Hirokazu Mizuno

Tissue-engineered composites of anulus fibrosus and nucleus pulposus for intervertebral disc replacement.

Study Design. By the technique of tissue engineering, composite intervertebral disc implants were fabricated as novel materials for disc replacement, implanted into athymic mice, and removed at times up to 12 weeks. Objectives. The goal of this study was to construct composite intervertebral disc structures consisting of anulus fibrosus cells and nucleus pulposus cells seeded on polyglycolic acid and calcium alginate matrices, respectively. Summary of Background Data. Previous work has documented the growth of anulus fibrosus cells on collagen matrices and nucleus pulposus cells cultured on multiple matrices, but there is no documentation of composite disc implants. Methods. Lumbar intervertebral discs were harvested from sheep spine, and the nucleus pulposus was separated from surrounding anulus fibrosus. Each tissue was digested in collagenase type II. After 3 weeks in culture, cells were seeded into implants. The shape of the anulus fibrosus scaffold was fabricated from polyglycolic acid and polylactic acid, and anulus fibrosus cells were pipetted onto the scaffold and allowed to attach for 1 day. Nucleus pulposus cells were suspended in 2% alginate and injected into the center of the anulus fibrosus. The disc implants were placed in the subcutaneous space of the dorsum of athymic mice and harvested at 4, 8, and 12 weeks. At each time point, 4 samples were stored in −70 C for collagen typing and analysis of proteoglycan, hydroxyproline, and DNA. Other samples were fixed in 10% formalin for Safranin-O staining. Results. The gross morphology and histology of engineered discs strongly resembled those of native intervertebral discs. Biochemical markers of matrix synthesis were present, increasing with time, and were similar to native tissue at 12 weeks. Tissue-engineered anulus fibrosus was rich in type I collagen but nucleus pulposus contained type II collagen, similar to the native disc. Conclusion. These results demonstrate the feasibility of creating a composite intervertebral disc with both anulusfibrosus and nucleus pulposus for clinical applications.

A composite tissue-engineered trachea using sheep nasal chondrocyte and epithelial cells

This study evaluates the feasibility of producing a composite engineered tracheal equivalent composed of cylindrical cartilaginous structures with lumens lined with nasal epithelial cells. Chondrocytes and epithelial cells isolated from sheep nasal septum were cultured in Hams F12 media. After 2 wk, chondrocyte suspensions were seeded onto a matrix of polyglycolic acid. Cell‐polymer constructs were wrapped around silicon tubes and cultured in vitro for 1 wk, followed by implanting into subcutaneous pockets on the backs of nude mice. After 6 wk, epithelial cells were suspended in a hydrogel and injected into the embedded cartilaginous cylinders following removal of the silicon tube. Implants were harvested 4 wk later and analyzed. The morphology of implants resembles that of native sheep trachea. H&E staining shows the presence of mature cartilage and formation of a pseudostratified columnar epithelium, with a distinct interface between tissue‐engineered cartilage and epithelium. Safranin‐O staining shows that tissue‐engineered cartilage is organized into lobules with round, angular lacunae, each containing a single chondrocyte. Proteoglycan and hydroxyproline contents are similar to native cartilage. This study demonstrates the feasibility of recreating the cartilage and epithelial portion of the trachea using tissue harvested in a single procedure. This has the potential to facilitate an autologous repair of segmental tracheal defects.—Kojima, K., Bonassar, L. J., Roy, A. K., Mizuno, H., Cortiella, J., Vacanti, C. A. A composite tissue‐engineered trachea using sheep nasal chondrocyte and epithelial cells. FASEB J. 17, 823–828 (2003)

Composite implantation of mesenchymal stem cells with endothelial progenitor cells enhances tissue-engineered bone formation

For successful tissue engineering, neovascularization of the implanted tissue is critical. Factors generated by endothelial cells are also considered crucial for the process of osteogenesis. The direct effects of supplementing tissue engineered constructs with cultured endothelial progenitor cells (EPCs) for enhancing bone regeneration have not been reported. In this study, we investigated the potential of EPCs to facilitate neovascularization in implants and evaluated their influence on bone regeneration. The influence of EPC soluble factors on osteogenic differentiation of mesenchymal stem cells (MSCs) was tested by adding EPC culture supernatant to MSC culture medium. To evaluate the influence of EPCs on MSC osteogenesis, canine MSCs-derived osteogenic cells and EPCs were seeded independently onto collagen fiber mesh scaffolds and co-transplanted to nude mice subcutaneously. Results from coimplant experiments were compared to implanted cells absent of EPCs 12 weeks after implantation. Factors from the culture supernatant of EPCs did not influence MSC differentiation. Coimplanted EPCs increased neovascularization and the capillary score was 1.6-fold higher as compared to the MSC only group (p < 0.05). Bone area was also greater in the MSC + EPC group (p < 0.05) and the bone thickness was 1.3-fold greater in the MSC + EPC group than the MSC only group (p < 0.05). These results suggest that soluble factors generated by EPCs may not facilitate the osteogenic differentiation of MSCs; however, newly formed vasculature may enhance regeneration of tissue-engineered bone.

Peri-implant soft tissue management through use of cultured mucosal epithelium

OBJECTIVE In implant therapy, peri-implant soft tissue management through use of mucosal grafting or skin grafting is necessary in some patients who do not have enough attached gingiva around the abutment. However, limitation of donor site size is a problem for the mucosal graft, and the different characteristics of skin, such as hair growth, are disadvantages in treatment that involves the use of skin graft. On the other hand, cultured epithelium fabricated with living mucosal cells has proved to be a good grafting material for any kind of mucosal defect. In this study, we used cultured mucosal epithelium for soft tissue management in implant therapy. STUDY DESIGN In the first surgical procedure of the implant therapy, a small segment of oral mucosa was sampled from a patient. The cultured epithelium was fabricated and then stored until it was grafted in the second surgery. RESULTS Twelve cases in which patients underwent peri-implant soft tissue management through use of cultured mucosal epithelium for implant therapy are presented, and the usefulness of this technique in the making of attached gingiva is analyzed. CONCLUSIONS From this study it was concluded that cultured mucosal epithelium can serve as a proper material for peri-implant soft tissue management.

Successful Culture and Sustainability in Vivo of Gene-Modified Human Oral Mucosal Epithelium

Human oral mucosal cells are an attractive site for tissue engineering because they are the most accessible cells in the body and easy to manipulate in vitro. They thus have possibilities for targeting by somatic gene therapy. We examined the efficiency of retrovirus-mediated gene transfer and the construction of mucosal epithelium in vivo. Human oral mucosal cells were transduced with a retroviral vector carrying the lacZ gene at high efficiency and constructed epithelium after G418 selection with 3T3 cells in vitro. The cultured oral mucosal epithelium membrane was then grafted onto immunodeficient mice. Beta-Gal expression was detected histochemically in vivo 5 weeks after grafting. Furthermore, we transduced factor IX cDNA into the mucosal epithelium membrane, and it was then transplanted into nude mice. Between 0.6 and 1.8 ng of human factor IX per milliliter was found in mouse plasma, and the production was continued for 23 days in vivo. These results confirmed that the oral mucosal epithelium is an ideal target tissue for gene therapy or tissue engineering.

In vitro analysis of distraction osteogenesis.

Distraction osteogenesis has been widely used for lengthening craniomaxillofacial bone. The healing process of distracted bone has been studied mainly by histological observation in vivo. To analyze the cellular response to the mechanical stress of distraction, we have established and evaluated an in vitro model of distraction osteogenesis using an organ culture technique. Five-week-old male Wistar rats were used for the experiments. The tibial bone was fractured by hand and fixed for 1 week by acrylic resin. After the initial healing period, the tibial bone was harvested and used for this study. A distraction instrument was devised to control the strain on the cultured bone using a micrometer. After distraction, the samples were histologically evaluated for ossification by hematoxylin and eosin stain and Alcian blue stain. As a result, the histological finding for the bone region at a slow rate of distraction (0.5 mm/day) was different from that at a rapid rate of distraction (1.0 mm/day). The proliferation of cartilage was inhibited at the rapid distraction rate. Thus, we hypothesized that mechanical stress regulated cartilaginous growth in tissue cultivation. Judging from this experiment, the model was useful for investigation of the mechanism of bone formation in distraction osteogenesis, because it was simple and served to isolate many factors.

Cultured oral epithelium as an effective biological dressing using for palatal wounds after palatoplasty

Abstract Cultured allografts derived from mucosal tissue provide a potent stimulus to wound healing in a wide variety of wounds. In the field of oral surgery, mucoperiosteal defect of the hard palate after palatoplasty causes scar contraction, leading to poor growth of the maxilla. The promotion of wound healing in these cases through cultured epithelial allografting has been reported. Cultured epithelial allografting was done using a strangers cultured cells. We grafted cultured oral epithelium in the hope of improving growth of maxilla. Clefts of the soft and hard palate (seven patients), and a cleft of the soft palate (two patients) were present. Average patient age was 1 year 4 months. Palatoplasty was done by a conventional pushback operation. Oral epithelial cells in healthy adults were cultured using 3T3 cells as the feeder layer. After 3 weeks, cultured oral mucosal epithelium was grafted on a raw surface following palatoplasty. In all patients, the grafted areas underwent re-epithelialization after about 1 week and did not exhibit any side effects of graft rejection. Grafted areas healed completely after 2–3 weeks in all cases. Cultured epithelial allografts serve as a temporary biological dressing, and accelerates epithelialization and wound healing. Allografting by cultured oral epithelium has proved to be a very useful therapeutic modality in palatoplasty, as well as effective augmentation materials in cases of oral mucosal defects.

An ex vivo model for gene therapy of hemophilia B using cultured human oral mucosal epithelium

Abstract Human oral mucosal cells are important target tissues for regenetic engineering and are sources of epithelial sheets. In this study, we examine the possibility of mucosal epithelial sheet as a target tissue for gene therapy. Human oral mucosal cells were transduced with a retroviral vector carrying human factor IX gene and constructed epithelium after G418 selection with 3T3 cells in vitro. The cultured oral mucosal epithelium membrane was then grafted onto immunodeficient mice. Between 0.6 and 1.8 ng of human factor IX per milliliter was found in mouse plasma, and the production was continued for 23 days in vivo. These results demonstrate a model of ex vivo gene therapy for hemophilia B using gene-modified oral mucosal epithelium.