Short-term effects of mechanical loading on the transdifferentiation of condylar chondrocytes.

INTRODUCTION: Transdifferentiation of chondrocytes into bone cells explains most of the prenatal and early postnatal condylar growth, but its role during later postnatal growth and the mechanisms regulating transdifferentiation remain unknown. This study aimed to quantify the effects of mechanical loading on chondrocyte-derived osteogenesis during late postnatal condylar growth using a short-term mandibular laterotrusion model. METHODS: Thirty 4-week-old Aggrecan-CreERT2, R26RtdTomato, and 2.3Col1a1-GFP compound mice received tamoxifen injections and were divided into control and experimental groups. Appliances were bonded to shift the mandibles of the experimental mice for 5 days, causing protrusion and retrusion of the right and left condyles, respectively. Radiographic, microcomputed tomographic, and histomorphometric analyses were performed. RESULTS: The experimental and control groups showed substantial transdifferentiation of chondrocytes into bone cells. The experimental mice developed asymmetric mandibles, with the protrusive side significantly longer than the retrusive side. The protrusive condyles showed significantly increased chondrogenesis and greater numbers of chondrocyte-derived osteogenic cells, especially in the posterior third. The opposite effects were seen on the retrusive side. CONCLUSIONS: Transdifferentiation of chondrocytes into bone cells occurs during late postnatal condylar growth. Laterotrusion regulates condylar chondrogenesis and chondrocyte transdifferentiation, which alters the amount and direction of condylar growth. Our study demonstrated that chondrocytes are key players in condylar bone formation and should be the focus of studies to control and further understand condylar growth.

Biomimetic Tubular Matrix Induces Periodontal Ligament Principal Fiber Formation and Inhibits Osteogenic Differentiation of Periodontal Ligament Stem Cells.

Periodontal ligament (PDL) is assembled from highly organized collagen fiber bundles (PDL principal fibers) that are crucial in supporting teeth and buffering mechanical force. Therefore, regeneration of PDL needs to reconstruct these well-ordered fiber bundles to restore PDL functions. However, the formation of PDL principal fibers has long been a challenge due to the absence of an effective three-dimensional (3D) matrix to guide the growth of periodontal ligament stem cells (PDLSCs) and to inhibit the osteogenic differentiation of PDLSCs during the PDL principal fibers deposition. In this work, we designed and fabricated a bio-inspired tubular 3D matrix to guide the migration and growth of human PDLSCs and form well-aligned PDL principal fibers. As a biomimetic 3D template, the tubular matrix controlled PDLSCs migration inside the tubules and aligned the cells to the designated direction. Inside the tubular matrix, the PDLSCs expressed PDL markers and formed oriented fiber bundles with the same size and density as those of natural PDL principal fibers. Furthermore, the tubular matrix downregulated the osteogenic differentiation of PDLSCs. A mechanism study revealed that the Yap1/Twist1 signaling pathway was involved in the inhibition of PDLSCs osteogenesis within the tubular matrix. This work provides an effective approach to induce PDLSCs to form principal fibers and gives insight into the underlying mechanism of inhibiting the osteogenic differentiation of PDLSCs in biomimetic tubular matrices.

Evaluation of a peptide motif designed for protein tethering to polymer surfaces.

In search for peptide motifs that allow us to efficiently tether fusion proteins onto polymer surfaces, we designed a KLKLKLKLKL (KL5) decapeptide in which basic and hydrophobic amino acids were alternately linked. By means of genetic engineering technology together with a bacterial expression system, the KL5 fusions of epidermal growth factor (EGF), basic fibroblast growth factor, and stromal cell-derived factor-1α were prepared together with their control counterparts without KL5. The adsorption experiments were performed for these fusion proteins on the surface of polystyrene, hydrophilized polystyrene, and polycaprolactone by surface plasmon resonance analysis. To understand the results of the binding assays, the structure of the fusion proteins was predicted by ab initio computer simulation and analyzed empirically by circular dichroism spectroscopy. The result of structural analyses suggested that the KL5 peptide is exposed to the outside and has a negligible effect on the structure of the protein partners. However, it was found that the efficiency of KL5 as a peptide motif greatly depends on protein partners. Our results showed that KL5 exerts most effectively its function as a peptide motif when fused to acidic proteins such as EGF. Indeed, the number of living human mesenchymal stem cells determined after 7-day culture was larger on the polystyrene and polycaprolactone surfaces with EGF tethered through the KL5 peptide than control surfaces. According to the results obtained in this study, we conclude that KL5 is useful as a peptide motif for tethering a specific class of protein partners.

Oriented immobilization of basic fibroblast growth factor: Bioengineered surface design for the expansion of human mesenchymal stromal cells.

E. coli expressed recombinant basic fibroblast growth factor (bFGF) with histidine-tag (bFGF-His) was immobilized onto the surface of a glass plate modified with a Ni(II)-chelated alkanethiol monolayer. The immobilization is expected to take place through the coordination between Ni(II) and His-tag. The bFGF-immobilized surface was exposed to citrate buffer solution to refold in situ the surface-immobilized bFGF. The secondary structure of immobilized bFGF-His was analyzed by solid-phase circular dichroism (CD) spectroscopy. Immortalized human mesenchymal stromal cells (hMSCs) were cultured on the bFGF-His-immobilized surface to examine their proliferation. CD spectroscopy revealed that the immobilized bFGF initially exhibited secondary structure rich in α-helix and that the spectrum was gradually transformed to exhibit the formation of ß-strands upon exposure to citrate buffer solution, approaching to the spectrum of native bFGF. The rate of hMSC proliferation was 1.2-fold higher on the bFGF-immobilized surface treated with in situ citrate buffer, compared to the polystyrene surface. The immobilized bFGF-His treated in situ with citrate buffer solution seemed to be biologically active because its secondary structure approached its native state. This was well demonstrated by the cell culture experiments. From these results we conclude that immobilization of bFGF on the culture substrate serves to enhance proliferation of hMSCs.

Dental caries prevalence, oral health knowledge and practice among indigenous Chepang school children of Nepal.

BACKGROUND: Chepang communities are one of the most deprived ethnic communities in Nepal. According to the National Pathfinder Survey, dental caries is a highly prevalent childhood disease in Nepal. There is no data concerning the prevalence of caries along with knowledge, attitude and oral hygiene practices among Chepang schoolchildren. The objectives of this study were to 1) record the prevalence of dental caries 2) report experience of dental pain 3) evaluate knowledge, attitude and preventive practices on oral health of primary Chepang schoolchildren. METHOD: A cross sectional epidemiological study was conducted in 5 government Primary schools of remote Chandibhanjyang Village Development Committee (VDC) in Chitwan district. Ethical approval was taken from the Institutional Review Board within the Research Department of the Institute of Medicine (IOM) Tribhuvan University. Consent was obtained from parents for conducting clinical examination and administrating questionnaire. Permission was taken from the school principal in all schools. Data was collected using a pretested questionnaire on 131 schoolchildren aged 8-16-year- olds attending Grade 3-5. Clinical examination was conducted on 361 school children aged 5-16 -year-olds attending grade 1-5. Criteria set by the World Health Organization (1997) was used for caries diagnosis. The questionnaires, originally constructed in English and translated into Nepali were administered to the schoolchildren by the researchers. SPSS 11software was used for data analysis. RESULTS: Caries prevalence for 5-6 -year-old was above the goals recommended by WHO and Federation of Dentistry international (FDI) of less than 50% caries free children. Caries prevalence in 5-6-year-olds was 52% and 12-13-year-olds was 41%. The mean dmft/DMFT score of 5-6 -year-olds and 12 -13-year -olds was 1.59, 0.31 and 0.52, 0.84 respectively. The DMFT scores increased with age and the d/D component constituted almost the entire dmft/DMFT index. About 31% of 8-16-year-olds school children who participated in the survey reported having suffered from oral pain. Further, the need for treatment of decayed teeth was reported at 100%. About 76% children perceived teeth as an important component of general health and 75% reported it was required to eat. A total 93% children never visited a dentist or a health care service. Out of 56% children reporting cleaning their teeth daily, only 24% reported brushing their teeth twice daily. About 86% of the children reported using toothbrush and toothpaste to clean their teeth. Although 61% children reported to have received oral health education, 82% children did not know about fluoride and its benefit on dental health. About 50% children reported bacteria as the main cause of tooth decay and 23% as not brushing teeth for gingivitis. Frequency of sugar exposure was low; 75% of children reported eating sugar rich food once daily. CONCLUSIONS: Caries prevalence of 5-6 -year- old Chepang school children is above the recommended target set by FDI/WHO. The study reported 31% schoolchildren aged 8-16-year old suffered oral pain and decayed component constituted almost the entire dmft/DMFT index. The brushing habit was reportedly low with only 24% of the children brushing twice daily. A nationwide scientifically proven, cost effective school based interventions is needed for prevention and control of caries in schoolchildren in Nepal.