Ectopic bone formation in rats: the importance of the carrier

Much research has been done to develop the ideal bone graft substitute (BGS). One approach to develop this ideal BGS is the use of growth factors, but for this approach osteoprogenitor cells are needed at the site of reconstruction. An alternative is a cell-based approach, where enough cells are provided to form bone in a carrier material. In previous studies of our group, titanium (Ti) carriers have been used, because of the excellent mechanical properties and the bone-compatibility of this material. On the other hand, calcium phosphate (CaP) ceramics are known for their excellent osteoconductivity. The aim of this study is to investigate the influence of the carrier in a cell-based bone regeneration approach, whereby we hypothesize that CaP-ceramic implants will induce more bone formation than Ti-fiber implants, in the same animal model as our previous experiment. Ti-fiber mesh implants and ceramic implants were seeded with rat bone marrow cells (RBM) and implanted subcutaneously. Histological analysis after one, three and six weeks showed differences in the way of bone formation in the two groups: bone appeared to grow from the center to the periphery of the implant in the titanium group, while bone formation in the ceramic group occurred through the whole implant. Histomorphometrical analysis after one week showed very limited bone formation for both the titanium and ceramic group. At three weeks, the amount of bone formation was increased till about 10% for the titanium group and 18% for the ceramic group. No significant difference between the two groups could be observed. In the six week group, the bone formation was 6% (Ti) and 23% (CaP), respectively (P < 0.001). Further, bone formation started earlier in the CaP-ceramic scaffolds than in the Ti scaffolds. Our hypothesis could be confirmed: ceramic implants induce more bone formation than titanium implants.     

Fibrin glue as an osteoinductive protein in a mouse model

Fibrin sealant or fibrin glue (FG) has been found to be effective as a wound-healing substance in surgery. However, its role in bone fracture healing and osseous tissue response is not fully understood. This ambiguity questions the potential of FG as an inductive protein. The present study was undertaken to evaluate the osteoinductive property of FG when coated with calcium phosphate and glass ceramics and implanted in the extraskeletal site of male Swiss albino mice. Implant materials used for this study were hydroxyapatite (HA) porous granules (300-350 microm), bioactive glass system (BGS)-AW type and calcium phosphate calcium silicate system (HABGS) non-porous granules (300-350 microm). Uncoated granules (control) and coated granules with 2.5 mg FG and 5 mg FG were implanted in the quadriceps muscle of mice and sacrificed after 28 days. Histologically, HA, BGS and HABGS implanted animal groups showed good healing response. However, neo-osteogenesis was observed only in the BGS and HABGS granules impregnated with FG. Furthermore, bone formation was observed to be more conspicuous in 5 mg FG coated BGS and HABGS granules when compared with 2.5 mg FG coated BGS and HABGS granules. Fluorochrome labeling proved that mineralization had already started by day 15 with FG preadsorbed BGS and HABGS granules. On the contrary, the uncoated granules did not show any de novo bone formation. This experimental study provides an evidence of the positive role of FG as a potential osteoinductive biologic tissue adhesive.     

骨诱导性磷酸钙陶瓷支架在脂肪组织中构建体内组织工程骨

背景：作为体内组织工程骨的构建场所,非骨组织的选择至关重要。早期研究大多选用肌肉作为异位骨移植物的构建区域,但其位置较深,可利用面积小、手术操作复杂,不利于临床推广。 目的：采用体内骨组织工程的方法,探索骨诱导性磷酸钙陶瓷支架在不同非骨组织中构建骨移植物的可行性。 方法：选取家犬的背部肌肉组织和脂肪组织为构建区,分别植入骨诱导性磷酸钙陶瓷支架以构建体内组织工程骨移植物。于移植后4,8,12,16周取样进行单光子计算机断层扫描及组织学检测,观察其构建过程,比较各个观测时间内,不同构建区的骨移植物中新骨组织的形成情况,评价不同非骨构建区域对体内骨组织骨移植物形成的影响。 结果与结论：骨诱导性磷酸钙陶瓷支架在肌肉组织和脂肪两处非骨组织中均可形成体内组织工程骨移植物,在构建初期,肌肉组中新骨形成的时间比脂肪组早,骨量也较多,但随着构建时间的延长,两组的新生骨量差异逐渐减小。提示,应用体内骨组织工程的方法在肌肉和脂肪组织均可构建出具有生命活性的自体骨移植物。与肌肉组织比较,脂肪组织面积宽广,位置浅表,因此在脂肪组织中构建体内组织工程骨移植物更有临床应用前景。     

In vivo evaluation of a porous hydroxyapatite/poly-DL-lactide composite for bone tissue engineering

As reported previously, a porous composite of uncalcined hydroxyapatite (u-HA) and poly-DL-lactide (PDLLA) showed excellent osteoconductivity and biodegradability as a bone substitute in rabbit model. In this study, to investigate the usefulness of this composite as a scaffold loaded with cells, we estimated whether this material showed osteogenesis on implantation to extraosseous site. On loading with syngeneic bone marrow cells and implantation into rat dorsal subcutaneous tissue, osteogenesis with enchondral ossification was seen both on and in the material at 3 weeks after implantation. The osteogenesis in the u-HA/PDLLA had progressed, and newly formed bone tissue was found in the material by 6 weeks. To investigate the osteoinductive properties of the material, we implanted this porous composite material into extraosseous canine dorsal muscle. At 8 weeks, osteogenesis was seen in the pores of the material. Newly formed bone could be observed adjacent to the material. In addition, cuboidal osteoblasts adjacent to the newly formed bone were evident. Neither cartilage nor chondrocytes were found. These results might indicate that the material induced osteogenesis by intramembranous ossification. Conversely, similar porous PDLLA did not induce osteogenesis during the observation period. Therefore, porous HA/PDLLA, which has osteoconductive and osteoinductive properties, might be a useful material for use as a bone substitute and cellular scaffold.     

成骨诱导脂肪基质细胞在骨组织工程体内成骨中的作用

背景：以往研究认为,经过成骨诱导后的脂肪基质细胞通过转化为成骨细胞分泌骨基质进而修复骨缺损,然而并没有明确结论证实。 目的：将经过体外成骨诱导的脂肪基质细胞复合支架材料分别植入骨缺损区和非骨区,根据是否成骨,验证经过成骨诱导后的脂肪基质细胞是否转化为成骨细胞。 方法：取12月龄犬背部皮下脂肪,经胶原酶消化法获得单个核细胞,将培养的第3代细胞与双相磷酸钙陶瓷形成复合物。在犬下颌骨两侧制备长20 mm、高10 mm的箱状缺损,拔除术区牙齿,将细胞支架复合物植入一侧术区,空白侧留作对照;另外在犬背部皮下肌肉区植入细胞支架复合物及骨诱导性磷酸钙陶瓷材料,术后6周及12周经组织学检测骨缺损修复情况。 结果与结论：脂肪基质细胞复合双相磷酸钙陶瓷在骨缺损区成骨,在肌肉区未形成新骨;骨诱导性磷酸钙陶瓷在肌肉区形成新骨。提示成骨诱导并不能将脂肪基质细胞转化为成骨细胞,其确切机制有待进一步研究。     

In vivo bone formation following transplantation of human adipose-derived stromal cells that are not differentiated osteogenically

A number of studies have shown in vivo bone regeneration by transplantation of osteogenic cells differentiated in vitro from adipose-derived stromal cells (ADSCs). However, the in vitro osteogenic differentiation process requires an additional culture period, and the dexamethasone that is generally used in the process may be cytotoxic. Here, we tested the hypothesis that ADSCs that are not differentiated osteogenically in vitro prior to transplantation would extensively regenerate bone in vivo when exogenous bone morphogenetic protein-2 (BMP-2) is delivered to the transplantation site. We fabricated a poly(dl-lactic-co-glycolic acid)/hydroxyapatite (PLGA/HA) composite scaffold with osteoactive HA that is highly exposed on the scaffold surface. This scaffold was able to release BMP-2 over a 4-week period in vitro. Human ADSCs cultured on BMP-2-loaded PLGA/HA scaffolds for 2 weeks differentiated toward osteogenic cells expressing alkaline phosphatase (ALP), osteopontin (OPN), and osteocalcin (OCN) mRNA, while cells on PLGA/HA scaffolds without BMP-2 expressed only ALP. To study in vivo bone formation, PLGA/HA scaffolds (group 1), BMP-2-loaded PLGA/HA scaffolds (group 2), undifferentiated ADSCs seeded on PLGA/HA scaffolds (group 3), and undifferentiated ADSCs seeded on BMP-2-loaded PLGA/HA scaffolds (group 4) were implanted into dorsal, subcutaneous spaces of athymic mice. Eight weeks after implantation, group 4 exhibited a 25-fold greater bone formation area and 5-fold higher calcium deposition than group 3. Bone regeneration by transplanted human ADSCs in group 4 was confirmed by expression of human-specific osteoblastic genes, ALP, collagen type I, OPN, OCN, and bone sialoprotein, while group 3 expressed much lower levels of collagen type I and OPN mRNA only. This study demonstrates the feasibility of extensive in vivo bone regeneration by transplantation of ADSCs without prior in vitro osteogenic differentiation, and that a PLGA/HA composite BMP-2 delivery system stimulates bone regeneration following transplantation of undifferentiated human ADSCs.     

The collagen component of biological bone graft substitutes promotes ectopic bone formation by human mesenchymal stem cells

Synthetic bone substitutes are attractive materials for repairing a variety of bone defects. They are readily available in unlimited quantities, have a defined composition without batch variability and bear no risk of disease transmission. When combined with mesenchymal stem cells (MSCs), bone healing can be further enhanced due to the osteogenic potential of these cells. However, human MSCs showed considerable donor variability in ectopic bone formation assays on synthetic bone substitutes, which may limit clinical success. This study addresses whether bone formation variability of MSCs is cell-intrinsic or biomaterial-dependent and may be improved using biological bone substitutes with and without collagen. Ectopic bone formation of MSCs from nine donors was tested in immune-deficient mice on biological bone substitutes of bovine and equine origin, containing collagen (bHA-C; eHA-C) or not (bHA; eHA). Synthetic β-TCP was used for comparison. Histology of 8-week explants demonstrated a significant influence of the bone graft substitute (BGS) on donor variability of ectopic bone formation with best results seen for eHA-C (15/17) and β-TCP (16/18). Bone was of human origin in all groups according to species-specific in situ hybridization, but MSCs from one donor formed no bone with any bone substitute. According to histomorphometry, most neo-bone was formed on eHA-C with significant differences to bHA, eHA and β-TCP (p < 0.001). Collagen-free biological BGSs were inferior to biological BGSs with collagen (p < 0.001), while species-origin was of little influence. In conclusion, BGS composition had a strong influence on ectopic bone formation ability of MSCs, and biological BGSs with a collagen component seem most promising to display the strong osteogenic potential of MSCs.     

Tissue-engineered growth of bone by marrow cell transplantation using porous calcium metaphosphate matrices

In this study we investigated not only osteoblastic cell proliferation and differentiation on the surface of calcium metaphosphate (CMP) matrices in vitro but also bone formation by ectopic implantation of these cell-matrix constructs in athymic mice in vivo. Interconnected porous CMP matrices with pores 200 microm in size were prepared to use as scaffolds for rat-marrow stromal-cell attachment. Cell-matrix constructs were cultured in vitro, and cell proliferation and ALPase activities were monitored for 56 days. In addition to their being cultured in vitro, cell-matrix constructs were implanted into subcutaneous sites of athymic mice. In vitro these porous CMP matrices supported the proliferation of osteoblastic cells as well as their differentiation, as indicated by high ALPase activity. In vivo the transplanted marrow cells gave rise to bone tissues in the pores of the CMP matrices. A small amount of woven bone formation was detected first at 4 weeks; osteogenesis progressed vigorously with time, and thick lamellar bones that had been remodeled were observed at 12 weeks. These findings demonstrate the potential for using a porous CMP matrix as a biodegradable scaffold ex vivo along with attached marrow-derived mesenchymal cells for transplantation into a site for bone regeneration in vivo.     

Fate of bone marrow stromal cells in a syngenic model of bone formation

Bone marrow stromal cells (BMSCs) have been demonstrated to induce bone formation when associated to osteoconductive biomaterials and implanted in vivo. Nevertheless, their role in bone reconstruction is not fully understood and rare studies have been conducted to follow their destiny after implantation in syngenic models. The aim of the present work was to use sensitive and quantitative methods to track donor and recipient cells after implantation of BMSCs in a syngenic model of ectopic bone formation. Using polymerase chain reaction (PCR) amplification of the Sex determining Region Y (Sry) gene and in situ hybridization of the Y chromosome in parallel to histological analysis, we have quantified within the implants the survival of the donor cells and the colonization by the recipient cells. The putative migration of the BMSCs in peripheral organs was also analyzed. We show here that grafted cells do not survive more than 3 weeks after implantation and might migrate in peripheral lymphoid organs. These cells are responsible for the attraction of host cells within the implants, leading to the centripetal colonization of the biomaterial by new bone.     

Intramuscular bone induction by the simultaneous administration of recombinant human bone morphogenetic protein 2 and bisphosphonate for autobone graft

An ideal substitute for bone graft is autobone tissue, of which there is an ample supply of the required form and with vascularity. Our strategy is to generate intramuscular autogenous bone by administering recombinant human bone morphogenetic protein 2 (rhBMP-2) with beta-tricalcium phosphate (beta-TCP) as a carrier, and to transplant this bone as a muscle-pedicled autograft. However, in a previous study (Jingushi et al., J. Orthop. Sci. 7, 490, 2002), bone resorption occurred early after bone induction. This study was conducted to determine whether rhBMP-2-induced bone tissue could be maintained by simultaneous administration of bisphosphonate, and to investigate whether the induced bone could be used for bone grafting. In this study, we first applied rhBMP-2 alone to a beta-TCP disk and inoculated it into rat quadriceps muscle. Bone area and the number of tartrate-resistant acid phosphatase (TRAP)-positive cells in the induced bone disk peaked at 2 weeks, and induced bone resorption occurred later. Bisphosphonate and rhBMP-2 were then simultaneously applied to a beta-TCP disk and inoculated as in the first experiment. The addition of bisphosphonate decreased the number of TRAP-positive cells and increased the bone area and compression strength at 4 weeks. In the last experiment, a rhBMP-2 applied beta-TCP disk treated with or without bisphosphonate was free-grafted to parietal bone 4 weeks after inoculation. Both bone disks united similarly. We concluded that the concurrent use of bisphosphonate prevented bone absorption attributed to osteoclast activity after bone induction by rhBMP-2. The bisphosphonate application did not disturb the union of induced bone to host bone.     

Mesenchymal stem cell-dependent formation of heterotopic tendon-bone insertions (osteotendinous junctions)

Ligament-to-bone and tendon-to-bone interfaces (entheses, osteotendinous junctions [OTJs]) serve to dissipate stress between soft tissue and bone. Surgical reconstruction of these interfaces is an issue of considerable importance as they are prone to injury and the integration of bone and tendon/ligament is in general not satisfactory. We report here the stem cell-dependent spontaneous formation of fibrocartilaginous and fibrous entheses in heterotopic locations of the mouse if progenitors possess a tenogenic and osteo-/chondrogenic capacity. This study followed the hypothesis that enhanced Bone Morphogenetic Protein (BMP)-signaling in adult mesenchymal stem cells that are induced for tendon formation may overcome the tendon-inherent interference with bone formation and may thus allow the stem cell-dependent formation of tendon-bone interfaces. The tenogenic and osteo-/chondrogenic competence was mediated by the adeno- and/or lentiviral expression of the biologically active Smad8 signaling mediator (Smad8ca) and of Bone Morphogenetic Protein 2 (BMP2). Modified mesenchymal progenitors were implanted in subcutaneous or intramuscular sites of the mouse. The stem cell-dependent enthesis formation was characterized histologically by immunohistological approaches and by in situ hybridization. Transplantation of modified murine stem cells resulted in the formation of tendinous and osseous structures exhibiting fibrocartilage-type OTJs, while, in contrast, the viral modification of primary human bone marrow-derived mesenchymal stromal/stem cells showed evidence of fibrous tendon-bone interface formation. Moreover, it could be demonstrated that Smad8ca expression alone was sufficient for the formation of tendon/ligament-like structures. These findings may contribute to the establishment of stem cell-dependent regenerative therapies involving tendon/ligaments and to the improvement of the insertion of tendon grafts at bony attachment sites, eventually.     

不同组织部位诱导膜血管化及成骨因子表达的比较

文题释义 诱导膜技术：又称膜诱导技术、Masquelet技术,1986年由Masquelet等最先提出并使用,是一种重建超临界尺寸骨缺损的有效方法。该技术在清创后所形成的骨缺损处植入骨水泥占位器,以形成一层生物膜,并在所形成的膜内进行植骨对骨缺损进行二次重建。该技术修复大段骨缺损具有重建时间短、骨愈合率高、并发症发生率较低等优势,其疗效已经被广泛证实。 血管内皮生长因子：是一种生长因子,在血管生成中发挥重要作用。血管内皮生长因子与血管内皮表面的特异性受体结合,可以促进内皮细胞的增殖、迁移,并可增加局部毛细血管的通透性,促使纤维蛋白原渗出,为血管内皮细胞的迁移和血管形成提供基质。在促进骨愈合方面,血管内皮生长因子通过促进血管增生,协调软骨细胞的消长、细胞外基质的改建,加快软骨内成骨。血管内皮生长因子还可以趋化并促进成骨细胞的分化,使碱性磷酸酶活性增强,局部钙盐沉积增加,促进骨愈合。 背景：研究发现在皮下、肌肉等部位植入聚甲基丙烯酸甲酯骨水泥也可形成诱导膜,膜内同样具有微血管的形成并分泌多种成骨因子。 目的：比较皮下、肌肉、股骨骨缺损处诱导膜内血管化程度和成骨因子表达的差异。 方法：将36只雄性SD大鼠(购自广州中医药大学实验动物中心)随机分为3组,分别在后肢皮下、肌肉内、股骨骨缺损处植入聚甲基丙烯酸甲酯抗生素骨水泥占位器,每组12只。植入6周后,取出骨水泥周围的诱导膜,苏木精-伊红染色观察诱导膜组织形态结构变化,Western Blot印迹方法、RT-qPCR法、免疫组织化学染色法检测诱导膜中骨形态发生蛋白2、转化生长因子β1、血管内皮生长因子的表达情况。实验获得广州中医药大学动物实验伦理委员会批准,批准号：20181101006。 结果与结论：①苏木精-伊红染色显示3组均可形成诱导膜,但骨缺损组诱导膜组织切片外层血管数量较肌肉组、皮下组多,靠近骨水泥侧的内层成纤维细胞和肌纤维细胞数量较肌肉组、皮下组多;②免疫组织化学染色显示,骨缺损组骨形态发生蛋白2、转化生长因子β1、血管内皮生长因子阳性表达最多,皮下组最少;③Western Blot与RT-qPCR检测显示,骨缺损组骨形态发生蛋白2、转化生长因子β1、血管内皮生长因子表达均多于肌肉组、皮下组(P < 0.001);④结果表明,不同的周围组织条件对诱导膜的组织结构和成骨因子表达有重要影响,在骨缺损处植入聚甲基丙烯酸甲酯骨水泥可提高诱导膜的形成质量,膜内新生血管更丰富,骨生长因子的表达量更多。ORCID: 0000-0003-1405-0765(李树源) 中国组织工程研究杂志出版内容重点：组织构建;骨细胞;软骨细胞;细胞培养;成纤维细胞;血管内皮细胞;骨质疏松;组织工程     

Osteoinduction by biomaterials--physicochemical and structural influences

Osteoinduction by biomaterials has been shown to be a real phenomenon by many investigators in the last decade. The exact mechanism of this phenomenon is, however, still largely unknown. This in vivo study in goats was performed to get insight into processes governing the phenomenon of osteoinduction by biomaterials and had four main goals: (i) to further investigate the influence of physicochemical properties and structure on biomaterial osteoinductive potential, (ii) to investigate the influence of implant size on the amount of induced bone, (iii) to investigate implantation site dependence, and (iv) to investigate changes occurring on the surface of the material after implantation. Intramuscular implantations of four different biphasic calcium phosphate ceramics, consisting of hydroxyapatite and beta-tricalcium phosphate and a carbonated apatite ceramic, indicated that, for a maximal osteoinductive potential, there is an optimal specific surface area for each material type. It was further shown that a decrease of the implant size with a half significantly decreased the relative amount of induced bone. In addition, subcutaneous implantation did not give rise to ectopic bone formation in any of the animals, while bone was induced in most animals intramuscularly. Analysis of the surfaces of the materials after subcutaneous implantation inside diffusion chambers indicated that the increased specific surface area leads to more surface reactivity, which is hypothesized to be essential for osteoinductivity by biomaterials.     

A comparison of ProOsteon, DBX, and collagraft in a rabbit model

Many bone graft substitutes (BGSs) have been developed and are commercially available. These products differ in the tailoring of their properties, including size, form, osteoconductivity, osteoinductivity, and resorption kinetics. Differential enhancement of these properties may optimize the performance of these materials for varying applications. BGSs offer an opportunity to lessen morbidity of harvesting and use of autogenous and/or allograft bone. The purpose of this study is to quantitatively compare the magnitude of bony ingrowth and biodegradation of different commercially available BGS materials in a rabbit femoral defect model. BGSs from each of three classes (ceramic (ProOsteon), demineralized bone matrix (DBX), and composite (Collagraft)) were implanted in cylindrical defects in bilateral femoral condyles of 12 adult New Zealand White rabbits. Each of the three BGS materials and the empty controls were compared. The specimens were harvested at 3 months postimplantation for radiographic and histologic evaluation. Histomorphometry yielded resorption of graft material remaining in the index defect. Magnitude of bony ingrowth was assessed based on an 8-bit 256 densitometry model. Histomorphometric analysis of the data demonstrated statistical differences in the resorption and magnitude of bony ingrowth of the three BGS materials. The three BGS were significantly different for ingrowth (p = 0.046) when using the Wilcoxon Test. The ceramic graft material averaged 47% bony ingrowth. Rabbit-based DBX material showed extensive osseous ingrowth (35%) and the composite graft material demonstrated significant bony ingrowth (56%). The control, as anticipated, showed the least amount of bony ingrowth (29%). Fisher's Exact Test yielded statistical differences (p = 0.0003) when comparisons for resorption were conducted. An ideal BGS material should be biocompatible, be able to withstand the local load environment for a given application, degrade in concert with bony replacement, and be both osteoinductive and osteoconductive. This in-vivo, head-to-head comparison of three commercially available BGS materials in an animal model compares these characteristics and demonstrates differences between them, which may act as a guide in the use of these products in human applications.     

A novel strategy for prefabrication of large and axially vascularized tissue engineered bone by using an arteriovenous loop

The repair of bone defects remains a major clinical challenge because none available reconstruction methods and biomaterials have been proved completely satisfactory. As a promising approach for bone regeneration, tissue engineered bone has become a technically feasible method to repair small to moderate sized bone defects in clinical practice, but it is difficult to repair large one, particularly when the recipient site is scarred by infection or radiation injury. Construction of large and vascularized tissue engineered bone may overcome the problems since vascularization is an essential prerequisite for the constructs to survive and integrate with existing host tissue. On the other hand, prefabrication large artificial bone in vivo bioreactor and axial vascularization by means of arteriovenous loop model in soft tissue have been proved to be feasible. Therefore, we hypothesize that combination of cells, solid scaffold, growth factors, and arteriovenous loop may eventually generate a large and vascularized tissue engineered bone flap in vivo bioreactor. Like vascularized autologous bone grafts, the new constructs could be transferred to the defect site by using microsurgical techniques. The strategy would facilitate clinical translation in bone tissue engineering and offer new therapeutic strategies for reconstruction of extended bone defects if the hypothesis proved to be practical.     

Comparison of hydrogels in the in vivo formation of tissue-engineered bone using mesenchymal stem cells and beta-tricalcium phosphate

Availability of grafts and morbidity at the donor site limit autologous transplantation in patients requiring bone reconstruction. A tissue-engineering approach can overcome these limitations by producing bone-like tissue of custom shape and size from isolated cells. Several hydrogels facilitate osteogenesis on porous scaffolds; however, the relative suitability of various hydrogels has not been rigorously assessed. Fibrin glue, alginate, and collagen I hydrogels were mixed with swine bone marrow-derived differentiated mesenchymal stem cells (MSCs), applied to 3-dimensionally printed porous beta-tricalcium phosphate (beta-TCP) scaffolds and implanted subcutaneously in nude mice. Although noninvasive assessment of osteogenesis in 3 dimensions is desirable for monitoring new bone formation in vivo, correlations with traditional histological and mechanical testing need to be established. High-resolution volumetric computed tomography (VCT) scanning, histological examination, biomechanical compression testing, and osteonectin (ON) expression were performed on excised scaffolds after 1, 2, 4, and 6 weeks of subcutaneous implantation in mice. Statistical correlation analyses were performed between radiological density, stiffness, and ON expression. Use of collagen I as a hydrogel carrier produced superior bone formation at 6 weeks, as demonstrated using VCT scanning with densities similar to native bone and the highest compression values. Continued contribution of the seeded MSCs was demonstrated using swine-specific messenger ribonucleic acid probes. Radiological density values correlated closely with the results of histological and biomechanical testing and ON expression. High-resolution VCT is a promising method for monitoring osteogenesis.     

Brief review of models of ectopic bone formation

Ectopic bone formation is a unique biologic entity--distinct from other areas of skeletal biology. Animal research models of ectopic bone formation most often employ rodent models and have unique advantages over orthotopic (bone) environments, including a relative lack of bone cytokine stimulation and cell-to-cell interaction with endogenous (host) bone-forming cells. This allows for relatively controlled in vivo experimental bone formation. A wide variety of ectopic locations have been used for experimentation, including subcutaneous, intramuscular, and kidney capsule transplantation. The method, benefits and detractions of each method are summarized in the following review. Briefly, subcutaneous implantation is the simplest method. However, the most pertinent concern is the relative paucity of bone formation in comparison to other models. Intramuscular implantation is also widely used and relatively simple, however intramuscular implants are exposed to skeletal muscle satellite progenitor cells. Thus, distinguishing host from donor osteogenesis becomes challenging without cell-tracking studies. The kidney capsule (perirenal or renal capsule) method is less widely used and more technically challenging. It allows for supraphysiologic blood and nutrient resource, promoting robust bone growth. In summary, ectopic bone models are extremely useful in the evaluation of bone-forming stem cells, new osteoinductive biomaterials, and growth factors; an appropriate choice of model, however, will greatly increase experimental success.     

Bone marrow-derived recipient cells in murine transplanted hearts: potential roles and the effect of immunosuppression

Currently, there is intense debate regarding the origin of reparative cells in injured hearts and vasculature. To determine the contribution of recipient bone marrow (BM)-derived cells to the regeneration of cells in the vasculature of transplanted hearts and to examine the effect of immunosuppression on this phenomenon, we evaluated the fate of green fluorescent protein (GFP)-positive recipient BM cells in non-GFP-expressing cardiac allografts. C57BL/6 BM-GFP chimeric recipients underwent cardiac transplantation. Allografts were immunosuppressed with tacrolimus for 14 or 30 days post-transplantation or were saline treated. Hearts were excised and stained with markers for endothelial cells (EC) or smooth muscle cells (SMC). Colocalization with BM-derived recipient cells was evaluated using confocal microscopy with three-dimensional image analysis. Immunosuppression with tacrolimus did not affect the frequency of recipient BM-derived cell chimerism as EC or SMC phenotypes. A higher frequency of EC chimerism was found at 14 days as compared to 30 days post-transplantation in allograft hearts. BM-derived recipient cells are recruited to areas of donor vascular injury with intercalation of recipient EC and SMC in the setting of ongoing alloimmune recognition of the allograft. Our findings confirm that immunosuppression with tacrolimus does not affect the frequency of recipient BM-derived cell repopulation at an early time point 14 days post-transplantation. EC repopulation by BM-derived recipient cells was found to be an early event in transplanted allograft hearts, which decreased in frequency over time.     

Teratoma formation by human embryonic stem cells is site dependent and enhanced by the presence of Matrigel

When implanted into immunodeficient mice, human embryonic stem cells (hESCs) give rise to teratoma, tumor-like formations containing tissues belonging to all three germ layers. The ability to form teratoma is a sine qua non characteristic of pluripotent stem cells. However, limited data are available regarding the effects of implantation site and the methods employed for implantation on the success rate of teratoma formation. In this study, the rate of teratoma formation in immunodeficient mice was site dependent: subcutaneous (25-100%), intratesticular (60%), intramuscular (12.5%), and under the kidney capsule (100%). Co-injecting the hESCs with Matrigel increased subcutaneous teratoma formation efficiency from 25-40% to 80-100%. We did not observe site-specific differences in the teratoma composition at the histological level. However, subcutaneous teratomas were quite distinct, easy to remove, and caused minimal discomfort to the mice. Also, subcutaneous teratomas displayed larger proportion of solid tissues as opposed to cyst formation that dominated the teratomas formed at the other sites. Interestingly, a chromosomally abnormal hESCs with trisomy 20 formed teratomas where the ratio of differentiated to undifferentiated tissues was significantly decreased suggesting defective pluripotency of the cells. In conclusion, subcutaneous implantation of hESCs in presence of Matrigel appears to be the most efficient, reproducible, and the easiest approach for teratoma formation by hESCs. Also, teratoma formation can be employed to study the development defects exhibited by the chromosomally abnormal hESC lines.     

Vascular smooth muscle cells of recipient origin mediate intimal expansion after aortic allotransplantation in mice

Intimal expansion by vascular smooth muscle cells (SMCs) is a characteristic feature of graft vascular disease. Whether graft intimal SMCs arise from donor or recipient tissue is not well established but has important pathogenetic implications. We examined for the presence of male cells in the expanded intima of sex-mismatched mouse aortic allografts (C57BL/6-to-BALB/c) at 30 or 60 days after transplant by in situ hybridization using a Y-chromosome probe. Study groups included male-to-female allografts, female-to-male allografts, and female-to-female allografts in recipients previously engrafted with male bone marrow. Although intimal expansion developed in all allografts, male-to-female allografts lacked Y-chromosome-positive intimal cells. In contrast, such cells were abundant in female-to-male allografts and most of these cells co-labeled for smooth muscle alpha-actin by immunostain. Female-to-female allografts in recipients with male bone marrow showed a limited number of intimal Y-chromosome-positive cells. However, none of these clearly co-labeled for smooth muscle alpha-actin and their numbers declined throughout time, consistent with graft-infiltrating inflammatory cells. We conclude that intimal expansion of mouse aortic allografts is mediated by SMCs that originated from the recipient. There was little evidence of their derivation from the bone marrow, suggesting instead the adjacent host aorta as the primary source of intimal SMCs.