Mesenchymal stem cell-mediated tissue regeneration is a promising approach for regenerative medicine for a wide range of applications. Here we report a new population of stem cells isolated from the root apical papilla of human teeth (SCAP, stem cells from apical papilla). Using a minipig model, we transplanted both human SCAP and periodontal ligament stem cells (PDLSCs) to generate a root/periodontal complex capable of supporting a porcelain crown, resulting in normal tooth function. This work integrates a stem cell-mediated tissue regeneration strategy, engineered materials for structure, and current dental crown technologies. This hybridized tissue engineering approach led to recovery of tooth strength and appearance.

Periodontitis is a periodontal tissue infectious disease and the most common cause for tooth loss in adults. It has been linked to many systemic disorders, such as coronary artery disease, stroke, and diabetes. At present, there is no ideal therapeutic approach to cure periodontitis and achieve optimal periodontal tissue regeneration. In this study, we explored the potential of using autologous periodontal ligament stem cells (PDLSCs) to treat periodontal defects in a porcine model of periodontitis. The periodontal lesion was generated in the first molars area of miniature pigs by the surgical removal of bone and subsequent silk ligament suture around the cervical portion of the tooth. Autologous PDLSCs were obtained from extracted teeth of the miniature pigs and then expanded ex vivo to enrich PDLSC numbers. When transplanted into the surgically created periodontal defect areas, PDLSCs were capable of regenerating periodontal tissues, leading to a favorable treatment for periodontitis. This study demonstrates the feasibility of using stem cell-mediated tissue engineering to treat periodontal diseases.

Compared with small animal models such as rodents, large animal models are superior in many aspects for the study of human diseases and pre-clinical therapies. Since the development of the Minnesota miniature pig in 1949 at the Hormel Institute (USA), miniature pigs have been used as a large animal model in medical studies for scientific, economic, and ethical reasons. The oral maxillofacial region of miniature pigs is similar to that of humans in anatomy, development, physiology, pathophysiology, and disease occurrence. In this review, we describe the anatomical characteristics of the oral maxillofacial system of the miniature pig, established models of oral diseases in this animal, and other uses of the miniature pig in orofacial research.

Mesenchymal stem cells (MSCs) are able to differentiate into a variety of cell types, offering promising approaches for stem cell-mediated tissue regeneration. Here, we explored the potential of utilizing MSCs to reconstruct orofacial tissue, thereby altering the orofacial appearance. We demonstrated that bone marrow MSCs were capable of generating bone structures and bone-associated marrow elements on the surfaces of the orofacial bone. This resulted in significant recontouring of the facial appearance in mouse and swine. Notably, the newly formed bone and associated marrow tissues integrated with the surfaces of the recipient bones and re-established a functional bone marrow organlike system. These data suggested that MSC-mediated tissue regeneration led to a body structure extension, with the re-establishment of all functional components necessary for maintaining the bone and associated marrow organ. In addition, we found that the subcutaneous transplantation of another population of MSCs, the human periodontal ligament stem cells (PDLSCs), could form substantial amounts of collagen fibers and improve facial wrinkles in mouse. By contrast, bone marrow MSCs failed to survive at 8 weeks post-transplantation under the conditions used for the PDLSC transplantation. This study suggested that the mutual interactions between donor MSCs and recipient microenvironment determine long-term outcome of the functional tissue regeneration.

The mechanism underlying jaw osteoradionecrosis (ORN) is not fully understood, particularly in the early stages. To investigate bone and vessel pathogenesis in the early stages of jaw ORN, we generated a mandibular ORN model in miniature pigs (minipigs) by applying a combination of single-dose 25-Gy irradiation (IR) and tooth extraction. We studied 6 ORN model minipigs and 6 control, non-irradiated minipigs. We measured dynamic morphological changes, bone-remodeling-associated gene expression, sphingomyelinase activity, and local blood flow. Bone remodeling, including bone resorption and new bone formation, was observed within 15 days post-IR. Later, an ORN-related imbalance in bone metabolism gradually occurred, with loss of bone regeneration capacity, collagen collapse, and microvascular obliteration. Within 24 hrs post-IR, sphingomyelinase significantly increased in irradiated tissues. At 1 wk post-IR, local blood flow increased, but at 15 days post-IR, it significantly decreased to 50% below normal levels. This study provided details of the sequential occurrences in early-stage ORN in a large animal model. Our results suggested that reduced local blood flow and consequent hypovascularity may have caused an imbalance in bone remodeling. This suggested that microvessel damage may play a key role in the initiation of ORN.

Jaw osteoradionecrosis (ORN) is a common and serious complication of radiation therapy for head and neck cancers. Bone marrow mesenchymal stromal cells (BMMSCs) are multipotent postnatal stem cells and have been widely used in clinical therapies. In the present study, we generated the mandibular ORN model in swine using a combination of single-dose 25-Gy irradiation and tooth extraction. A typical ORN phenotype, including loss of bone regeneration capacity and collagen collapse with the obliteration of vessels, gradually appeared after irradiation. After autologous BMMSC transplantation, new bone and vessels were regenerated, and the advanced mandibular ORN was treated successfully. In summary, we developed a swine model of jaw ORN, and our results indicate that autologous BMMSC transplantation may be a promising therapeutic approach for ORN.

Abstract:The objectives of this study were to screen for the better Oligonucleotide (ODN) that promotes the proliferation of human bone-marrow mesenchymal stem cells (hBMSCs) and to investigate the mechanism of action of the ODN that has the greatest effect on the hBMSCs cell cycle. hBMSCs were isolated, cultured to the third passage and subjected to osteogenic, adipogenic and chondrogenic induction in order to examine their capacities for multi-differentiation. The hBMSCs were seeded at different plating densities (3.0×10 ) and tested for seven consecutive days to determine the better plating density. A total of 12 experimental groups and 1 control group of hBMSCs (4 replicate wells in each group) were established and treated with ODN types MT01, FC003, or SAT05f at concentrations of 0.5mg/l, 1.0 mg/l, 2.0 mg/l or 4.0 mg/l; the control group was treated with an equal volume of PBS. Proliferation of hBMSCs was determined for 3 consecutive days after treatment via CCK-8 assay. The type and concentration of ODN that had a significant facilitatory effect on hBMSCs proliferation was selected and cell cycle analysis was done on days 1, 2 and 3 after ODN treatment; control groups were treated with an equal amount of PBS. The expressions of cyclin A, cyclin D1, cyclin dependent kinase (CDK) 2 and CDK 4 in hBMSCs were measured on day 2 after treatment using fluorescent quantitative real-time PCR. The isolated and cultured hBMSCs were found to have osteogenic, adipogenic and chondrogenic differentiation capacities. The better cell growth curve was found to occur at a plating density of 6.0×10 3 /cm 2 . Optical density was significantly increased in hBMSCs treated with 0.5 mg/L FC003 on day 1 (P<0.01) compared to the control group. Optical density was significantly decreased on day 1 after treatment with 1.0 mg/L and 4.0 mg/l of SAT05f (P<0.01 and P<0.05, respectively). Optical density was significantly increased on days 1, 2 and 3 after treatment with a final concentration of 2.0 mg/L of MT01 (P<0.01, P<0.05, P<0.05, respectively). The percentage of cells in phase G0/G1 was significantly reduced, and the percentage of cells in phases S and G2/M was significantly increased (P<0.01) after treatment with 2.0 mg/ l MT01 compared to the control group. Furthermore, the expressions of cyclin A, cyclin D1, CDK 2 and CDK 4 were significantly elevated (P<0.01) compared with the control group. In conclusion, a 2.0 mg/l concentration of MT01 significantly promotes hBMSCs proliferation as evidenced by the decrease in the percentage of cells in phase G0/G1 and the increase in the percentage of cells in phases S and G2/M. The underlying molecular mechanisms may include, but are not limited to, elevated expressions of cyclin A, cyclin D1, CDK 2 and CDK 4.