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Acellular porcine small intestinal submucosa (SIS) has been successfully used for reconstructing esophagus with half circumferential defects. However, repairing full circumferential esophageal defects with SIS has been restricted due to the latter's poor mechanical properties. In the present study, synthetic polyesters biomaterial poly(3-hydroxybutyrate-co-3-hydroxyhexanoate) (PHBHHx) and poly(lactide-co-glycolide) (PLGA) have been used to improve the mechanical properties of SIS. Feasibility of SIS/PHBHHx-PLGA composite material scaffold for esophageal tissue engineering has been assessed through a series of testing. The appropriate mixing ratio of PHBHHx and PLGA polymers has been determined as 5:5 by mechanical testing and in vitro degradation experiment. The morphology of constructed membranous and tubular scaffolds was also characterized. As confirmed by enzyme-linked immunosorbent assay, the contents of VEGF and TGF-β have respectively reached 657 ± 18 ng mL(-1) and 130 ± 4 pg mL(-1) within the SIS/PHBHHx-PLGA specimens. Biocompatibility of the SIS/PHBHHx-PLGA specimens with rat bone marrow mesenchymal stem cells (MSCs) was also evaluated by scanning electron microscopy and a live-dead cell viability assay. Actin filaments of MSCs on the composite materials were labeled. Biological safety of the extract from SIS/PHBHHx-PLGA specimens, measured as hemolysis rate, was all lower than 5%. Compared with SIS and SIS/PHBHHx-PLGA specimens, inflammatory reaction provoked by the PHBHHx-PLGA specimens in rats was however more severe. Our results have suggested that SIS/PHBHHx-PLGA composite material can offer a new approach for esophageal tissue engineering.

We hereby report on a case in which a huge chest wall defect generated by resection of a massive aggressive tumor (desmoplastic fibroma) was repaired with osteogenic-induced mesenchymal stem cells embedded in a bone-derived biomaterial. In this case, there were three challenges to overcome: reconstruction of the soft tissue, repair of the skeletal defect of the thoracic wall and repair of the defect in the pleural cavity. The defects of soft tissue and pleural cavity were reconstructed, respectively, with an ipsilateral abdominal flap and a diaphragm muscular flap. The huge defect in the chest wall was successfully repaired with the tissue-engineered ribs, which was confirmed by long-term follow-up with computerized tomography and histological and immunohistochemical evaluations. In view of its effectiveness and safety, tissue-engineered bones may have a broad application for the repair of large skeletal defects and bone regeneration.

Purpose: Acellular porcine small intestinal submucosa (SIS) has been successfully used for esophagoplasty in dogs. However, this has not led to complete epithelialization and muscular regeneration. We undertook the present study to assess the effect of tissue-engineered esophagus generated by seeding bone marrow mesenchymal stem cells (BMSCs) onto an SIS scaffold (BMSCs-SIS) in a canine model., Methods: We cultured, passaged, and measured autologous BMSCs and myoblasts with cell proliferation and immunohistochemical assays. We labeled the third passage of BMSCs with PKH-26, a fluorescent dye, before seeded it onto the SIS. We resected canine cervical esophagus to generate a defect 5 cm in length and 50% in circumference, which we repaired with BMSCs-SIS or SIS alone., Results: Four weeks later, barium esophagram demonstrated that esophageal lumen surface of the patch graft was smoother in the BMSCs-SIS group compared with the SIS group. Histological examination suggested a strong similarity between BMSCs and esophageal myoblasts in terms of morphology and function. Although both BMSCs-SIS and SIS repaired the esophageal defects, we noted complete re-epithelialization with almost no inflammation only in the former group. By 12 wk after the surgery, we observed long bundles of skeletal muscles only in the BMSCs-SIS group, where the microvessel density was also much greater., Conclusions: Bone marrow mesenchymal stem cells on an SIS scaffold can promote re-epithelialization, revascularization, and muscular regeneration. This approach may provide an attractive option for esophageal regeneration., (Copyright © 2013 Elsevier Inc. All rights reserved.)

Tissue-engineered bones (TEBs) constructed with bone-marrow-derived mesenchymal stem cells (BMSCs) seeded on biomaterial scaffolds have achieved good results for bone defect repair in both animal experiments and clinical trials. This has been limited, however, by the source and quantity of BMSCs. We here explored TEBs constructed by placenta-derived mesenchymal stem cells (PMSCs) and compared their effect for the repair of critical-sized segmental osteoperiosteal defects with TEBs constructed with BMSCs. PMSCs were isolated from rabbit placenta by gradient centrifugation and in vitro monolayer culturing, and BMSCs were isolated from the hindlimb bone marrow of newborn rabbit. Primary cultured PMSCs and BMSCs were uniformly in a spindle shape. Immunocytochemistry indicated that both types of cells are positive for CD44 and CD105, and negative for CD34 and CD40L, confirming that they are mesenchymal stem cells. BrdU-labeled PMSCs and BMSCs were respectively co-cultured with bio-derived bone materials to construct TEBs in vitro. Critical-sized segmental osteoperiosteal defects of radii were created in 24 rabbits by surgery. The defects were repaired with TEBs constructed with PMSCs and BMSCs. The results showed that TEBs constructed by both PMSCs and BMSCs could repair the osteoperiosteal defects in a 'multipoint' manner. Measurement of radiography, histology, immunohistochemistry, alkaline phosphatase activity, osteocalcin assaying and biomechanical properties have found no significant difference between the two groups at 2, 4, 8 and 12 weeks after the transplantation (P > 0.05). Taken together, our results indicate that PMSCs have similar biological characteristics and osteogenic capacity to BMSCs and can be used as a new source of seeding cells for TEBs., (© 2012 The Authors Journal compilation © 2012 FEBS.)

To develop a naturally derived tendon tissue engineering scaffold with the preservation of the native ultrastructure, tensile strength, and biochemical composition of the tendon extracellular matrix (ECM), decellularized tendon slices (DTSs) were prepared using repetitive freeze/thaw of the intact Achilles tendons, frozen section, and nuclease treatment. The DTSs were characterized in the native ultrastructure, mechanical properties, biochemical composition, and cytocompatibility. Histological examination and DNA quantification analysis confirmed that cells were completely removed from tendon tissue by repetitive freeze/thaw in combination with nuclease treatment 12 h. The intrinsic ultrastructure of tendon tissue was well preserved based on scanning electron microscopy examination. The tensile strength of the DTSs was retained 85.62% of native tendon slice. More than 93% of proteoglycans (fibromodulin, biglycan) and growth factors (TGF-β1, IGF-1, VEGF, and CTGF) inherent in tendon ECM were preserved in the DTSs according to ELISA analysis. Furthermore, the DTSs facilitated attachment and repopulation of NIH-3T3 fibroblasts in vitro. Overall, the DTSs are sheet scaffolds with a combination of elemental mechanical strength and tendon ECM bioactive factors that may have many potential applications in tendon tissue engineering., (Copyright © 2012 Wiley Periodicals, Inc.)

Porcine small intestinal submucosa (SIS) has been widely used in repairing various tissues and organs. Despite this, some SIS products have the capacity to cause variable inflammatory responses after implantation resulting in severe adverse effects due to porcine cell existence. In this study, we described a multi-step method including mechanical disassociation, degrease, enzyme digestion, detergent treatment, freeze-drying and sterilization by irradiation for preparation of SIS. The efficacy of acellularization was evaluated by histological observation and the content of porcine immunoreceptor DAP12 gene. The change of growth factors contents within SIS accompanying with decellularization was quantitatively assessed by ELISA. Inflammatory reaction of SIS implanted subcutaneously in a rat was investigated. The histological examination revealed no remaining cells after enzyme digestion. Moreover, qPCR analysis demonstrated that the content of a porcine immunoreceptor gene DAP12 DNA in final SIS product (SISv) was only 1.05% of that in SIS samples (SISi) prepared by mechanical disassociation. Degrease with methanol/chloroform dramatically reduced the contents of VEGF, b-FGF, TGF-β, and TNF-α within SISii, but further treatment could not significantly reduced the contents of growth factors. SIS implanted into rats showed that inflammatory cells was more accumulated surrounded to SISi at 1, and 2 weeks, but reduced in SISv samples. The degree of inflammatory reaction for SISv was significantly less than that of SISi., (Copyright © 2010 Elsevier Ltd. All rights reserved.)

Myocardial infarction (MI) remains a common and deadly disease. Using tissue-engineered cardiac grafts to repair infarcted myocrdium is considered to be a therapeutic approach. This study tested the feasibility of using MSCs-seeded SIS to repair chronic myocardial infarction in a rabbit model. MI in rabbits was created by ligation of the left anterior descending artery. BrdU-labeled mesenchymal stem cells (MSCs) were seeded on the small intestinal submucosa and cultured for 5-7 days prior to implantation. Four weeks after myocardial infarction, cardiac grafts were implanted onto the epicardial surface of infarcted myocardium. Four weeks after implantation of the membranes, a serial of tests including echocardiography, hemodynamics, histology and immunohistochemistry were undertaken to evaluate the effect of the implanted grafts on recovery of the infarcted myocardium. It was shown that left ventricular contractile function and dimension, the capillary density of the infarcted region, and myocardial pathological changes were significantly improved in rabbits implanted either SIS or MSCs-seeded SIS. But the MSCs-seeded SIS was more effective. Immunofluorescence staining demonstrated the migration of Brdu-labeled MSCs from the membrane into the infarcted area and their differentiation to cardiomyocytes and smooth muscle cells. Taken together, these results suggest that MSCs-seeded SIS can be used to repair chronic myocardial infarction, which enhances myocardial regeneration.

The acellular porcine small intestinal submucosa (SIS) has been successfully used for esophagoplasty. However, it does not lead to a complete epithelialization in a canine model. A cellular component may be required for better reconstruction. The present study was undertaken to investigate the feasibility and effectiveness of the combination of SIS and autologous oral mucosal epithelial cells (OMECs) for esophageal reconstruction. The OMECs harvested from beagle dogs were cultured and propagated, and the 3rd passage cells were seeded on a single-layer SIS. Male beagle dogs were subjected to surgical resection to produce cervical esophageal defects (5 cm in length, 180 degrees in range). SIS with or without OMECs was patched on the esophageal defects. Barium esophagram, immunohistochemistry, and histology were performed to evaluate the therapeutic effectiveness. Four weeks after surgery, the histological examination showed a complete re-epithelialization and almost no inflammation in the SIS with OMECs group. But in the SIS group, only a partial epithelialization was observed along with inflammation. Eight weeks after surgery, the squamous epithelium was found to cover the entire graft surface in both groups; however, the muscular regeneration was observed in the SIS with OMECs, but not in the SIS group. The graft of SIS combined with autologous OMECs promotes re-epithelialization and muscular regeneration. It is an effective alternative method for esophageal repair.

Objective: To develop a method for reconstructing urothelial structures using tissue engineering techniques., Methods: The bladder mucosa of a puppy dog was mechanically separated, cutted into pieces, and digested with trypsin. The obtained bladder transitional epithelia were cultured with DKSFM and then transplanted to the surface of the small intestinal submucosa (SIS) framework. The compound of the epithelia and small intestinal submucosa (SIS) materials were planted under the back skin of the nude rats. The rats were sacrificed at week 1, 2, 4 and 8 after the transplant. Histological and immunohistochemical examinations were performed to the implanted samples., Results: The bladder transitional epithelia adhered to, and grew and proliferated on the surface of the SIS framework. The bladder transitional epithelia covered the entire surface of the framework after nine days of culture in vitro, showing a single-layer cellular structure. The bladder transitional epithelia planted on the SIS framework formed a multi-layer structure after four weeks or eight weeks of transplant to the nude rats. The brown cellular plasma staining was observed, indicating a positive reaction to the immunohistochemical staining with cytokeratin., Conclusion: Urothelial structures can be reconstructed in vitro and in vivo with tissue engineering technologies, which lays a technical foundation for further urinary tract reconstructing experiments.

Objective: To investigate the feasibility of acellular porcine small intestinal submucosa (SIS as bioscaffold of tissue engineering skin., Methods: The second passage keratinocytes were seeded on SIS, after seeded for 11, 13, 15, 17 days, the keratinocytes/SIS composites were observed by dye directly, histopathology, immunohistochemical studies with monoclonal antibodies against laminin and transmission electron microscope (TEM)., Results: At eleventh day, keratinocytes were growth very well on the surface of SIS, there are 2-3 cell layers on partial of the SIS surface, the continued expression of laminin can be detected between the keratinocytes and the surface of SIS. After 13, 15, 17 days this stratified structure increased, cells contact more closely, the tonofibrils in cells, desmosome between cells and the basal membrane between the keratinocytes and the surface of SIS can be seen with TEM., Conclusions: SIS is a kind of good bioscaffold in the culture of porcine keratinocytes in vitro.

Objective: To study the effect of bio-derived bones, as substitutes of autogenous bone grafts and demineralized cadaver bones, on the attachment, spreading and proliferation of isolated osteoblasts., Methods: Osteoblasts were isolated from the calvaria of a fetal rabbit through sequential collagenase digestion. In the attachment study, the osteoblasts labeled with 3H-leucine were incubated with the bio-derived bone materials in sterile microcentrifugable tubes for 15, 90 and 180 minutes, and 24 hours, respectively. The attached cells were collected and the radioactivity was measured with liquid scintillation spectrometry. In the proliferation study, the osteoblasts were cultured with the bio-derived bone materials for 24 hours and 3H-thymidine was added during the last 2 hours of the incubation. The attached cells were collected and the radioactivity was measured with liquid scintillation spectrometry. Osteoblasts were seeded on the bone graft materials for 60 or 120 minutes, 24 or 48 hours, and 3 or 7 days, then the co-culture was processed for scanning electron microscopy to observe the interaction of osteoblasts and the bio-derived bone materials., Results: Osteoblasts attached to the bio-derived bone materials in a time-dependent manner. There were significantly (P<0.05) more attached cells after 180 minutes than after 15 and 90 minutes of incubations (P<0.05). Osteoblasts were proliferated in a large amount on the surface and in the materials. Osteoblasts seeded onto 100 mg bio-derived bones resulted in significantly (P<0.05) more measurable proliferation than those seeded onto 10 mg bones. Osteoblasts appeared round as they attached to the materials, then flattened and spread over with time passing., Conclusions: Bio-derived bones can provide a good environment for the attachment and proliferation of osteoblasts.

Objective: To prepare human acellular amniotic membrane (HAAM) and to measure its cytocompatibility and biocompatibility., Methods: HAAM were prepared by chemical detergent-enzymatic extraction. Fresh human amnion was crosslinked with glutaradehyde, shaken in 0.5% SDS for 24 hours, and then treated with 0.25% trypsin for 4 hours. The production were freeze-drying and sterilized using ethylene oxide. Human fibroblasts were isolated from embryo and expanded in vitro. The fibroblasts were seeded in HAAM. HAAM and specimen were stained with HE and Mallory, and observed grossly, under light microscopy and scanning electron microscopy. The HAAM were implanted in the back of SD rats., Results: There were no residues of cells in the HAAM (HE, Mallory staining). One side of HAAM had reticular and porous structure, the other side had compact fibrous structure. Pore size was from 10 to 80 nm. The HAAM could be seeded with expanded fibroblasts in vitro, and fibroblasts had the potential of spread and proliferation. The SD rat in the implant test had no death, convulsions and other abnormal response., Conclusion: The detergent-enzymatic extraction process can remove cells and solvable components effectively and preserve the tissue matrix well and keep the reticular structure. The HAAM can be used as an ideal scaffold of biological membrane for tissue engineering.

Objective: To investigate the ability to repair goat tibia defect with marrow stromal stem cells (MSCs) and bio-derived bone, and the feasibility of the compounds as bone substitute material., Methods: MSCs were cultured with the bio-derived bone in vitro, and the 20 mm tibia defect of goat was made and fixed with plate. Eighteen goats were divided into experimental group, control group and blank group. The defects were not filled with anything in blank group, with tissue engineering bone in experimental group and bio-derived bone in control group. The repair capability was assessed by physical, X-ray and bone mineral density examinations 8, 12, 16, and 24 weeks after operation., Results: In experimental group, the defects were partially repaired 8 weeks, and completely repaired 12 and 16 weeks; there was significant difference in bone density between experimental group and control group (P < 0.05) 8, 12 and 16 weeks, but no significant difference 24 weeks. The defects of blank group were not repaired 24 weeks., Conclusion: The tissue engineering bone can efficiently repair bone defect, and its repair capability is better than that of bio-derived bone alone both in quantity and quality of bone formation.

Objective: To review the research advance of the preparation and characteristics of small intestinal submucosa(SIS)., Methods: Recent original articles related to such aspects of small intestinal submucosa were reviewed extensively., Results: Small intestinal submucosa was an easily obtained biomaterial. SIS was a bio-absorbable and degradable material. SIS had tissue specific regeneration properties., Conclusion: SIS is a suitable bio-derived material for tissue engineering of blood vessel, muscle tendon, urinary bladder and abdomen.

Objective: To construct tissue engineering bone with bio-derived materials and bone marrow stromal cells (MSCs), and to investigate the effect of allogeneic engineering bone implants on healing of segmental bone defects., Methods: MSCs being aspirated aseptically from tibial tuberosities of young rhesus monkeys were induced into osteoblasts in vitro and then were cultured and marked with 5-bromo-2-deoxyuridine (BrdU). Tissue engineering bones were constructed with these labeled osteoblasts being seeded onto bio-derived materials made from fresh human bones which were treated physically and chemically, Then the constructs were implanted in 15 allogeneic monkeys to bridge 2.5 cm segmental bone defects of left radius as experimental groups, bio-derived materials only were implanted to bridge same size defects of right radius as control group. and, 2.5 cm segmental bone defects of both sides of radius were left empty in two rhesus monkeys as blank group. Every 3 monkeys were sacrificed in the 1st, 2nd, 3rd, 6th and 12th weeks postoperatively and both sides of the implants samples were examined macroscopically, histologicaly, and immunohistochemicaly. The two monkeys in blank group were sacrificed in the 12th week postoperatively., Results: Apparent inflammatory reactions were seen around both sides of the implants samples in the 1st, 2nd, 3rd weeks, but it weakened in the 6th week and disappeared at the 12th week. The labeled osteoblasts existed at the 6th week but disappeared at the 12th week. The bone defects in experimental group were repaired and the new bone formed in multipoint way, and osteoid tissue, cartilage, woven bone and lamellar bone occurred earlier when compared with control group in which the bone defects were repaired in 'creep substitution' way. The bone defects in blank group remained same size at the 12th week., Conclusions: Engineering bones constructed with bio-derived materials and MSCs were capable of repairing segmental bone defects in allogeneic monkeys beyond 'creep substitution' way and making it healed earlier. Bio-derived materials being constituted with allogeneic MSCs may be a good option in construction of bone tissue engineering.

Objective: To study the adhesion, migration and three-dimentional growth of Schwann cells on PLA (polylactic acid) nonspinning fibre cloth and polyglycolic/polylactic acid (PLGA) fibres., Methods: Schwann cells/ECM gel solution and PLA nonspinning fibre cloth and PLGA fibres pretreated by collagen, polylysine and ECM were co-cultured. Then the migration and three-dimensional growth of Schwann cells on the fibres were observed under phase contrast microscope and laser scanning confocal microscope., Results: Schwann cell/ECM solution was compounded with PLA nonspinning fibre cloth. With formation of gel, most Schwann cells resided in the fibre net holes, and adhered to the fibres to form a multiplayer-arranged Schwann cell column like Büngner band. Schwann cells could adhere to PLGA fibres and grew and migrated along the fibres. ECM gel could significantly increase the adhering and migrating cell number., Conclusions: ECM gel can facilitate the adhesion, growth and migration of Schwann cells on the seteroframe. It is a good integrating material for tissue engineering bioartificial nerve.

Objective: To investigate the effects of three-dimentional culture in bioderived material modified by Pluronic F-127 on the growth and function of rabbit periosteal osteoblast in vitro., Methods: Bio-derived materials were from fresh pig ribs and were modified by Pluronic F-127. Then rabbit periosteal osteoblasts were cultured in bio-derived materials(group A), in the modified bio-derived materials(group B) and on the plastic surfaces as a control (group C), respectively. During a 7-day period, the status of growth, cell viability and alkaline phosphatase(ALP) activity were measured., Results: Osteoblasts attached, elongated and grew well on the modified bio-derived materials. There were no significant difference in osteogenesis and ALP activity between group A and group B(P > 0.05). The osteogenesis and ALP activity in groups A, B were less than those in group C (P < 0.01)., Conclusion: Pluronic F-127 can be used for a carrier for bioactive factors to modify bio-derived material.

Objective: To investigate a cryophylactic agent (CPA) to protect tissue engineered tendon (TET) in deep low temperature., Methods: Sixty-four BALB/C inbred nude mice were chosen, which included 4 as blank control group, left sides of 60 as experimental group and their right sides as control group. Transformed human embryonic tendon cells of the 54th passage and artificial materials of carbon fiber (CF) and polyglycolic acid (PGA) were co-cultured in vitro to construct TET. TET was frozen in liquid nitrogen with four kinds of CPA (groups A, B, C, and D) for 2 months. They were thawed quickly and transplanted into hind limbs of nude mice to repair the defects of Achilles tendon, which was 5 mm in length and 65.7% of total Achilles tendon. In control group, no cryopreservation treatment was taken. The morphological, histological, ultrastructure, and immunohistochemistry examinations were made and short tandem repeat loci were detected 2, 4, 6, 8, and 12 weeks later., Results: In the experimental group, the morphological properties of tendon cells resumed gradually and the capability of synthesizing collagen enhanced by degrees. Tendon cells survived and could secret type I collagen and there was less difference between experimental and control groups 12 weeks after transplantation. In group A, vacuole in mitochondrion of tendon cell decreased, tendon cell arranged in order and abundant collagen fibers were found and linked., Conclusion: The cryopreservation agent in group A can protect TET in deep low temperature.

Osteoblasts isolated from the cranium of Zelanian rabbit, after being transfected with green fluorescent protein, were inoculated onto the bio-derived bone to investigate the 3D fabrication of engineering bone in rotating wall vessel bioreactor (RWVB). As a control, the cell-scaffold constructs were cultured in T-flasks simultaneously. During one week's culture, the samples were collected every 12 h. The histological and immunohistochemical assays were operated respectively. Furthermore, the cells' metabolism of nutrients was monitored and analyzed during the whole culture process. The results show that the cell number fabricated in RWVB was five times of that in T-flasks. Bone tissue fabricated in RWVB with two different densities grew well, and osteoblasts maintained normal chromosome. It is demonstrated that with the stress stimulation inside the fluid in RWVB, the active expression of ALP can be increased, a rapid proliferation and differentiation of osteoblasts are possible and the 3D fabrication of engineering bone could be realized. [ABSTRACT FROM AUTHOR]

Abstract: As mesenchymal stem cells (MSCs) are capable of self-renewal and multilineage differentiation, the feasibility and efficacy of co-culturing human MSCs (hMSCs) with rabbit articular chondrocytes (rACs) to promote chondrogenic and osteogenic differentiation of hMSCs for clinical osteoarthritic therapy were investigated in the present study. The two distinct cell types were encapsulated in alginate hydrogels singly or in one of three ratios (2:1, 1:1, 1:2 of hMSCs to rACs) and cultured under chondrogenic conditions for 28 days. The results demonstrated that newly synthesized cartilaginous extracellular matrix (ECM) and type II collagen (col-2) gene signal were upregulated with greater hMSC ratios and longer culture periods. However, a specific col-2 gene probe for human was found only in single hMSC group but absent in all co-culture groups, which indicate that the enhanced cartilaginous phenotype originated from the co-cultured rACs. Osseous phenotype was histologically detected only in the 2:1 group on day 28; and xenogenic osteocalcin assay showed that it originated from hMSCs. This suggests that variations in the ratios of co-cultured hMSC and rAC regulated the cartilaginous and osseous phenotype as well as the differentiation of hMSCs in alginate constructs. The study provides new insights into the role of cell–cell interactions in regulating both cell differentiation and cell function and highlights the importance of developing appropriate differentiation protocols for tissue engineering therapies. [Copyright &y& Elsevier]