Cell-scaffold Complexes Research Articles

Cytokinetic abscission in Toxoplasma gondii is governed by protein phosphatase 2A and the daughter cell scaffold complex

Cytokinetic abscission marks the final stage of cell division, during which the daughter cells physically separate through the generation of new barriers, such as the plasma membrane or cell wall. While the contractile ring plays a central role during cytokinesis in bacteria, fungi and animal cells, the process diverges in Apicomplexa. In Toxoplasma gondii, two daughter cells are formed within the mother cell by endodyogeny. The mechanism by which the progeny cells acquire their plasma membrane during the disassembly of the mother cell, allowing daughter cells to emerge, remains unknown. Here we identify and characterize five T. gondii proteins, including three protein phosphatase 2A subunits, which exhibit a distinct and dynamic localization pattern during parasite division. Individual downregulation of these proteins prevents the accumulation of plasma membrane at the division plane, preventing the completion of cellular abscission. Remarkably, the absence of cytokinetic abscission does not hinder the completion of subsequent division cycles. The resulting progeny are able to egress from the infected cells but fail to glide and invade, except in cases of conjoined twin parasites.

Regenerated silk fibroin based on small aperture scaffolds and marginal sealing hydrogel for osteochondral defect repair

BackgroundOsteochondral defects pose an enormous challenge without satisfactory repair strategy to date. In particular, the lateral integration of neo-cartilage into the surrounding native cartilage is a difficult and inadequately addressed problem determining tissue repair’s success.MethodsRegenerated silk fibroin (RSF) based on small aperture scaffolds was prepared with n-butanol innovatively. Then, the rabbit knee chondrocytes and bone mesenchymal stem cells (BMSCs) were cultured on RSF scaffolds, and after induction of chondrogenic differentiation, cell-scaffold complexes strengthened by a 14 wt% RSF solution were prepared for in vivo experiments.ResultsA porous scaffold and an RSF sealant exhibiting biocompatibility and excellent adhesive properties are developed and confirmed to promote chondrocyte migration and differentiation. Thus, osteochondral repair and superior horizontal integration are achieved in vivo with this composite.ConclusionsOverall, the new approach of marginal sealing around the RSF scaffolds exhibits preeminent repair results, confirming the ability of this novel graft to facilitate simultaneous regeneration of cartilage–subchondral bone.

DFCs/TDM based artificial bio-root to obtain long-term functional root regeneration in non-human primate

Stem cell/scaffold-based tissue engineering technology is expected to regenerate tooth root, thus replace dental implant. Although numerous patterns of biological tooth root have been constructed, most of them failed for the reasons involved the type of selected stem cells, scaffold materials, and the recombination strategy of cell-scaffold complexes. Here we introduced a novel functional biological root (FBR) with a sandwich structure, by which occlusal function and long-term masticatory function were gradually restored during two years functional evaluation in rhesus monkeys. FBR complex was constructed based on dental follicle cell sheets (DFCSs) and treated dentin matrix (TDM) under in vitro 3D suspension culture. Compared with other reported systems, DFCs displayed a more suitable seeding cell potential for bio-root construction due to the eminent odontogenic potential to develop cementum, periodontal ligament, and alveolar bone in vivo. Meanwhile, TDM also exhibited unique superiority of reserving native dentin tubules which can release numerous odontogenic proteins and factors comparing with other materials. Another unique characteristic of this work was that the favorable regeneration potential of FBR was proved in non-human primate, whose tooth morphology, number and development was more similar to humans, and the resulting FBRs closely resembled the natural tooth root in terms of their anatomical and physiological features, especially the “sandwich” structure of periodontal tissue, adequately demonstrating its potential application in tooth root regeneration.

Study on the Effect of PDA-PLGA Scaffold Loaded With Islet Cells for Skeletal Muscle Transplantation in the Treatment of Diabetes.

At present, islet cells transplantation was limited by the way in which islet cells are implanted into the body, their ability to adapt to the microenvironment and the maintenance time for relieving diabetic symptoms. In order to solve this problem, we made PDA-PLGA scaffold loaded with islet cells and used it for skeletal muscle transplantation to investigate its therapeutic effect in the treatment of diabetes. The PLGA scaffold was prepared by the electrospinning method, and modified by polydopamine coating. A rat diabetic model was established to evaluate the efficacy of PDA-PLGA scaffold loaded with RINm5f islet cells through skeletal muscle transplantation. The results showed that the PDA-PLGA scaffold has good biosafety performance. At the same time, transplantation of the stent to the skeletal muscle site had little effect on the serum biochemical indicators of rats, which was conducive to angiogenesis. The PDA-PLGA scaffold had no effect on the secretory function of pancreatic islet cells. The PDA-PLGA scaffold carrying RINm5f cells was transplanted into the skeletal muscle of type I diabetic rats. 1 week after the transplantation of the PDA-PLGA cell scaffold complex, the blood glucose of the treatment group was significantly lower than that of the model group (p < 0.001) and lasted for approximately 3 weeks, which further indicated the skeletal muscle transplantation site was a new choice for islet cell transplantation in the future.

Female Bioengineering: Primordial Germ Cell Differentiation of Mesenchymal Stem Cells onto Placental Scaffolds

Introduction: Female infertility is a condition that is currently treated through the maximization of existing reserves; a necessity due to the fact that a true reversal of the processes leading to infertility is not yet technologically possible. This experiment examined the ability of mesenchymal stem cells to differentiate into primordial germ cells (PGC) when cultured onto the placental scaffold. Methods: To produce the scaffolds, the cotelydons were collected and decellularized by umbilical vessel SDS perfusion. Adipose derived cells were isolated based on rapid adherence to the plastic. Results: The isolated cells displayed the markers CD90 and 105, while lacked CD34 and CD45. When seeded onto the scaffold, the cells successfully differentiated into PGC like cells, displaying typical PGCs markers, including STELLA, OCT4, DAZL and VASA. Discussion: These materials were chosen for their low cost and wide availability. The data in this study show the promising potential of cell-scaffold complex to support the development of female tissue engineering-based regenerative medicine therapies.

Recapitulating Developmental Condensation and Constructing Self-organised Cartilaginous Tissue for Cartilage Regeneration.

To develop a novel chondrocyte condensation culture strategy recapitulating developmental condensation and construct self-organised cartilaginous tissue for cartilage regeneration. Cell-condensation aggregate (CCA) was generated using the condensation culture method by sequential cell seeding. The chondrification capacities and biocompatibilities of CCA were assessed by comparison with the cell-scaffold complex (CSC), which was constructed by cell-scaffold coculture. Preclinical studies including implantation into nude mice subcutaneously and cartilage defect repair in rabbits were performed. CCA constructed by condensation culture exhibited a morphology of self-organised cartilaginous tissue. Meanwhile, the condensation culture inhibited or abolished expression of HOX genes including HOXC4 and HOXD8, which was partially consistent with developmental HOX gene expression patterns and associated with enhanced regeneration capacities. Compared with CSC, CCA showed a higher capacity for chondrification and regeneration of rabbit cartilage defects. The therapeutic assessments indicate that CCA is an efficient therapeutic tool for cartilage regeneration, providing a new strategy for tissue engineering by mimicking developmental events.

In vivo ectopic osteogenesis of adipose-derived mesenchymal stem cells/osteoblasts combined with PLLA/HAp nanostructured aerogel scaffolds

<h3>Objectives</h3> Osteoblast deficiency is a key problem in bone tissue engineering, and transplant of mesenchymal stem cells combined with osteoblasts can achieve ideal results. We sought to investigate the in vivo ectopic osteogenesis of adipose-derived mesenchymal stem cells (ADSCs) and osteoblasts (OBs) combined with poly(l-lactic acid) (PLLA)/hydroxyapatite (HAp) nanostructured aerogel scaffolds. <h3>Methods</h3> We tested a new supercritical fluid-assisted technique for the formation of nanostructured aerogel scaffolds. We obtained PLLA aerogel scaffolds and PLLA/HAp aerogel scaffolds with high level of porosity (>90%), interconnectivity, and mechanical properties (compressive modulus up to 2 kPa). The fibrous nanostructure of these scaffolds was joined to micronic cells of controllable size. ADSCs and OBs were obtained by using an adherent method and an enzymatic digestion method. ADSCs, OBs, and the mixture of ADSCs and OBs (at a ratio of 1:1) were cultured with PLLA/HAp nanostructured aerogel scaffolds, respectively. After 48 hours of in vitro culture, cell-scaffold complexes were subcutaneously implanted into the back of Sprague-Dawley rats in corresponding groups, and PLLA/HAp nanostructured aerogel scaffolds without cells were implanted in a control group. The rats in each group were killed at 8 weeks postoperatively. The macroscopic and histopathological observations were performed to assess the ectopic osteogenesis potential. <h3>Results</h3> After adipogenic, chondrogenic, and osteogenic induction, ADSCs were positive for Oil Red O, toluidine blue, and alizarin red staining. Results of flow cytometry showed that ADSCs were positive for CD147, CD90, CD105, and CD44, with the rate of positivity being >80%, but negative for CD117, CD34, CD131, and CD45, with the rate of positivity being <5%. Passage 3 OBs were positive for both alizarin red staining and alkaline phosphatase staining. At 8 weeks after implantation, soft tissues grew into the complexes under gross observation. At 8 weeks after implantation, ectopic bone formation was visible in each group. The bone formation was more visible in the ADSC-PLLA/HAp nanostructured aerogel scaffold group than in the other groups with a significant difference (<i>P</i> < .05). <h3>Conclusions</h3> To conclude, ADSCs can promote the ectopic bone formation of OBs in vivo in combination with PLLA/HAp nanostructured aerogel scaffolds.

Effect of nano-biomaterials silk-collagen sponge scaffold on the tendon and ligament healing of patients with anterior cruciate ligament injuries

With the rapid development of nanotechnology, the application of nanomaterials is more and more extensive, and the contact between humans, animals and plants and nanomaterials is inevitable. Anterior cruciate ligament (ACL) is an important intra-articular ligament of the knee joint, which plays a very significant role in the normal movement and functional stability of the knee joint. ACL is generally difficult to heal after injury and the main treatment method currently is use auto-graft or allograft and synthetic materials for ACL reconstruction, but there exist problems such as large demand but limited sources. Nanotechnology development provides new ideas and approaches for ACL reconstruction. This technology inoculates certain cells from autologous tissues into scaffold materials, and then implants the grown cell scaffold complex into defect or injury sites for completing the tissue or organ defect repair and functional reconstruction. On the basis of summarising and analysing previous research results, this paper selected silk and collagen sponges as experimental materials to prepare nano-biological scaffolds, then observed the surface structure of the prepared scaffolds under scanning electron microscope and conducted histological analysis and biomechanical testing, and finally applied this nano-silk-collagen sponge biomaterial scaffold (NNSCSBS) to the tendon ligament repair surgery of patients with ACL injury. The results showed that collagen fills fibrous spaces with relatively uniform distribution after NSCSBS hybridisation, and the NSCSBS cultured in vitro had more abundant skin fibroblasts growth; the NSCSBS had the porosity of (77.63 ± 1.82)%, the moisture of (78.24 ± 1.19)%, the maximum load of (122.48 ± 12.66) N, the stiffness of (26.07 ± 2.58) N/mm without statistically significant differences (P > 0.05); postoperative ACL's bone mineral density and collagen fibre composition both increased; the graft tendon was surrounded by new bone tissue, and the tendon and bone were connected by fibrous collagen with narrower interface gap and more mature new bone tissue. Therefore, the NSCSBS can provide mechanical support with silk scaffolds as the core, increase the scaffold's three-dimensional space with composite collagen as bio-component, promote more cell adhesion and improve biocompatibility of nano-biomaterial scaffold, and better repair ligament-osseous junction to form a bionic structure, having a good application prospect.

Application of 3D biological nano scaffold materials in the health treatment of foot and ankle sports

Bone tissue is an important part of the human body and plays an indispensable physiological function. Damage to bone tissue will have a serious impact on the health of patients. Three types of MGPC scaffolds were prepared using 3D printing technology. The macroscopic, microstructure, and histology of the three scaffolds were observed under an electron microscope. The cells grew normally and spread well. Rat L-929 fibroblasts were cultured in three scaffold extracts, and the cells were cultured with DMEM as a control. Cell proliferation was detected by cell counting. Scanning electron microscopy of the cell-scaffold complex revealed that a large number of chondrocytes adhered and grew on the surface of the MGPC scaffold. With the increase of culture time, the number of cells in each group showed an increasing trend. MGPC has no cytotoxicity, excellent physical and chemical properties, and is expected to become a biological scaffold material.

Versatile Cell-Specific Ligand Arrangement System onto Desired Compartments of Biodegradable Matrices for Site-Selective Cell Adhesion Using DNA Tags.

A promising approach for the regeneration of tissues or organs with three-dimensional hierarchical structures is the preparation of scaffold-cell complexes that mimic these hierarchical structures. This requires an effective technique for immobilizing cell-specific ligands at arbitrarily chosen positions on matrices. Here, we report a versatile system for arranging cell-specific ligands onto desired compartments of biodegradable matrices for site-selective cell arrangement. We utilized the specific binding abilities of specific DNAs, immobilizing them as tags to arrange cell-recognition ligands at desired areas of the matrices by specific binding with cell-recognition ligand-DNA conjugates. We synthesized poly(l-lactide) (PLLA), a biodegradable polymer, with an oligo-DNA (trimer of deoxyguanosine: dG3) attached via a poly(ethylene glycol) (PEG) spacer to generate dG3-PEG-b-PLLA. The peptides Arg-Gly-Asp-Ser (RGDS) and Arg-Glu-Asp-Val (REDV) were chosen as cell-recognition ligands and were attached to an adapter DNA (aDNA), which can specifically bind to the dG3 moiety through G-quadruplex formation. The obtained dG3-PEG-b-PLLA was deposited on a small spot of the PLLA film, and the aDNA-RGDS or aDNA-REDV conjugate was added on the film to immobilize these ligands at the spot. We confirmed the specific adhesion of L929 cells (a mouse fibroblast cell line) and human umbilical vein endothelial cells (HUVECs) on the small areas coated with dG3-PEG-b-PLLA in the presence of aDNA-RGDS and aDNA-REDV, respectively, even after applying shear stress by flowing medium across the spot. Cell-specific attachment of the target cells was effectively achieved in a spatially controlled manner. This technique has the potential for the construction of cell-scaffold complexes that mimic the hierarchical structures of natural organs and may represent a breakthrough in realizing regenerative medicine and tissue engineering of complex organs.

Optimization of the theoretical model for growth rate of mesenchymal stem cells on three-dimensional scaffold under fluid shear stress

Bone tissue engineering is considered as one of the most promising way to treat large segmental bone defect. When constructing bone tissue engineering graft in vitro, suitable bioreactor is usually used to incubate cell-scaffold complex under perfusion to obtain bone tissue engineering graft with good repair efficiency. However, the theoretical model for growth rate of single cell (especially for stem cell) during this process still has many defects. The difference between stem cells and terminally differentiated cells is always ignored. Based on our previous studies, this study used self-made perfusion apparatus to apply different modes and strengths of fluid shear stress (FSS) to the cells seeded on scaffolds. The effects of FSS on the proliferation and osteogenic differentiation of mesenchymal stem cells (MSCs) were investigated. The regression analysis model of the effect of FSS on the single-cell growth rate of MSCs was further established. The results showed that 0.022 5 Pa oscillatory shear stress had stronger ability to promote proliferation and osteogenic differentiation of MSCs, and the growth rate of a single MSC cell under FSS was modified. This study is expected to provide theoretical guidance for optimizing the perfusion culture condition of bone tissue engineering grafts in vitro.

Fabrication of poly (lactic-co-glycolic acid)/decellularized articular cartilage extracellular matrix scaffold by three-dimensional printing technology and investigating its physicochemical properties

To manufacture a poly (lactic-co-glycolic acid) (PLGA) scaffold by low temperature deposition three-dimensional (3D) printing technology, prepare a PLGA/decellularized articular cartilage extracellular matrix (DACECM) cartilage tissue engineered scaffold by combining DACECM, and further investigate its physicochemical properties. PLGA scaffolds were prepared by low temperature deposition 3D printing technology, and DACECM suspensions was prepared by modified physical and chemical decellularization methods. DACECM oriented scaffolds were prepared by using freeze-drying and physicochemical cross-linking techniques. PLGA/DACECM oriented scaffolds were prepared by combining DACECM slurry with PLGA scaffolds. The macroscopic and microscopic structures of the three kinds of scaffolds were observed by general observation and scanning electron microscope. The chemical composition of DACECM oriented scaffold was analyzed by histological and immunohistochemical stainings. The compression modulus of the three kinds of scaffolds were measured by biomechanical test. Three kinds of scaffolds were embedded subcutaneously in Sprague Dawley rats, and HE staining was used to observe immune response. The chondrocytes of New Zealand white rabbits were isolated and cultured, and the three kinds of cell-scaffold complexes were prepared. The growth adhesion of the cells on the scaffolds was observed by scanning electron microscope. Three kinds of scaffold extracts were cultured with L-929 cells, the cells were cultured in DMEM culture medium as control group, and cell counting kit 8 (CCK-8) was used to detect cell proliferation. General observation and scanning electron microscope showed that the PLGA scaffold had a smooth surface and large pores; the surface of the DACECM oriented scaffold was rough, which was a 3D structure with loose pores and interconnected; and the PLGA/DACECM oriented scaffold had a rough surface, and the large hole and the small hole were connected to each other to construct a vertical 3D structure. Histological and immunohistochemical qualitative analysis demonstrated that DACECM was completely decellularized, retaining the glycosaminoglycans and collagen typeⅡ. Biomechanical examination showed that the compression modulus of DACECM oriented scaffold was significantly lower than those of the other two scaffolds ( P<0.05). There was no significant difference between PLGA scaffold and PLGA/DACECM oriented scaffold ( P>0.05). Subcutaneously embedded HE staining of the three scaffolds showed that the immunological rejections of DACECM and PLGA/DACECM oriented scaffolds were significantly weaker than that of the PLGA scaffold. Scanning electron microscope observation of the cell-scaffold complex showed that chondrocytes did not obviously adhere to PLGA scaffold, and a large number of chondrocytes adhered and grew on PLGA/DACECM oriented scaffold and DACECM oriented scaffold. CCK-8 assay showed that with the extension of culture time, the number of cells cultured in the three kinds of scaffold extracts and the control group increased. There was no significant difference in the absorbance ( A) value between the groups at each time point ( P>0.05). The PLGA/DACECM oriented scaffolds have no cytotoxicity, have excellent physicochemical properties, and may become a promising scaffold material of tissue engineered cartilage.

In vitro study of bone morphogenetic protein 2 gelatin/chitosan hydrogel sustained-release system composite hydroxyapatite/zirconium dioxide foam ceramics and induced pluripotent stem cells derived mesenchymal stem cells

To construct bone morphogenetic protein 2 (BMP-2) gelatin/chitosan hydrogel sustained-release system, co-implant with induced pluripotent stem cells (iPS) derived mesenchymal stem cells (MSCs) to hydroxyapatite (HA)/zirconium dioxide (ZrO 2) bio porous ceramic foam, co-culture in vitro, and to explore the effect of sustained-release system on osteogenic differentiation of iPS-MSCs. BMP-2 gelatin/chitosan hydrogel microspheres were prepared by water-in-oil solution. Drug encapsulation efficiency, drug loading, and in vitro sustained release rate of the microspheres were tested. HA/ZrO 2 bio porous ceramic foam composite iPS-MSCs and BMP-2 gelatin/chitosan hydrogel sustained release system co-culture system was established as experimental group, and cell scaffold complex without BMP-2 composite gelatin/chitosan hydrogel sustained release system as control group. After 3, 7, 10, and 14 days of co-culture in the two groups, ALP secretion of cells was detected; gene expression levels of core binding factor alpha 1 (Cbfa1), collagen type Ⅰ, and Osterix (OSX) were detected by RT-PCR; the expression of collagen type Ⅰ was observed by immunohistochemical staining at 14 days of culture; and cell creep and adhesion were observed by scanning electron microscopy. BMP-2 gelatin/chitosan hydrogel sustained-release system had better drug encapsulation efficiency and drug loading, and could prolong the activity time of BMP-2. The secretion of ALP and the relative expression of Cbfa1, collagen type Ⅰ, and OSX genes in the experimental group were significantly higher than those in the control group at different time points in the in vitro co-culture system ( P<0.05). Immunohistochemical staining showed that the amount of fluorescence in the experimental group was significantly more than that in the control group, i.e. the expression level of collagen type Ⅰ was higher than that in the control group. The cells could be more evenly distributed on the materials, and the cell morphology was good. Scanning electron microscopy showed that the sustained-release system could adhere to cells well. iPS-MSCs have the ability of osteogenic differentiation, which is significantly enhanced by BMP-2 gelatin/chitosan hydrogel sustained-release system. The combination of iPS-MSCs and sustained-release system can adhere to the materials well, and the cell activity is better.

Experimental studies on the repair and restitution of cartilage by cartilage acellular extracellular matrix and adipose tissue-derived stem cells

Objective: To investigate the effects of the repair and restitution of ear-shaped cartilage by adipose tissue-derived stem cells(ADSC) and cartilage acellular extracellular matrix. Methods: ADSC were extracted by digesting with collagenase type II from the adipose tissue from 32 patients with adiposity whose fats were drawn, and were cultured and subcultured in vitro. The natural biological scaffolds were prepared by acellular method using porcine ear cartilage, and then the second generation ADSC(5.0×10(7)/ml) were inoculated on the preformed natural bio-scaffold scaffold by culturing in vitro for 3 days to form a cell scaffold complex. 32 New Zealand white rabbits were randomly divided into the experimental groups, the control group A, the control group B and the control group C. All New Zealand white rabbits were modeled by ear cartilage defects. The cell scaffolds composite was implanted into the experimental group of the ear cartilage defects of rabbits, the ADSC were implanted into the control group A, the cartilage acellular extracellular matrix scaffold was implanted into the control group B and the control group C was modeled only by ear cartilage defects. After 16 weeks, the animals were sacrificed and the repair effect was observed by gross appearance and histological examinations including HE, Toluidine blue staining, Safranin O and typeⅡ collagen staining. Its were quantitatively analyzed by positive staining results of type Ⅱ collagen. Ear cartilage tissue elasticity was detected. SPSS 17.0 software was used to analyze the data. Results: The cartilage defects in the experimental group were repaired well by general shape observation and those in the control group was filled in with granulation tissue. There were significant differences between the experimental group and the control group in the wet weight(P<0.05). HE staining showed that cartilage cavities formed in articular cartilage defects, and only the fibrous tissue was filled with the ear cartilage defect in the control groups. In the repair area, Toluidine blue staining, Safranin O and type Ⅱ collagen staining were positive in the experimental group, and negative in the control groups. There was no significant difference between the experimental group and the normal ear cartilage in the ear cartilage elastic constant detection(P>0.05). Conclusions: The mechanics and histology of rabbit ear neonatal cartilage constructed by ADSC combined with cartilage acellular matrix are close to normal ear cartilage. Cartilage acellular matrix material combined with adipose-derived stem cells has good repair and reconstruction ability for ear cartilage defects, which possesses potential clinical application value.

Single‐cell Raman microscopy of microengineered cell scaffolds

AbstractStudying cells in a three‐dimensional (3D) environment has great potential in understanding cell behaviours such as morphology, proliferation, differentiation, and migration. Microengineered 3D cell scaffolds with precise defined geometries have offered a new approach to study cell behaviour and its interactions with scaffolds. The use of Raman spectroscopy to characterise biomolecules is a rapidly expanding area and has been implemented in numerous fields including pharmacology, microbiology, toxicology, and single‐cell studies. However, one area where it remains unexploited despite the vast potential of the technique is in the investigation of 3D cell scaffolds. A combination of Raman microscopy and chemometric approaches have employed to investigate the structure and biochemistry of nanofabricated scaffolds and a cell–scaffold complex. The 3D Raman mapping combined with the use of nanofabricated 3D scaffolds offers a unique opportunity to assess the influence of scaffold architecture on cell body and cell nuclei morphology and biochemistry. For the first time, we have cultured a human epithelial colorectal adenocarcinoma cell line on OrmoComp scaffolds and determined the structure and biochemistry of nanofabricated scaffolds and a cell–scaffold complex with the use of Raman microscopy combined with appropriate data analysis protocols. The results demonstrate the potential of 3D Raman mapping for identifying biochemical and physical variation within single cells as they grow and adhere to 3D scaffolds.

Bone marrow stem cells combined with polycaprolactone-polylactic acid-polypropylene amine scaffolds for the treatment of acute liver failure

Physical elasticity is a major mechanical property of several tissue types, and enormous efforts have been invested into the development of elastomeric biomaterials that mimic properties of native tissue. In the present study, a polycaprolactone-polylactic acid scaffold was photografted with polyacrylamide, tumor necrosis factor-alpha, and insulin-like growth factor 1 to construct a novel scaffold, which was named the polycaprolactone-polylactic acid-polypropylene amine-insulin-like growth factor 1/tumor necrosis factor-α (PCL-PLA-PAAm-IGF-1/TNF-α) scaffold. Its inhibitory effect on cell senescence of bone marrow mesenchymal stem cells was studied. When the PCL-PLA-PAAm-IGF-1/TNF-α scaffolds were transplanted into a rat model of acute hepatic failure, we found that the scaffolds clearly contributed to liver repair. The simple method presented here may be useful for the preparation of cell-scaffold complexes for liver tissue engineering.

Mechanical Loading Improves Engineered Tendon Formation with Muscle-Derived Cells: An In Vivo Analysis.

The authors' previous study showed that muscle-derived cells could regenerate strong engineered tendon with better tissue structure. However, little was known about the mechanism of neotendon built by muscle-derived cells, and the development and maturation of the cells. The authors hypothesized that mechanical loading modulated this process. The aim of this study was to investigate whether mechanical loading could regulate muscle-derived cell-based engineered tendon formation and maturation. Muscle-derived cells were isolated, expanded, and seeded onto polyglycolic acid fibers that formed a cell-scaffold complex. After in vitro culture for 2 weeks, half of them were implanted without loading and the other half were sutured to mouse fascia that could provide a natural dynamic loading. At 12 and 24 weeks after implantation, histologic examinations, ultrastructure, and biomechanical characteristics were evaluated. Gross observation results showed that under mechanical loading, neotendon tissue could be generated with muscle-derived cells and the tissue structure became more mature with the increase of culture time. Well-organized aligned collagen fibers and elongated morphologic cells were observed on histologic examination under mechanical loading. In contrast, the nonload group failed to form neotendon, but formed disorganized fibrous tissue with significantly worse mechanical properties and poor collagen fibril structure. This study demonstrates that mechanical loading is indispensable in tendon tissue engineering with muscle-derived cells. Although muscle-derived cells have a potential advantage in neotendon regeneration, stress deprivation resulted in a distinctly inferior maturity level of engineered tendon.

Effects of Endothelial Progenitor Cells on Vascularization and Osteogenesis of Tissue-engineered Bones in Beagle Dogs

Objective The explore the effects of endothelial progenitor cells(EPCs)on vascularization and osteogenesis of tissue-engineered bones in Beagle dogs.Methods Bone marrow mesenchymal stem cells(BMSCs)and EPCs from bone marrow of Beagle dogs were isolated,cultured,and expanded and then seeded in β-tricalcium phosphate(TCP)scaffolds. The cell scaffold complexes(EPCs/ BMSCs/TCP group and BMSCs/TCP group)and the blank TCP scaffold were transplanted into the limb muscles of Beagle dogs,respectively. The relative CT values were calculated in the 3rd and 6th month through CT scan after operation. The vascularization and the osteogenesis were detected 6 months after operation by histological examination.Results Three months after the surgery,the relative CT value in the EPCs/BMSCs/TCP group was(366.67±19.51)HU,significantly higher than that[(163.00±30.81)HU] in the BMSCs/TCP group(t=2.10,P=0.0006);6 months after surgery,the relative CT value in the EPCs/BMSCs/TCP group was(553.34±26.86)HU,which was also significantly higher than that[(241.34±21.57)HU] in the BMSCs/TCP group(t=2.11,P=0.0006). The relative CT values at 6 months after operation were significantly higher than those at 3 months after operation in both BMSCs/TCP group(t=2.10,P=0.0255)and EPCs/BMSCs/TCP group(t=2.10,P=0.0006). Six months after the operation,HE staining showed no bone formation or calcium deposition in the blank TCP group,and osteoblasts,mature bone trabeculae,and calcium deposition were observed in EPCs/BMSCs/TCP group and BMSCs/TCP group. Immunohistochemical staining showed that the number of peripheral,central,and whole vessels in the EPCs/BMSCs/TCP group was 21.67±1.45,23.33±2.60,and 45.00±1.16,which were significantly higher than those in the BMSCs/TCP group[8.67±0.88(t=2.07,P=0.0016),9.33±0.67(t=2.07,P=0.0065),and 18.00±1.00(t=2.07,P=0.001),respectively]. The number of vessels was not significantly different between the BMSCs/TCP group and the blank TCP group(18.00±1.00 vs. 16.67±2.40;t=2.07,P=0.636). The osteogenic area of the EPCs/BMSCs/TCP group was(1 322 000±141 300)pixel,significantly higher than that of the BMSCs/TCP group[(874 900±49 430)pixel;t=2.10,P=0.04]. BMSCs/TCP group showed significantly higher bone area in the peripheral area than in the central region[(170 000±42 320)pixel vs.(613 900±90 290)pixel;t=2.10,P=0.02];in contrast,there was no statistically significant difference in bone area between the peripheral region and the central region in the EPCs/BMSCs/TCP group[(376 400±20 160)pixel vs.(310 400±6917)pixel;t=2.10,P=0.07].Conclusion EPCs as seed cells combined with BMSCs can promote vascularization and osteogenesis of tissue-engineered bone in Beagle dogs.

The impact of collagen sponge composite bone marrow mesenchymal stem cells (BMSCs) in inducing interbody fusion.

To assess the effectiveness of bone mesenchymal stem cells (BMSCs) in the induction of interbody fusion. The 3rd generation BMSCs were seeded on collagen sponge scaffold and cultured in osteoblast induction medium for 3 weeks to prepare cell-scaffold complex. Thirty patients were randomly divided into three groups to establish the L4/L5 interbody fusion model. The cell-scaffold complex was implanted in the intervertebral space in group I, the collagen sponge scaffold was implanted in group II, and the autologous iliac crest spongy bone was implanted in group III. Palpation, radiography, micro-CT, and histology were performed on the 12th weeks after operation to evaluate osteogenesis and spinal fusion. BMSCs differentiated into osteoblasts in the cell-scaffold complex after osteogenic induction for 3 weeks. The spinal fusion rates in group I, II, and III were 40%, 0%, and 70%, respectively. Micro-CT and histological examination showed mature bone marrow and trabecular bone formation in the fusion segments. The new bone was integrated with the upper and lower vertebral body. The bone trabecula in group III was stronger than group I. The surgical segments in group II was scar tissue without collagen sponge residue. BMSCs can induce osseous fusion in the lumbar vertebra.

Construction of tissue engineered cartilage based on acellular cartilage extracellular matrix oriented scaffold and chondrocytes

To observe the feasibility of acellular cartilage extracellular matrix (ACECM) oriented scaffold combined with chondrocytes to construct tissue engineered cartilage. Chondrocytes from the healthy articular cartilage tissue of pig were isolated, cultured, and passaged. The 3rd passage chondrocytes were labeled by PKH26. After MTT demonstrated that PKH26 had no influence on the biological activity of chondrocytes, labeled and unlabeled chondrocytes were seeded on ACECM oriented scaffold and cultivated. The adhesion, growth, and distribution were evaluated by gross observation, inverted microscope, and fluorescence microscope. Scanning electron microscope was used to observe the cellular morphology after cultivation for 3 days. Type Ⅱ collagen immunofluorescent staining was used to check the secretion of extracellular matrix. In addition, the complex of labeled chondrocytes and ACECM oriented scaffold (cell-scaffold complex) was transplanted into the subcutaneous tissue of nude mouse. After transplantation, general physical conditions of nude mouse were observed, and the growth of cell-scaffold complex was observed by molecular fluorescent living imaging system. After 4 weeks, the neotissue was harvested to analyze the properties of articular cartilage tissue by gross morphology and histological staining (Safranin O staining, toluidine blue staining, and typeⅡcollagen immunohistochemical staining). After chondrocytes that were mainly polygon and cobblestone like shape were seeded and cultured on ACECM oriented scaffold for 7 days, the neotissue was translucency and tenacious and cells grew along the oriented scaffold well by inverted microscope and fluorescence microscope. In the subcutaneous microenvironment, the cell-scaffold complex was cartilage-like tissue and abundant cartilage extracellular matrix (typeⅡcollagen) was observed by histological staining and typeⅡcollagen immunohistochemical staining. ACECM oriented scaffold is benefit to the cell adhesion, proliferation, and oriented growth and successfully constructes the tissue engineered cartilage in nude mouse model, which demonstrates that the ACECM oriented scaffold is promise to be applied in cartilage tissue engineering.