend, there were also no significant differences in structural properties including maximum force (62.9 N vs. 27.0 N; p=0.070), stiffness (24.9 N/mm vs. 12.0 N/mm; p=0.111), and strain energy (87.2 N mm vs. 31.4 N mm; p=0.061). A subset of the rabbits (n=4 1 yrMSC, n=2 4 yrMSC) showed the presence of ectopic bone in the repair region and were not included in the mechanical analyses. We conclude that in the rabbit model MSCs do not lose their benefit as a tendon repair therapy with aging and that MSCs can be cryogenically stored for 3 years and still effectively repair soft tissue injuries.