Research progress of cell sheet technology and its applications in tissue engineering and regenerative medicine / 生物医学工程学杂志

Cell sheet engineering is an important technology to harvest the cultured cells in the form of confluent monolayers using a continuous culture method and a physical approach. Avoiding the use of enzymes, expended cells can be harvested together with endogenous extracellular matrix, cell-matrix contacts, and cell-cell contacts. With high efficiency of cell loading ability and without using exogenous scaffolds, cell sheet engineering has several advantages over traditional tissue engineering methods. In this article, we give an overview on cell sheet technology about its applications in the filed of tissue regeneration, including the construction of soft tissues (corneal, mucous membrane, myocardium, blood vessel, pancreas islet, liver, bladder and skin) and hard tissues (bone, cartilage and tooth root). This techonoly is promising to provide a novel strategy for the development of tissue engineering and regenerative medicine. And further works should be carried out on the operability of this technology and its feasibility to construct thick tissues.

Role of activating transcription factor 2 in the growth of mandible condyle chondrocytes / 实用口腔医学杂志

Objective: To study the role of activating transcription factor 2 (ATF-2) in the growth of mandibular condyle cartilage. Methods: Primary chondrocytes of condyle were cultured. Expression plasmid of ATF-2 and plasmid bcl-2 promoter were transfected into chondrocytes. Luciferase assay and Western blot were used. Results: The absence of ATF-2 in mandibular condyle chondrocytes resulted in a decline in bcl-2 promoter activity, reduction in bcl-2 protein level. Conclusion: The results strongly imply that ATF-2 is required for adequate bcl-2 expression, and play a significant role in controlling growth plate chondrocyte progression.

The preparation of a new hydroxyapatite and the study on its cytocompatibility / 生物医学工程学杂志

The cuttlebones, harvested from cuttles, undergo the chemical reaction in high temperature and high pressure for a certain time. The products are qualitatively analysed, and spacial structure observation and cytocompatibility are tested. The results show that the chemical component of the cuttlebone is CaCO3 and the crystal type is aragonite. Cuttlebones undergo a hydro-thermal reaction, and thus transform into hydroxyapatite-that is, the cuttlebone-transformed hydroxyapatite(CBHA). The CBHA materials have the interconnected microporous network structures. Under the high magnification, CBHAs appear to have many micro-spheres, thus construct a new self-assembled nano-material system. The marrow stromal osteoblasts can adhere to and proliferate well on the surface of the CBHAs. These results show that CBHAs have good biocompatibility. Therefore, it can be a potential candidate scaffold for bone tissue engineering.

Experimental study of artificial bone composite of bicoral, rhBMP-2 and PLA in repairing calvarial defects / 华西口腔医学杂志

<p><b>OBJECTIVE</b>To study the effects of artificial bone composite of bicoral, rhBMP-2 and PLA in repairing calvarial critical-size defects.</p><p><b>METHODS</b>Calvarial defects in 24 rabbits were surgically made and then half of the defects were repaired with the artificial composite bone. Another half of them were repaired with bicoral/PLA composite and served as controls. Four rabbits in each group were sacrificed at 4, 8, 12 weeks after operation, respectively. The treatment effects were evaluated with scanning electron microscopy and mechanical strength testing.</p><p><b>RESULTS</b>New bone was observed not only in the periphery, but also inside the artificial bone in both groups, but earlier and more new bone formation was observed in treatment group compared with control group. The mechanical strength test showed that the artificial bone in two groups, which had same mechanical strength before implantation, had significant different mechanical strength after operation. The strength of the artificial composite bone was higher than that of controls and was same with normal rabbit calvarial bone.</p><p><b>CONCLUSION</b>The artificial composite bone possess a highly repairing ability, and the healing in bone defects may be accomplished by both osteoinductive and osteoconductive mechanism. The material may be used as a good substitute for bone grafting.</p>

Experimental study on the chondrogenesis potentiality of marrow stromal cell under the induction of transforming growth factor-beta / 华西口腔医学杂志

<p><b>OBJECTIVE</b>Seed cell study is an essential area in the research of tissue engineering. To evaluate the potentiality of marrow stromal cell(MSCs) as seed cell in the regeneration of tissue engineered cartilage, formation of cartilage nodules by culture expanded MSCs pellets under the induction of TGF-beta was investigated.</p><p><b>METHODS</b>MSCs were cultured and expanded in vitro. Cell pellets containing 1 x 10(6) MSCs were obtained by centrifuging MSCs solution at 1,000 r/min in 5 ml centrifugation tube. Pellets were exposed to cell culture media containing 20 ng/ml TGF-beta for 7 days and then cultured for another 7 and 21 days. The nodules were moved out of the tube and cartilage formation was observed by stereomicroscope, light microscope and electronic microscope.</p><p><b>RESULTS</b>10 days after exposure to TGF-beta, pellets contracted and formed small and round nodules on the bottom of the tubes. The nodules grew bigger slowly and reached maximal diameter of 1.8 mm in 28 days. The surface of the nodules was smooth and bright white. Histological examination showed that extra cellular matrix formed in 14 days and in some areas cells situated in lacuna. In 28 days' specimens, a lot of cells situated in lacuna could be observed and the histological appearance looked much similar to cartilage. Electronic microscope observation demonstrated that in 28 days' specimens a large amount of collagen fiber existed.</p><p><b>CONCLUSION</b>Under the induction of TGF-beta, MSCs could differentiate into chondrogenesis cell and form cartilaginous nodules in vitro. This indicated that MSCs could be the potential seed cells in the regeneration of cartilage employing method of tissue engineering.</p>

Experimental study on subcutaneous bone formation by marrow stromal osteoblast-cancellous bone matrix compound artificial bone / 中华口腔医学杂志

<p><b>OBJECTIVE</b>To investigate the feasibility of using marrow stromal osteoblast (MSO) as bone derived cell and using cancellous bone matrix (CBM) as scaffold for bone tissue engineering, the subcutaneous osteogenesis of MSO-CBM compound artificial bone (MCCAB) was observed in the experiment.</p><p><b>METHODS</b>The marrow stromal cells of adult New Zealand rabbits cultivated and induced in vitro were used to form MCCAB by mixing, seeding and solidifying methods assisted by alginate. The MCCABs were auto-transplanted subcutaneously into the rabbits for 4 to 8 weeks. The alginate-cancellous bone matrix composites or the cancellous bone matrix alone were implanted as control. The effectiveness of bone formation was assessed by means of roentgenography, histology and computerized histomorphometry.</p><p><b>RESULTS</b>The osteogenesis of MCCABs was better than that of the alginate-cancellous bone matrix composites and of the cancellous bone matrixes. In the MCCABs, both intramembranous and cartilaginous osteogeneses were seen but the former was obvious. In the control, only slight cartilaginous osteogeneses were seen.</p><p><b>CONCLUSIONS</b>The osteogeneses of the MCCABs constructed by using tissue engineering method were obvious when transplanted subcutaneously. The MSO and CBM can be used as good bone-derived cell and scaffold material respectively for tissue-engineered bone construction.</p>

Experimental studies on cranial defect repair with marrow stromal osteoblast-cancellous bone matrix compound artificial bone / 中华整形外科杂志

<p><b>OBJECTIVE</b>To investigate the feasibility of using marrow stromal osteoblast-cancellous bone matrix compound artificial bone (MCCAB) as tissue-engineered bone, the osteogenesis of MCCAB in the cranial defect was observed in the experiment.</p><p><b>METHODS</b>The in vitro cultivated and induced marrow stromal cells of adult New Zealand rabbits were seeded into the alginate-cancellous bone matrix to form MCCAB. The MCCAB was then implanted into the cranial defect for 4 to 8 weeks. The cancellous bone matrix (CBM) alone or the marrow stromal osteoblasts (MSOs) alone was implanted as the control. The effectiveness of bone formation was assessed by histological and roentgenographic analysis.</p><p><b>RESULTS</b>The osteogenesis of MCCAB was better than CBM or MSOs and superior to the blank group.</p><p><b>CONCLUSION</b>MCCAB can effectively repair cranial defect. It could be used clinically to restore large bone defects.</p>

Fabrication of tissue engineered bone by seeding osteoblasts into titanium coral scaffolds / 实用口腔医学杂志

砄bjective:To fabricate bone tissue that has similar structural and mechanical characters with normal bone.Methods: Titanium meshes were molded into the shape of column in the length of 12 mm and in the diameter of 8 mm. The column was filled with natural coral granduls.4?10 7 marrow derived osteoblasts in 200 ?l cell culture medium were seeded into each of five scaffolds and incubated in vitro for 2 d to ensure that cells adhere well on the scaffolds. Then the scaffolds were implanted subcutaneously into the back of nude mice. Two months after implantation, the animals were sacrificed and the implanted materials were investigated by gross specimen inspection, X ray examination and histological observation. Results:2 months after in vivo incubation, the newly formed tissue was red and had the gross appearance of bone, and kept the original shape of column. Titanium mesh situated in the surface area. X ray examination showed that large amount of new bone formed in the scaffolds, there was no space between new bone and titanium mesh. Most of coral granduls had been absorbed. Histological observation demonstrated that in the surface area, new bone integrated well with titanium mesh and was enforced by titanium mesh(like cortical bone), and in the middle area large amount of lamellar bone formed.Conclusion: Newly formed bone in this experiment has similar structural with normal cortical bone.

Absorbability and bone-conductivity of nano-hydroxyapatite/collagen composite implanted under the calvarial periosteum / 实用口腔医学杂志

Objective:To study the bone-conductivity and absorbability of nano-hydroxyapatite/collagen(nHAC) composite implanted under the calvarial periosteum in rabbits.Methods:24 nHAC samples and 24 HA samples were prepaired in the shape of round disk with the diameter of 8 mm and thickness of 3 mm.nHAC samples were implanted under calvarial periosteum on the left side and HA samples on the right of 24 rabbits.The bone-conductivity and absorbability of the samples were examined by new bone height measuring and residual implant materia measuring 2,4,8 and 12 weeks after operation. Results:2,4,8 and 12 weeks after implantation new bone height(mm) in nHAC group was 0.54?0.09,0.72?0.12,1.83?0.14 and 2.63?0.07,that in HA group 0.13?0.11,0.31?0.12,1.23?0.05 and 1.75?0.14,respectively(P

Culture of rat Schwann cells derived from degenerated peripheral nerve on ZQ membrane / 实用口腔医学杂志

?Objective: To study the methodology of the culture of Schwann cells derived from degenerated peripheral nerve. Method: Sciatic nerve of adult rats was surgically cut. 14 days after operation, the degenerated nerve tissue was obtained and treated with trypsin and collagenase typeⅡ to prepare single cell suspension,the cells were purified by different speed of attachment and digestion, and incubated on ZQ membrane in the presence of 10 -5 mol/L cytosine arabinoside. The growth of the cells of passage 2 was studied by MTT assay. Schwann cells were identified with anti S100 immumohistochemistry. Results: The cultured cells were spindly in shape and 95% of them were S100 positive. The population doubling time of passage 2 cells was 72 h.The cells attached and stretched on ZQ membrane as well as on the culture vessel surface. Conclusion: Schwann cells can be cultured and purified by predegeneration of the peripheral nerve,different speed of attachment and digestion and the presence of cytosine arabinoside. The cells can grow well on ZQ membrane.

Tissue engineering of bone and cartilage composite / 实用口腔医学杂志

砄bjective: To fabricate tissue engineered bone cartilage composite. Method: Rabbit marrow stem cells (MSCs) were in vitro cultured, expanded and induced to differeciate to osteoblasts. Chondrocytes were obtained by collagenase type Ⅱ digestion of rabbit ear cartilage. Osteoblasts and chondrocytes were co seeded into different part of natural coral scaffold, and then implanted subcutaneously into the back of nude mice. Two months after implantation,the specimens were harvested and bone cartilage composites formation was observed by gross inspection and histologic observation. Results: The newly formed tissue was composed of two parts. One part was glisteringly white and another part was dark red. There was an obvious boundary between the two parts. Microscopic observation revealed successful restoration of bone cartilage composite. Conclusion:Bone cartilage composite can be prepared by co deeding of osteoblasts and chondrocytes into natural coral scaffold.

Fabrication of trachea cartilage ring by tissue engineering / 实用口腔医学杂志

砄bjective: To study the feasibility of fabrication of trachea cartilage ring by tissue engineering.Methods : PGA non woven mesh was put into the mold of trachea cartilage ring and enforced with polylactic acid. Rabbit chondrocytes were harvested by collagenase type Ⅱdigestion of ear cartilage and seeded into PGA scaffold in the density of 5?10 7/ml.The cell polymer complexes were incubated in vitro for 1 week and then implanted subcutaniously into the back of nude mice. The formation of trachea cartilage ring was observed by gross inspection and histological examination 2 months after implantation. Results: New cartilage tissue in the shape of trachea ring was found 2 months after implantation. The specimens showed the appearance of glisteringly white with good flexibility. Histological examination demonstrated that newly restored tissue was constituted of cartilage. Conclusion: It may be an efficient method to fabricate trachea cartilage ring by seeding chondrocytes in PGA scaffold.

Injectable tissue-engineered bone using calcium alginate as carriers / 实用口腔医学杂志

objective: To develop injectable tissue engineered bone through injection of osteoblasts/alginate composite in rabbits. Methods: Bone marrow cells isolated from iliac bone of New Zealand rabbits were cultured and induced to differentiate into osteoblasts.The osteoblasts were mixed with 25 g/L sodium alginate solution to generate osteoblasts/alginate composite with final cellular density of 5?10 6/ml. 0.17 g of sterilized CaSO 4 powder was then added to 2 ml osteoblasts/alginate. The mixture was injected into the dorsal subcutaneous tissue at left side of 6 New Zealand rabbits. The alginate composite without osteoblasts was injected into the right side as the control. 4 and 8 weeks after implantation, the bone formation was evaluated by means of gross, X ray and histological observation. Results: 4 weeks after implantation, cartilage formation was observed and 8 weeks after implantation,new mature bone was observed in the osteoblasts/alginate composites. No new bone formation was observed in all of the control specimens. Conclusion: Calcium alginate can be used as a carrier in injectable bone tissue engineering, and new bone can be created through injection of osteoblasts/alginate composites in immune animals.

Construction of mouse MyoD vector and its expression in Escherichia coli / 实用口腔医学杂志

Objective:To identify the cDNA gene of mouse MyoD by restriction enzyme analysis, and to express the gene in Escherichia coli (E coli) using a protein expression vector.Methods: After the cDNA gene of mouse MyoD had been amplified and identified,it was inserted into expression vector pBV220 in which exogenous gene was controlled by R RP L promoters.The recombinant plasmid pBV-my was transformed into E coli DH5? and the bacteria were induced at 42 ℃ to express encoded protein.Results:The cDNA of mouse MyoD was sequenced correctly.When the engineered bacteria had been induced an anticipated 55 ku protein band from the bacteria was observed on SDS-PAGE gel and amounted to 30% of total bacterial protein. Conclusion:The cDNA of mouse MyoD has been successfully coloned and efficiently expressed in E coli.

Bone formation induced by purified BMP and by recombinant human BMP-2 / 实用口腔医学杂志

Objective: To study the biodegradable of coral PLA composite artifical bone combined with bBMP or rhBMP as a new kind of bone substitute material. Methods: The composites were implanted into the muscle pouches of mice after combined with rhBMP-2 or bBMP respectively. Ectopic osteoinductive activity of rhBMP-2 or bBMP was examined and compared by histology and histo-morphometry.Results: rhBMP-2 and bBMP had different osteoinductivety. rhBMP-2 appeared to induce less bone and more angioid tissue and marrow. While bBMP seemed to have opposite effects. Conclusion: bBMP is more osteoinductive than rhBMP-2.

Repair of bone defect with chitin/rhBMP2/collagen complex / 实用口腔医学杂志

Objective: To investigate the feasibility of chitin as bone substitute material and carrier of rhBMP2.Methods: Porous chitin and chitin/rhBMP2/collagen complex were implanted into calvarial defects in 8 rabbits. Bone repairing ability was assessed by radiographic and histological observation. 2 rabbits without implantation were served as controls. Results: Chitin had certain bone conductive ability. When combined with rhBMP2,a complex possessing both bone conductive activity and bone inductive activity was produced. The complex had greater bone repairing ability than chitin alone. Conclusion: Chitin may be used as a bone substitute material and carrier of BMP. But its mechanical strength and surface activity should be improved.

Preparation of tissue engineered bone graft loading titanium dental implant / 实用口腔医学杂志

Objective: To prepare tissue engieered bone graft loading titanium dental implant. Methods: Titanium dental implant (3 mm in diameter) was inserted into porous natural coral column((5 mm in diameter). Bone marrow derived osteoblasts were cultured and expanded in vitro. Cells were induced by recombinant human bone morphogenetic protein-2 for three days and then harvested and seeded into porous coral and onto dental implant at the density of 2 ?108/ml. Four cell-coral-implant complexs were incubated in vitro for 2 days and then implanted subcutaniously into nude mice. New bone formationre and new bone integration with dental implant were evaluated by gross inspection, X-ray examination and hitologic observation 1 and 2 months after implantation. Results: By gross observation, specimen of 1 month was red and white. X-ray examination showed that there was little radiodense shadow around the dental implant. Specimen of 2 months was red and had the gross appearance of bone. Dental implant could be observed situating in the newly formed bone graft. X-ray examination showed that coral scaffold was absorbed completely. Large amount of X -ray blocking shadow could be observed around the dental implant. Histologic examination showed that bone-like tissue formed in the pores and on the surface of natural coral and in some area new bone could be observed integrating with implant in 1 month specimen. In 2 months specimen, large amount of new bone formed around the implant and integrated well with the implant. Conclusions: Tissue engineered bone graft may integrate well with titatium dental implant.

Tissue engeneered bone in the shape of human TMJ condyle with marrow stem cells and porous natural coral / 实用口腔医学杂志

Objective: To prepare tissue engeneered bone in the shape of human TMJ condyle. Methods: Rabbit marrow stem cells (MSCs) were in vitro cultured and induced by rhBMP2.107 Cells were seeded into each piece of natural porous coral (NC) in the shape and in the size of 4 -year-old-child mandibular condyle. After two days in vitro incubation, six cell-coral complexes were implanted subcautanrously into the back of nude mice. Two months after operation, bone formation was observed by gross inspection,X-ray examination,scanning electronic microscope observation and histological observation. Results: New bone grafts in the shape of human mandibular condyle were successfully restored two months after implantation in all the samples. X-ray examination showed large amount of X-ray blocking shadow. NC was partially absorbed. New bone formation could be observed by electronic microscope observation and hostological observation on the surface and in the pores of NC. Conclusion: It is an effective method to fabricate bone graft in specific shape by seeding osteogenesis cells into natural coral in the wanted shape.

STUDY ON BONE TISSUE ENGINEERING / 解放军医学杂志

To investigate the feasibility of using coral and other materials as scaffolds for bone tissue engineering, coral, coral hydroxyapatite(CHA), cancellous bone matrix and other natural biomaterials served as culture scaffolds of osteoblasts were manufactured. The results showed, in addition to PLA, PGA, PLGA and other synthetic polymers, some natural biomaterials are also ideal scaffolds materials for bone tissue engineering.

INTRAMUSCULAR BONE FORMATION BY IMPLANTATION OF COMPOSITE MARROW STROMAL OSTEOBLAST-CANCELLOUS BONE MATRIX ARTIFICIAL BONE / 解放军医学杂志

The marrow stromal cells of adult New Zealand rabbits cultivated and induced in vitro were used to form MCCAB by mixing,seeding and solidifying methods with the aid of alginate. The MCCABs were auto-transplanted intramuscularly into the rabbits for 4 to 8 weeks. The alginate-cancellous bone matrix composites or the cancellous bone matrix alone were implanted as control. The effectiveness of bone formation was assessed by means of roentgenography and histology.The results showed that the osteogeneses of MCCABs were better than those of the alginate-cancellous bone matrix composites and of the cancellous bone matrix. In the MCCABs, both intramembranous and cartilaginous osteogenesis were seen with the former predominating. In the control, only slight cartilaginous osteogenesis was seen. The results suggested that the osteogenesis of the MCCABs constructed by using tissue engineering method was obvious when transplanted intramuscularly, therefore, this kind of tissue-engineered bone could be an effective way for clinical application.