[Research progress of cell sheet technology and its applications in tissue engineering and regenerative medicine].

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.

Locally injection of cell sheet fragments enhances new bone formation in mandibular distraction osteogenesis: a rabbit model.

Effective methods to shorten the treatment period of distraction osteogenesis (DO) are needed. To investigate whether injections of osteogenic bone marrow stromal cell (BMSC) sheet fragments could be used to facilitate new bone formation during DO, 30 rabbits underwent bilateral mandibular osteotomy and their mandibles were lengthened at a rate of 0.75 mm/12 h for 6 days after a 5-day latency period. There were three treatment groups (n = 10 for each group): Serum-free medium, dissociated BMSCs, and BMSC sheet fragments. A local injection was conducted with a needle directly into the distracted areas immediately after distraction. Rabbits were sacrificed for examination at 3 and 6 weeks after injection. Gross examination, radiographic evaluation, and micro-CT scanning indicated a significant increase in bony union in the BMSC sheet fragment group, compared with the medium group and the dissociated cell group. The histomorphometric analysis showed more intensive bone formation in the sheet fragment group than the other two groups at each time point. Additionally, the peak load was significantly higher in the fragment group than those in the others. The results show that injection of BMSC sheet fragments promotes bone formation in DO and indicate a promising approach to shorten the treatment period of osteodistraction.

[Cell-based approaches to promote bone regeneration in distraction osteogenesis].

OBJECTIVE: To summarize the recent progress of cell-based approaches for promoting bone regeneration in distraction osteogenesis (DO). METHODS: Recent literature concerning enhancement of bone regeneration following DO using cell-based approaches was reviewed and analyzed. RESULTS: An overview of 4 different cell-based approaches was mainly provided: single cell injection, cell scaffold-based strategies/injectable tissue engineered bone, microtissue technology or cell aggregate technology, and stem cell gene therapy. Each has its advantages and disadvantages. Other methods are still in the experimental research except that compound injection of bone marrow mesechymal stem cells and platelet-rich plasma has been applied to clinical practice. CONCLUSION: The cell-based approach is a promising strategy in the field of bone regenerative medicine. These approaches have bright future in promoting bone regeneration and reducing the treatment period in DO in the clinical application. However, well-designed preclinical studies are required to establish safe and effective guidelines for cell-based approaches to promoting bone regeneration during DO.

Prefabrication of axial vascularized tissue engineering coral bone by an arteriovenous loop: a better model.

The most important problem for the survival of thick 3-dimensional tissues is the lack of vascularization in the context of bone tissue engineering. In this study, a modified arteriovenous loop (AVL) was developed to prefabricate an axial vascularized tissue engineering coral bone in rabbit, with comparison of the arteriovenous bundle (AVB) model. An arteriovenous fistula between rabbit femoral artery and vein was anastomosed to form an AVL. It was placed in a circular side groove of the coral block. The complex was wrapped with an expanded-polytetrafluoroethylene membrane and implanted beneath inguinal skin. After 2, 4, 6 and 8 weeks, the degree of vascularization was evaluated by India ink perfusion, histological examination, vascular casts, and scanning electron microscopy images of vascular endangium. Newly formed fibrous tissues and vasculature extended over the surfaces and invaded the interspaces of entire coral block. The new blood vessels robustly sprouted from the AVL. Those invaginated cavities in the vascular endangium from scanning electron microscopy indicated vessel's sprouted pores. Above indexes in AVL model are all superior to that in AVB model, indicating that the modified AVL model could more effectively develop vascularization in larger tissue engineering bone.

The role of the lateral pterygoid muscle in the sagittal fracture of mandibular condyle (SFMC) healing process.

The aim of this study was to examine the role of the lateral peterygoid muscle in the reconstruction of the shape of the condyle during healing of a sagittal fracture of the mandibular condyle. Twenty adult sheep were divided into 2 groups: all had a unilateral operation on the right side when the anterior and posterior attachments of the discs were cut, and an oblique vertical osteotomy was made from the lateral pole of the condyle to the medial side of the condylar neck. Ten sheep had the lateral pterygoid muscle cut, and the other 10 sheep did not. Sheep were killed at 4 weeks (n=2 from each group), 12 weeks (n=4), and 24 weeks (n=4) postoperatively. Computed tomograms (CT) were taken before and after operations. We dissected the joints, and recorded with the naked eye the shape, degree of erosion, and amount of calcification of the temporomandibular joint (TMJ). In the group in which the lateral peterygoid muscle had not been cut the joints showed overgrowth of new bone and more advanced ankylosis. Our results show that the lateral pterygoid muscle plays an important part in reconstructing the shape of the condyle during the healing of a sagittal fracture of the mandibular condyle, and combined with the dislocated and damaged disc is an important factor in the aetiology of traumatic ankylosis of the TMJ.

Report of two Chinese patients suffering from CLCN7-related osteopetrosis and root dysplasia.

Osteopetrosis is a group of genetic bone disorders. There are three types of osteopetrosis: autosomal recessive osteopetrosis (ARO), autosomal dominant osteopetrosis type II (ADO II), and intermediate autosomal recessive osteopetrosis (IARO). The prevalence of ADO II is about 1:100,000, while no more than 20 cases of IARO have been reported worldwide. We present the first Chinese IARO patient with a novel homozygous variant in CLCN7 gene (p. Pro470Leu) and an ADO II patient with a heterozygous variant in CLCN7 gene (p. Arg286Trp). In addition to general osteosclerosis, the striking features of these two patients are unerupted teeth with root dysplasia. We speculate that ClC-7 in different tooth cells may contribute directly to the root development, the defect of ClC-7 may have a dose dependent effect on the phenotype of root dysplasia, and the tooth position may also affect the root phenotype with dysfunctional ClC-7.

Surgical treatment of a giant neurofibroma.

Neurofibromatosis type 1, an autosomal dominant inherited disease, presents pathologic symptoms of multiple systems, including neurofibromatosis, skeletal dysplasia, café-au-lait spots in skins, and so on. A 45-year-old man with neurofibromatosis type 1 was reported in this article. The patient presented a giant neurofibroma in his head and neck, dysplasia of skull, facial bones and spinal columns, and multiple café-au-lait spots in systematic skins. Satisfactory curative effects were obtained in this case after tumor resection and prosthesis implantation.

A novel hydroxyapatite ceramic bone substitute transformed by ostrich cancellous bone: characterization and evaluations of bone regeneration activity.

Various biomaterials have been used for bone repair and reconstruction of bone defects. Inorganic xenogenic bone substitutes have been intensively studied because they possesses favorable regenerative properties. The purpose of this study was to evaluate the properties of a novel inorganic xenogenic bone substitute, sintered ostrich cancellous bone (SOCB). Bone regeneration capability was also comparing to that of other bone substitutes in rabbit calvarial defects. Biochemical and biomechanical properties of the SOCB ceramic closely resembled those of human bone. Bone regeneration was evaluated by radiograph, histology, and histomorphometry. Bone regeneration was significantly enhanced in defects treated with SOCB when compared with other bone substitutes. The biochemical and biomechanical properties of SOCB are favorable for bone regeneration. SOCB might be a promising biomaterial for the repair of bone defects.

Clinical and animal research findings in pycnodysostosis and gene mutations of cathepsin K from 1996 to 2011.

Cathepsin K (CTSK) is a member of the papain-like cysteine protease family. Mutations in the CTSK gene cause a rare autosomal recessive bone disorder called pycnodysostosis (OMIM 265800). In order to follow the advances in the research about CTSK and pycnodysostosis, we performed a literature retrospective study of 159 pycnodysostosis patients reported since 1996 and focused on the genetic characteristics of CTSK mutations and/or the clinical phenotypes of pycnodysostosis. Thirty three different CTSK mutations have been found in 59 unrelated pycnodysostosis families. Of the 59 families, 37.29% are from Europe and 30.51% are from Asia. A total of 69.70% of the mutations were identified in the mature domain of CTSK, 24.24% in the proregion, and 6.06% in the preregion. The hot mutation spots are found in exons 6 and 7. CTSK mutations result in total loss or inactivity of the CTSK protein, which causes abnormal degradation of bone matrix proteins such as type I collagen. Skeletal abnormalities, including short stature, an increase in bone density with pathologic fractures, and open fontanels and sutures, are the typical phenotypes of pycnodysostosis. Research on Ctsk(-/-) mouse models was also reviewed here to elucidate the biological function of Ctsk and the mechanism of pycnodysostosis. New evidence suggests that Ctsk plays an important role in the immune system and may serve as a valid therapeutic target in the future treatment of pycnodysostosis.

Enhancing bone formation by transplantation of a scaffold-free tissue-engineered periosteum in a rabbit model.

OBJECTIVES: The periosteum plays an important role in bone regeneration. However, the harvesting of autogenous periosteum is associated with disadvantages such as donor site morbidity and limited donor sources. This study uses an osteogenic predifferentiated cell sheet to fabricate a scaffold-free tissue-engineered periosteum (TEP). MATERIAL AND METHODS: We generated an osteogenic predifferentiated cell sheet from rabbit bone marrow stromal cells (BMSCs) using a continuous culture system and harvested it using a scraping technique. Then, the in vitro characterization of the sheet was investigated using microscopy investigation, quantitative analysis of alkaline phosphatase (ALP) activity, and RT-PCR. Next, we demonstrated the in vivo osteogenic potential of the engineered sheet in ectopic sites together with a porous ß-tricalcium phosphate ceramic. Finally, we evaluated its efficiency in treating delayed fracture healing after wrapping the cell sheet around the mandible in a rabbit model. RESULTS: The engineered periosteum showed sporadic mineralized nodules, elevated ALP activity, and up-regulated gene expression of osteogenic markers. After implantation in the subcutaneous pockets of the donor rabbits, the in vivo bone-forming capability of the engineered periosteum was confirmed by histological examinations. Additionally, when wrapping the engineered periosteum around a mandibular fracture gap, we observed improved bone healing and reduced amounts of fibrous tissue at the fracture site. CONCLUSION: The osteogenic predifferentiated BMSC sheet can act as a scaffold-free TEP to facilitate bone regeneration. Hence, our study provides a promising strategy for enhancing bone regeneration in clinical settings.

Engineering injectable bone using bone marrow stromal cell aggregates.

With the increasing popularity of minimally invasive surgery, to develop an injectable bone would be highly preferable for the repair of bone nonunions and defects. However, the use of dissociated cells and exogenous carriers to construct injectable bone faces several drawbacks. To circumvent these limitations, we first harvested a cell sheet from rabbit bone marrow stromal cells using a continuous culture method and a scraping technique. The obtained sheet was then cut into fragments of multicellular aggregates, each of which was composed of a certain number of cells, extracellular matrix, and intercellular connections. The aggregates showed apparent mineralization properties, high alkaline phosphatase activity, increased osteocalcin content, and upregulated bone markers, implying their in vitro osteogenic potential. Then, serum-free medium (the control group), dissociated cell suspension (the cell group), and suspension of multicellular aggregates (the aggregate group) were injected subcutaneously on the back of the nude mice to evaluate ectopic bone formation. The results revealed that the aggregate group showed significantly larger and denser bone at the injection sites than the cell group, whereas bone formation did not occur in the control group. Additionally, when injecting them locally into the mandibular fracture gap of delayed healing in a rabbit model, we observed the most improved bone healing in the aggregate group. More cells survive and retain at the injection sites in the aggregate group than that in the cell group postoperatively. Our study indicates that the multicellular aggregates might be considered a promising strategy to generate injectable bone tissue and improve the efficacy of cell therapy.

Reconstruction of rabbit critical-size calvarial defects using autologous bone marrow stromal cell sheets.

The reconstruction of bone defects remains a significant clinical problem. In this study, we constructed cell sheet from bone marrow stromal cells on normal culture plates by a simple method. The cell sheets showed evident mineralized nodules, high alkaline phosphatase activities, indicating their in vitro osteogenic potential. Then its osteogenic capability to heal critical-size rabbit calvarial defect was investigated. Forty adult New Zealand White rabbits were randomly divided into 4 groups of 10 animals each: (1) empty, (2) demineralized bone matrix (DBM) alone, (3) DBM/cell suspension, and (4) DBM/cell sheet. Specimens were harvested 6 and 12 weeks after implantation, respectively. Radiographic, histologic, and histomorphometric analyses were performed to evaluate the new bone formation inside the defect. The results revealed that the defect treated with DBM/cell sheet showed significantly more bone formation than other 3 groups (P < 0.05). Our study indicates that the cell sheet enhances bone regeneration in healing critical-size rabbit calvarial defect, and cell sheet-based engineered bone might be considered as potential substitutes for bone reconstruction.

Engineering scaffold-free bone tissue using bone marrow stromal cell sheets.

The use of exogenous scaffolds to engineer bone tissue faces several drawbacks including insufficient biological activity, potential immunogenicity, elevated inflammatory reaction, fluctuating degradation rate, and low cell-attachment efficiency. To circumvent these limitations, we sought to engineer large scaffold-free bone tissue using cell sheets. We harvested intact cell sheets from bone marrow stromal cells using a continuous culture method and a scraping technique. The cell sheets were then rolled and fabricated into large constructs. Finally, the constructs were implanted into the subcutaneous pockets of nude mice. The cells within the sheet maintained in vitro osteogenic potential after osteoblast differentiation. Computed tomography scans and histological examination confirmed new bone formation in vivo. Additionally, the engineered bone exhibited enhanced compressive strength. Our results indicate that the BMSC sheets can facilitate the formation of functional three-dimensional bone tissue without the use of exogenous scaffolds. Hence, the study provides an intriguing alternative strategy for bone repair.

Modified approach to construct a vascularized coral bone in rabbit using an arteriovenous loop.

The most important factor for the survival of thick three-dimensional tissues is the degree of vascularization. In this study, a modified arteriovenous loop (AVL) model was developed to prefabricate an axial vascularized tissue-engineered coral bone. In group A (n = 28), an arteriovenous fistula between rabbit femoral artery and vein was anastomosed to form an AVL. The AVL was placed in a coral block (6 x 8 x 10 mm (3)) as a vascular carrier. The complex was wrapped with polytetrafluoroethylene membrane and implanted subcutaneously. In group B (n = 20), there was no vascular carrier, and the same dimensional coral was directly implanted beneath inguinal skin. After 2, 4, 6, and 8 weeks, the rabbits were perfused with heparinized saline (for scanning electron microscopy), India ink (for histological examination), and ethylene perchloride (for vascular casts) via the abdominal aorta. In group A, histology showed that newly formed vasculature extended over the surfaces and invaded the entire coral blocks. The vascular density was significantly superior to that in group B. Vascular casts showed that new blood vessels robustly sprouted from the AVL. Scanning electron microscopy demonstrated that there were minute sprouting cavities in the vascular endangium. In this model, an axial vascularized coral bone could be effectively constructed.

Enhanced treatment of articular cartilage defect of the knee by intra-articular injection of Bcl-xL-engineered mesenchymal stem cells in rabbit model.

Direct intra-articular injection of mesenchymal stem cells (MSCs) has been proposed as a potential cell therapy for cartilage defects. This cell therapy relies on the survival of the implanted MSCs. However, the arduous local environment may limit cell viability after implantation, which would restrict the cells' regenerative capacity. Thus, it is necessary to reinforce the implanted cells against the unfavourable microenvironment in order to improve the efficacy of cell therapy. We examined whether the transduction of an anti-apoptotic protein, Bcl-xL, into MSCs could prevent cell death and improve the implantation efficiency of MSCs in a rabbit model. Our current findings demonstrate that the group treated with Bcl-xL-engineered MSCs could improve cartilage healing both morphologically and histologically when compared with the controls. These results suggest that intra-articular injection of Bcl-xL-engineered MSCs is a potential non-invasive therapeutic method for effectively treating cartilage defects of the knee.

Vitalized guided bone regeneration membrane from marrow stromal cells.

PURPOSE: Various materials have been used to make guided bone regeneration membranes. The purpose of this study was to create a novel osteogenic membrane without synthetic material. The osteogenic potential of the membrane was evaluated by both in vitro and in vivo testing. MATERIALS AND METHODS: The membrane was obtained by continuous culture of marrow stromal cells. The structure of the membrane was characterized by staining with hematoxylin-eosin, von Kossa, and carboxyfluorescein diacetate; immunohistochemical staining against collagen type I; electron microscopy; and energy-dispersive spectrometry. The osteogenic potential and bone augmentation effect of the membrane were investigated by implantation of the membrane and a membrane/natural coral composite into nude mice, respectively. RESULTS: The membrane was composed of living cells and a dense matrix of collagen type I. Mineral deposition was apparent through electronic microscopic observation and von Kossa staining. Energy-dispersive spectrometry indicated that the calcium:phosphorus ratio of mineral was 1.71 in the membrane. The membrane had formed a thin layer of bone 2 months after implantation subcutaneously. In the bone augmentation specimens, new bone was observed histologically on the surface and in the pores of natural coral in all specimens of membrane-coral composite. CONCLUSIONS: This study developed a novel strategy to produce a vital guided bone regeneration membrane without synthetic material. Membrane derived from marrow stromal cells was osteogenic and had an optimizing bone augmentation effect.

[Constructing tissue engineered trachea-like cartilage graft in vitro by using bone marrow stromal cells sheet and PLGA internal support: experimental study in bioreactor].

OBJECTIVE: To explore the feasibility of constructing tissue engineered trachea-like cartilage graft in vitro by using bone marrow stromal cells (BMSCs) sheet and PLGA internal support. METHODS: Rabbit BMSCs were expanded and induced by transforming growth factor-1 to improve chondrocyte phenotype of BMSCs. BMSCs sheets were obtained by continuous culture and wrapped the PGLA scaffold in the shape of cylinder. The constructs were incubated in spinner flask for 8 weeks and cartilage formation was investigated by gross inspection, histology, glycosaminoglycan and mechanical strength content. RESULTS: After in vitro culture, cartilage like tissue in cylindrical shape had been regenerated successfully. Stiff, shiny, pearly opalescence tissues were observed. Histological analysis showed engineered trachea cartilage consisted of evenly spaced lacunae embedded in matrix, cells stationed in the lacunae could be noticed clearly. Safranin-O staining on the sections showed homogenous and positive red staining, which demonstrated that the engineered tissue was rich in proteoglycans. CONCLUSIONS: Based on the cell sheet and internal support strategy, trachea-like cartilage in cylindrical shape could be successfully fabricated which provided a highly effective cartilage graft substitute and could be useful in many situations of trachea-cartilage loss encountered in clinical practice.

Novel approach to engineer implantable nasal alar cartilage employing marrow precursor cell sheet and biodegradable scaffold.

PURPOSE: Repair of nasal and auricular malformation remains an obstacle for clinicians because of poor regenerative capacity of cartilage and limitation of donor sites. In the current study, we developed a novel approach to regenerate implantable nasal alar cartilage by using marrow precursor cell (MPC) sheet and biodegradable scaffold of polylactic acid-polyglycolic acid copolymer (PLGA). MATERIALS AND METHODS: Rabbit MPCs were expanded and induced by transforming growth factor-beta to improve chondrocyte phenotype. MPC sheets were obtained by continuous culture and used to wrap PLGA scaffold in the shape of the human nasal alar. The constructs were incubated in a spinner flask for 4 weeks, and cartilage formation was investigated by gross inspection and histological examination. The constructs were then implanted subcutaneously into a nude mouse. Specimens were harvested and analyzed 4 weeks after implantation. RESULTS: The results showed that cartilaginous tissue formed and PLGA absorbed during in vitro incubation. Histological analysis showed engineered cartilage consisted of evenly spaced lacunae embedded in a matrix rich in proteoglycans, and kept the initial shape of the nasal alar. Based on this "MPC sheet combining polymer strategy," implantable nasal alar could be successfully regenerated. CONCLUSION: This strategy has the advantage of high cell transplantation efficiency and great potential for clinical application.

Platelet-rich plasma - A promising cell carrier for micro-invasive articular cartilage repair.

Due to the limited regenerative capacity of cartilage tissues, articular cartilage defect caused by various lesions remains a problem to be resolved. Tissue engineering provided a valuable alternative to current therapeutic approaches, which is expected to greatly reduce the need of joint replacement. Scaffold, acting as cell carrier, plays an important role in maintaining cells in defect sites, thus facilitates the chondrogenesis. However, an open operation is often needed to implant the cell/scaffold composite, to find a less invasive way to delivering the complex into the defect site would be desirable. Different from synthetic and other nature derived scaffold, platelet-rich plasma (PRP) is a fraction of plasma which contains multiple growth factors and could be clotted when mixed with thrombin. Therefore, we hypothesized that PRP could be used as an autologous cell carrier to inject and fix chondrocytes into the defect site of articular cartilage. With the assistance of arthroscope, the defect could be precisely located, and injectable PRP-Cell composite would make the operation micro-invasive and simple.