Design criteria for a modular tissue-engineered construct

A modular construct, created by the assembly of discrete microscale objects, has been proposed to enable the engineering of large, vascularized tissues containing multiple cell types. A simple theoretical analysis of the design constraints relevant to a modular construct was performed and used to define useable device operating ranges. The analysis assumed that the primary design constraint was the operating wall shear stress that would lead to a non-thrombogenic endothelial cell layer. At the lower end of the desirable shear range, oxygen depletion (over the length of the construct) limited the maximum allowed construct length, whereas at the upper end of this shear range, construct pressure difference limited maximum construct length. To compare with the theoretical analysis, real constructs were assembled, and construct porosity was assessed using superficial velocity-pressure difference profiles. Significant deviations from ideal construct porosity were observed for soft collagen gel constructs. Improvement of the module mechanical properties through the use of poloxamine instead of collagen as the module material enabled constructs closer to the ideal case to be assembled. With such improvements, modular tissue engineering offers a feasible strategy for the development of clinically significant whole-organ replacements.     

Magnetic resonance microscopy versus light microscopy in human embryology teaching

A study was carried out on the application of magnetic resonance microscopy (MRM) in teaching prenatal human development. Human embryos measuring 8 mm, 15 mm, 18.5 mm, and 22 mm were fixed in a 4% paraformaldehyde solution and sections obtained with magnetic resonance imaging (MRI) were compared to those prepared for light microscopy (LM), using the same embryos. The MRM and LM slices were of a similar quality. In the MRM sections, embryonic organs and systems were clearly visible, particularly the peripheral and central nervous systems, and the cardiovascular and digestive systems. The digitalization and clarity of the MRM images make them an ideal teaching aid that is suitable for students during the first years of a health-science degree, particularly medicine. As well as providing students with their first experience of MRM, these images allow students to access, at any time, all embryos used, to assess changes in the positions of different organs throughout their stages of development, and to acquire spatial vision, an absolute requirement in the study of human anatomy. We recommend that this technique be incorporated into the wealth of standard embryonic teaching methods already in use.     

Magnetic resonance microscopy of mouse embryos in utero

Magnetic resonance microscopy (MRM) was used to study mouse embryonic development in utero. MRM is a non-invasive imaging technique to study normal and abnormal embryonic development. To overcome image blurring as a result of embryonic movement, fast imaging sequences were used (less than 1 min scanning time). Clear morphologic proton images were obtained by diffusion spin echo and by rapid acquisition with relaxation enhancement (RARE), revealing living mouse embryos with great anatomical detail. In addition, functional information about embryonic blood flow could be obtained, in the absence of a contrast agent. This was achieved by combining two imaging sequences, RARE and very fast gradient echo. We expect that MRM will soon become a feasible method to study longitudinally both normal and abnormal (transgenic) mouse development.     

Chorioallantoic membrane for in vivo investigation of tissue-engineered construct biocompatibility

In tissue engineering approach, the scaffold plays a key role for a suitable outcome of cell-scaffold interactions and for the success of tissue healing and regeneration. As a consequence, the characterization of scaffold properties and the in vivo evaluation of tissue responses and effects result to be essential in the development of suitable implantable device. Among the in vivo methods, the chick embryo chorioallantoic membrane (CAM) assay represents a rather simple and cost-effective procedure to study the biocompatibility responses of graft materials. CAM is indeed characterized by low experiment costs, simplicity, relative speed in obtaining the expected results, limited ethical concern, no need of high-level technical skill, and the absence of a mature immune system, resulting in an inexpensive, simple, and practical method to evaluate and characterize tissue-engineered constructs. The results till now obtained suggest that CAM assay can be used as a pre-screening assay, before in vivo animal studies, to determine whether the scaffold is liable to cause an adverse reaction and to evaluate its future enhancement of existing materials for tissue engineering. A review of the more recent results related to the use of CAM for in vivo biomaterial property evaluation is herein reported.     

In vivo noninvasive monitoring of dissolved oxygen concentration within an implanted tissue-engineered pancreatic construct

The function of an implanted tissue-engineered pancreatic construct is influenced by many in vivo factors; however, assessing its function is based primarily on end physiologic effects. As oxygen significantly affects cell function, we established a dual perfluorocarbon method that utilizes (19)F nuclear magnetic resonance spectroscopy, with perfluorocarbons as oxygen concentration markers, to noninvasively monitor dissolved oxygen concentration (DO) in βTC-tet cell-containing alginate beads and at the implantation milieu. Beads were implanted in the peritoneal cavity of normal and streptozotocin-induced diabetic mice. Using this method, the feasibility of acquiring real-time in vivo DO measurements was demonstrated. Results showed that the mouse peritoneal environment is hypoxic and the DO is further reduced when βTC-tet cell constructs were implanted. The DO within cell-containing beads decreased considerably over time and could be correlated with the relative changes in the number of viable encapsulated cells. The reduction of construct DO due to the metabolic activity of the βTC-tet cells was also compatible with the implant therapeutic function, as observed in the reversal of hyperglycemia in diabetic mice. The importance of these findings in assessing implant functionality and host animal physiology is discussed.     

Novel pulse duplicating bioreactor system for tissue-engineered vascular construct

Cell culture in a biomimetic environment is known to improve the mechanical endurance of tissue-engineered cardiovascular components. Our goal was to generate a bioreactor that can reproduce a wide range of pulsatile flows with a completely physiological pressure profile. The morphology and biochemical properties of tissue-engineered products were also studied to test the usefulness of this novel bioreactor. The combination of an outflow valve, compliance chamber, and resistant clamps together with a balloon pumping system was able to successfully reproduce both physiological systolic and diastolic pressures. The compliance chamber was especially effective in transforming the original peaky pressure waveform into a physiological pressure profile. The tissues, cultured under a physiological pressure waveform with pulsatile flow, presented widely distributed cells in close contact with each other. They also showed significantly higher cell numbers, total protein content, and proteoglycan-glycosaminoglycan content than cultured tissues under a peaky pressure wave or under static conditions. This new bioreactor system is suitable for evaluating a favorable environment for tissue-engineered cardiovascular components.     

Cartilage repair using an in vitro generated scaffold-free tissue-engineered construct derived from porcine synovial mesenchymal stem cells

The objective was to in vitro generate a mesenchymal stem cell (MSC)-based tissue-engineered construct (TEC) to facilitate in vivo repair in a porcine chondral defect model. Porcine synovial MSCs were cultured in monolayer at high density and were subsequently detached from the substratum. The cell/matrix complex spontaneously contracted to develop a basic TEC. Immunohistochemical analysis showed that the basic TEC contained collagen I and III, fibronectin, and vitronectin. The basic TEC exhibited stable adhesion to the surface of a porcine cartilage matrix in an explant culture system. The TEC cultured in chondrogenic media exhibited elevated expression of glycosaminoglycan and chondrogenic marker genes. The TEC were implanted in vivo into chondral defects in the medial femoral condyle of 4-month-old pigs, followed by sacrifice after 6 months. Implantation of a TEC into chondral defects initiated repair with a chondrogenic-like tissue, as well as secure biological integration to the adjacent cartilage. Histologically, the repair tissue stained positively with Safranin O and for collagen II. Biomechanical evaluation revealed that repair tissue exhibited mechanical properties similar to those of normal porcine cartilage in static compression and friction tests. This technology is a unique and promising method for stem cell-based cartilage repair.     

Magnetic resonance microscopy of hamster olfactory bulb: A histological correlation

Background: Magnetic Resonance Imaging (MRI) has been widely used as a noninvasive diagnostic tool for obtaining morphological, metabolic, and functional information from tissue. However, its potential application in observing detailed structure comparable to that of the light microscope has not yet been fully explored. In order to evaluate the usefulness of MR microscopy, a high resolution three-dimensional (3-D) technique was applied to observe the laminar structure of the mammalian olfactory bulb (OB). Methods: Adult male hamsters (Mesocrecitus auratus) were used as an animal model. Hamster OB and the attached anterior olfactory nucleus were removed from the skull for the MRI examinations. The images were performed with a Bruker AMX-400 system equipped with microimaging accessories. T2 weighted 3-D spin echo sequence was used with a field of view of 9 mm and data matrix of 128*128*128. The in-plane resolution was 70*70*70 μm. Histological preparation, including vibratome sectioning at 40 μm and Nissl staining, were used for light microscopic evaluations and comparisons. Results: Five distinct layers from the superficial to the center of the OB were distinguished in the MR images of coronal, sagittal and horizontal slices. As compared to the histological sections at the corresponding cutting planes, the laminar structure of the OB displayed in the MR microscopic images correlated well with its counterparts. Conclusions: MR microscopy is capable of detecting cellular variation of unsectioned and unstained tissue. It can also be easily applied to obtain spatial information with good resolution. It appears to provide a great potential for diagnostic pathology. © 1995 Wiley-Liss, Inc.     

Initial intramembraneous osteogenesis in vitro

Calvariae of fetal mice 12 to 13 days in utero were placed in Rose chambers and cultured in BGJb medium supplemented with 20% fetal calf serum and antibiotics. After periods of 7 and 12 days, the explants were harvested and processed for light and electron microscopy. The mesenchymal cells, after a brief lag time, differentiated into osteoblasts which produced woven bone. Light and electron microscopic observations showed that in vitro, as well as in vivo, growth and development share all of the same characteristics of initial intramembraneous osteogenesis: (1) migration and differentiation of mesenchymal cells into osteoblasts, (2) the subsequent appearance of matrix vesicles in the extra-cellular space, (3) crystallization of hydroxyapatite within and about these vesicles, (4) growth of hydroxyapatite crystals into spheroidal nodules of bone, and (5) the subsequent fusion of these nodules into seams of woven bone. Thus cellular involvement in initial osteogenesis has been observed in a system where differentiation into osteoblasts and initial calcification takes place in vitro, suggesting that the events responsible for these phenomena have occurred within the cells prior to their morphological differentiation.     

体外构建血管化组织工程心肌

目的:探讨体外构建血管化组织工程心肌的可行性。方法:大鼠骨髓间充质干细胞以浓度为10μmol/L的5-氮胞苷诱导为心肌细胞并用DAPI标记、骨髓源内皮祖细胞以内皮细胞培养基EGM2-MV定向诱导为内皮细胞并用CM-dil标记。标记后的心肌细胞与内皮祖细胞按2:1比例,以4×10~6个/ml的密度种植于Matrigel基质胶支架上。同种密度心肌细胞种植为对照组。对复合体形态、细胞分布等进行观察。结果:H-E染色及荧光显微镜观察可见两组复合体的细胞分布均匀,生长状态良好;单纯心肌细胞种植组可见细胞黏附聚集生长,并未出现网状结构,而2:1混合培养组24 h后可见内皮细胞相互连接成网状结构,出现典型的成血管现象,且形成的血管样结构明显多于对照组;心肌细胞凋亡数较对照组少。结论:骨髓源性心肌细胞与骨髓源性内皮细胞联合种植于Matrigel基质胶共培养,可于体外成功构建血管化组织工程心肌。     

Radiofrequency coils for magnetic resonance microscopy

Given the several orders of magnitude fewer spins per voxel for MR microscopy than for conventional MRI, efficient coil design is important to obtain sufficient signal-to-noise within reasonable data acquisition times. As MR microscopy is typically performed using very high magnetic fields, coil design must also incorporate the effects of increased component losses and skin-depth-dependent resistance, as well as radiation losses and phase effects for coils when conductor dimensions constitute a substantial fraction of the electromagnetic wavelength. For samples much less than 1 mm in size, wire solenoids or microfabricated planar coils are used. For samples with diameters of several millimeters, saddle, birdcage, Alderman-Grant or millipede coils become the preferred choice. Recent advances in multiple-coil probes and phased arrays have been used to reduce data acquisition time and/or increase sample throughput, and small superconducting coils have shown significant improvements in signal-to-noise over equivalently sized room-temperature coils.     

A tissue-engineered muscle repair construct for functional restoration of an irrecoverable muscle injury in a murine model

There are no effective clinical treatments for volumetric muscle loss (VML) resulting from traumatic injury, tumor excision, or other degenerative diseases of skeletal muscle. The goal of this study was to develop and characterize a more clinically relevant tissue-engineered muscle repair (TE-MR) construct for functional restoration of a VML injury in the mouse lattissimus dorsi (LD) muscle. To this end, TE-MR constructs developed by seeding rat myoblasts on porcine bladder acellular matrix were preconditioned in a bioreactor for 1 week and implanted in nude mice at the site of a VML injury created by excising 50% of the native LD. Two months postinjury and implantation of TE-MR, maximal tetanic force was ～72% of that observed in native LD muscle. In contrast, injured LD muscles that were not repaired, or were repaired with scaffold alone, produced only ～50% of native LD muscle force after 2 months. Histological analyses of LD tissue retrieved 2 months after implantation demonstrated remodeling of the TE-MR construct as well as the presence of desmin-positive myofibers, blood vessels, and neurovascular bundles within the TE-MR construct. Overall, these encouraging initial observations document significant functional recovery within 2 months of implantation of TE-MR constructs and provide clear proof of concept for the applicability of this technology in a murine VML injury model.     

Noninvasive measurement of viable cell number in tissue-engineered constructs in vitro, using 1H nuclear magnetic resonance spectroscopy

Noninvasive monitoring of tissue-engineered constructs is of critical importance for accurate characterization of constructs and their remodeling in vitro and in vivo. This study investigated the utility of (1)H NMR spectroscopy to noninvasively quantify viable cell number in tissue-engineered substitutes in vitro. Agarose disk-shaped constructs containing betaTC3 cells were employed as the model tissue-engineered system. Two construct prototypes containing different initial cell numbers were monitored by localized, water-suppressed 1H NMR spectroscopy over the course of 13 days. (1)H NMR measurements of the total choline resonance at 3.2 ppm were compared with results from the traditional cell viability assay MTT and with insulin secretion rates. Results show a strong linear correlation between total choline and MTT (R (2) = 0.86), and between total choline and insulin secretion rate (R (2) = 0.90). Overall, this study found noninvasive measurement of total choline to be an accurate and nondestructive assay for monitoring viable betaTC3 cell numbers in tissue-engineered constructs. The applicability of this method to in vivo monitoring is also discussed.     

同种异体骨髓间充质干细胞在比格犬体内异位构建组织工程骨

BACKGROUND: Allogenic bone marrow mesenchymal stem cells as seed cells for tissue engineering have become the future trend of development. OBJECTIVE: To investigate the osteogenic effects of allogenic bone marrow mesenchymal stem cells and the outcome in vivo. METHODS: Bone marrow mesenchymal stem cells from beagle dogs were marked with chloromethylbenzoyl ammonia fluorescent dye (CM-Dil), and the proliferation of labeled cells was measured using MTT assay in vitro. Autologous or allogenic bone marrow mesenchymal stem cells were inoculated into coral and β-tricalcium phosphate scaffolds for 7 days osteogenic induction and then subcutaneously implanted into the back of beagle dogs. Dogs undergoing blank scaffold implantation served as negative controls. Hematoxylin-eosin staining was used to observe new bone formation at 3 days, 1, 2, 4, 8, 12 weeks after surgery. Bone formation area was statistically analyzed using ipp software. In the CM-Dil group, frozen sections were made to trace the in vivo outcome of bone marrow mesenchymal stem cells under a fluorescence microscope. RESULTS AND CONCLUSION:The osteogenesis speed in the allogenic bone tissue engineering group was faster than that in the autologous bone tissue engineering group at 4-8 weeks after implantation, but no significant difference between the two groups was found beginning at the 12th week. At 4 weeks after implantation, the expression of γ-carboxy glutamic acid protein in the autologous bone tissue engineering group was higher than that in the allogenic bone tissue engineering group, prompting the bone mineralization appeared earlier in the latter group than the former one. ELISA results showed that the expression of alkaline phosphatase and osteocalcin in the autologous bone tissue engineering group was higher than that in the allogenic bone tissue engineering group at 4 weeks after implantation, and then the expression showed no difference at 12 weeks. CM-Dil labeling results showed that the number of allogenic bone marrow mesenchymal stem cells was reduced significantly compared with that of autologous bone marrow mesenchymal stem cells. All these findings indicate that the ectopic osteogenesis of the allogenic tissue-engineered bone in large animals is found within 12 weeks after implantation, but the osteogenesis efficiency at early stage (within 8 weeks) is lower compared with the autologous tissue-engineered bone. This difference may be related to the post-implantation immunoreactions that lead to the reduction in cell number. 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     

Biomimetic microtopography to enhance osteogenesis in vitro

Biomimicry is being used in the next generation of biomaterials. Tuning material surface features such as chemistry, stiffness and topography allow the control of cell adhesion, proliferation, growth and differentiation. Here, microtopographical features with nanoscale depths, similar in scale to osteoclast resorption pits, were used to promote in vitro bone formation in basal medium. Primary human osteoblasts were used to represent an orthopaedically relevant cell type and analysis of adhesions, cytoskeleton, osteospecific proteins (phospho-Runx2 and osteopontin) and mineralisation (alizarin red) was performed. The results further demonstrate the potential for biomimicry in material design and show that the osteoblast response can be tuned from changes in feature size.     

Construction of tissue-engineered homograft bioprosthetic heart valves in vitro

Human mesenchymal stem cells (MSCs) differentiate into multiple cell-lineages and may serve as an alternative source of seed cells for tissue engineering. We investigated whether MSCs could be induced to differentiate into endothelial cells (ECs) and function as seed cells for the in vitro construction of tissue-engineered heart valves (TEHVs). Aortic or pulmonary valve homografts were decellularized with 0.1% sodium dodecylsulphate and used as scaffolds for TEHVs. The MSCs were isolated from human bone marrow by Percoll gradient centrifugation (1.073 g/ml), differentiated into ECs with vascular endothelial growth factor (10 ng/ml), and seeded onto a decellularized scaffold (high-density seeding, >10(5) cells/cm2) and grown in static culture for 14 days. Over 90% of the differentiated cells from MSCs stained positively for von Willebrand factor and Tie-2-related antigen. Additionally, Weibel-Palade corpuscle was observed in the cytoplasm of these cells. Levels of reendothelialization in static culture on days 7, 14, and 20, were 73%, 85%, and 95%, respectively. These results show that MSCs from human bone marrow can differentiate, in vitro, into ECs that can then be used to construct TEHVs. Reendothelialization in static culture can be used to provide the basic material for pulsatile-flow cultivation.     

Effect of low oxygen tension on tissue-engineered cartilage construct development in the concentric cylinder bioreactor

Cartilage is exposed to low oxygen tension in vivo, suggesting culture in a low-oxygen environment as a strategy to enhance matrix deposition in tissue-engineered cartilage in vitro. To assess the effects of oxygen tension on cartilage matrix accumulation, porous polylactic acid constructs were dynamically seeded in a concentric cylinder bioreactor with bovine chondrocytes and cultured for 3 weeks at either 20 or 5% oxygen tension. Robust chondrocyte proliferation and matrix deposition were achieved. After 22 days in culture, constructs from bioreactors operated at either 20 or 5% oxygen saturation had similar chondrocyte densities and collagen content. During the first 12 days of culture, the matrix glycosaminoglycan (GAG) deposition rate was 19.5 x 10(-9) mg/cell per day at 5% oxygen tension and 65% greater than the matrix GAG deposition rate at 20% oxygen tension. After 22 days of bioreactor culture, constructs at 5% oxygen contained 4.5 +/- 0.3 mg of GAG per construct, nearly double the 2.5 +/- 0.2 mg of GAG per construct at 20% oxygen tension. These data demonstrate that culture in bioreactors at low oxygen tension favors the production and retention of GAG within cartilage matrix without adversely affecting chondrocyte proliferation or collagen deposition. Bioreactor studies such as these can identify conditions that enhance matrix accumulation and construct development for cartilage tissue engineering.     

Interaction of mesenchymal stem cells and osteoblasts for in vitro osteogenesis

It has recently been reported that bone marrow-derived mesenchymal stem cells (MSCs), which are systemically administrated to different species, undergo site-specific differentiation. This suggests that the tissue specific cells may cause or promote the differentiation of the MSCs toward their cell type via a cell-to-cell interaction that is mediated not only by hormones and cytokines, but also by direct cell-to-cell contact. In this study, in order to assess the possible synergistic interactions for osteogenesis between the two types of cells, the MSCs derived from rabbit bone marrow were co-cultured with rat calvarial osteoblasts in direct cell-to-cell contact in a control medium (CM) and in an osteogenic medium (OM). The cell number, alkaline phosphatase activity, and amount of calcium deposition were assayed in the cultures of MSCs, osteoblasts, and co-cultures of them in either OM or CM for up to 40 days. The cell numbers and the alkaline phosphatase activities in the co-culture were somewhere in between those of the osteoblast cultures and the MSC cultures. The amounts of deposited calcium were lower in the co-culture compared to those of the other cultures. This suggests that there are little synergistic interactions during osteogenesis in vitro between the rat osteoblasts and rabbit MSCs.     

Fiber-based tissue-engineered scaffold for ligament replacement: design considerations and in vitro evaluation

The anterior cruciate ligament (ACL) is the major intraarticular ligamentous structure of the knee, which functions as a joint stabilizer. It is the most commonly injured ligament of the knee, with over 150,000 ACL surgeries performed annually in the United States. Due to limitations associated with current grafts for ACL reconstruction, there is a significant demand for alternative graft systems. We report here the development of a biodegradable, tissue-engineered ACL graft. Several design parameters including construct architecture, porosity, degradability, and cell source were examined. This graft system is based on polymeric fibers of polylactide-co-glycolide 10:90, and it was fabricated using a novel, three-dimensional braiding technology. The resultant micro-porous scaffold exhibited optimal pore diameters (175-233 microm) for ligament tissue ingrowth, and initial mechanical properties of the construct approximate those of the native ligament.     

The maturity of tissue-engineered cartilage in vitro affects the repairability for osteochondral defect

Cartilage tissue engineering using cells and biocompatible scaffolds has emerged as a promising approach to repair of cartilage damage. To date, however, no engineered cartilage has proven to be equivalent to native cartilage in terms of biochemical and compression properties, as well as histological features. An alternative strategy for cartilage engineering is to focus on the in vivo regeneration potential of immature engineered cartilage. Here, we used a rabbit model to evaluate the extent to which the maturity of engineered cartilage influenced the remodeling and integration of implanted extracellular matrix scaffolds containing allogenous chondrocytes. Full-thickness osteochondral defects were created in the trochlear groove of New Zealand white rabbits. Left knee defects were left untreated as a control (group 1), and right knee defects were implanted with tissue-engineered cartilage cultured in vitro for 2 days (group 2), 2 weeks (group 3), or 4 weeks (group 4). Histological, chemical, and compression assays of engineered cartilage in vitro showed that biochemical composition became more cartilagenous, and biomechanical property for compression gradually increased with culture time. In an in vivo study, gross imaging and histological observation at 1 and 3 months after implanting in vitro-cultured engineered cartilage showed that defects in groups 3 and 4 were repaired with hyaline cartilage-like tissue, whereas defects were only partially filled with fibrocartilage after 1 month in groups 1 and 2. At 3 months, group 4 showed striking features of hyaline cartilage tissue, with a mature matrix and a columnar arrangement of chondrocytes. Zonal distribution of type II collagen was most prominent, and the International Cartilage Repair Society score was also highest at this time. In addition, the subchondral bone was well ossified. In conclusion, in vivo engineered cartilage was remodeled when implanted; however, its extent to maturity varied with cultivation period. Our results showed that the more matured the engineered cartilage was, the better repaired the osteochondral defect was, highlighting the importance of the in vitro cultivation period.