Chitosan as a potential osteogenic factor compared with dexamethasone in cultured macaque dental pulp stromal cells.

Chitosan, a natural biopolymer derived from chitin, is considered a promising scaffold material for bone tissue engineering. The ability of chitosan to promote the osteogenic differentiation of dental pulp stromal/stem cells (DPSCs) is unknown. We have evaluated the potential of chitosan to induce the osteogenic differentiation of macaque DPSCs in comparison with that of dexamethasone. DPSCs were cultured in mineralizing medium supplemented with 5 or 10 µg/ml chitosan or with 1 or 10 nM dexamethasone. The metabolic activity of DPSCs was measured by MTT assay. Their osteogenic differentiation was determined by the number of transcripts of RUNX2, alkaline phosphatase (ALP), and COL1A1 by using real-time polymerase chain reaction, by alizarin red staining for mineral deposition, and by the ALP activity released into the medium for their ability to support biomineralizaton. Addition of chitosan to the mineralizing medium significantly increased DPSCs metabolism after 7 and 14 days of culture (P ≤ 0.0001). Chitosan at 5 µg/ml also significantly enhanced RUNX2 and ALP mRNA but not COL1A1 mRNA; chitosan tended to increase the release of ALP hydrolytic enzyme activity into the medium during the first week. Dexamethasone upregulated the osteogenic markers tested. Mineral deposition was similar in the chitosan and dexamethasone groups and was not statistically different from that of the mineralizing control group. Thus, the potential of chitosan to stimulate DPSCs proliferation and early osteogenic differentiation is comparable with that of dexamethasone, but mineralization remains unaffected by chitosan treatment. In addition to its role as a three-dimensional scaffold for osteogenic cells in vivo, chitosan might also stimulate DPSCs proliferation and early osteogenic differentiation in vitro.

In Vitro Hemostatic Activity of Novel Fish Gelatin-Alginate Sponge (FGAS) Prototype.

A hemostatic sponge prototype was successfully synthesized from fish gelatin as an alternative to mammalian gelatin; it was mixed with alginate in certain combinations, double cross-linked with calcium ions, and gamma irradiated at a dose of 20 kGy to improve the characteristics and effectiveness of its function as a local hemostatic agent. There were improvements in the physicochemical and mechanical properties, porosity index, absorption capacity, biodegradation properties, biocompatibility, and hemocompatibility of the fish gelatin-alginate sponge (FGAS) prototypes compared with the pure fish gelatin sponge. Hemostatic activity tests showed that the means for clotting time, prothrombin time, and activated partial thromboplastin time were shorter in the FGAS prototype than in the negative control, and there was no significant difference compared with the commercial gelatin sponge. The hemostatic mechanism of the FGAS prototype combined a passive mechanism as a concentrator factor and an active mechanism through the release of calcium ions as a coagulation factor in the coagulation cascade process.

Characterization of Polyvinyl Alcohol-Collagen-Hydroxyapatite Composite Membrane from Lates calcarifer Scales for Guided Tissue and Bone Regeneration.

OBJECTIVE: To determine the chemical structure, tensile strength, porosity, and degradability of polyvinyl alcohol (PVA)-collagen-hydroxyapatite (HA) composite membranes for guided tissue and bone regeneration. MATERIALS AND METHODS: The PVA-collagen-HA composite membrane was divided into three groups: the group without irradiation, the group with 15 kGy irradiation, and 25 kGy irradiation. Each group was tested for chemical structure with Fourier-transform infrared (FT-IR) at a wavelength of 400 to 4,000 cm-1. Tensile strength test was tested in dry and wet conditions with the standard method of American Standard Testing Mechanical (ASTM) D638, and porosity using scanning electron microscope and analyzed using ImageJ software. Degradability test immersed in a solution of phosphate-buffered saline. Data were analyzed using analysis of variance (ANOVA) and Tukey's test. RESULTS: FT-IR test before and after storage for 30 days on three media showed a stable chemical structure with the same functional groups. ANOVA analysis showed a significant difference (p < 0.05) in the dry condition (p = 0.006), Tukey's test showed a significant difference in the 15 kGy and 25 kGy irradiated groups (p = 0.005), but the groups without irradiation had no significant difference with the 15 kGy (p = 0.285) and 25 kGy (p = 0.079) irradiation groups. In wet conditions, there was no significant difference (p > 0.05) in each group (p = 373). The size of the porosity in the group without irradiation, 15 kGy irradiation, and 25 kGy irradiation showed a size of 4.65, 6.51, and 8.08 m, respectively. The degradability test showed a decrease in weight in each group, with the total weight of the membrane being completely degraded from the most degraded to the least: the groups without irradiation, 15 kGy irradiation, and 25 kGy irradiation. The ANOVA test on the degradability test shows significant (p < 0.05) in the PVA-collagen-HA composite membrane group over time intervals (p = 0.000). Tukey's post hoc test showed a significant difference (p < 0.05) after 1 week between the groups without irradiation with 15 kGy (p = 0.023). CONCLUSION: PVA-collagen-HA composite membrane has a stable chemical structure, optimal tensile strength, porosity, and ideal degradability as guided bone regeneration and guided tissue regeneration.

The Effect of Gamma-Ray Irradiation on the Physical, Mechanical, and Morphological Characteristics of PVA-Collagen-Chitosan as a Guided Tissue Regeneration (GTR) Membrane Material.

OBJECTIVE: The aim of this study was to evaluate the effect of gamma-ray irradiation on the physical, mechanical, and morphological characteristics of the polyvinyl alcohol (PVA)-collagen-chitosan membranes as a guided tissue regeneration membrane material. MATERIAL AND METHOD: The membrane was fabricated by mixing PVA, collagen, and chitosan using the film casting method. PVA-collagen-chitosan membranes were irradiated with various radiation dose (0, 15, and 25 kGy). Furthermore, it is characterized using Fourier-transform infrared (FTIR) for functional group identification, morphological test was performed using scanning electron microscopy (SEM), and mechanical properties (i.e., tensile strength and elongation) were evaluated using universal testing machine and swelling studies. STATISTICAL ANALYSIS: Statistical analysis was performed based on analysis of variance and post hoc with p-value < 0.05. RESULT: The FTIR spectrum shows various peaks of functional groups from the PVA-collagen-chitosan membrane. The result of the statistical analysis show changes in tensile strength (p = 0.0004) and membrane elongation (p = 0.000451) at different radiation doses of 0, 15, and 25 kGy. The membrane absorption obtains p-value of 0.0193, while the SEM results show that the PVA-collagen-chitosan membrane homogeneously mixed. CONCLUSION: There is an effect of gamma-ray irradiation on tensile strength, elongation, and water absorption of the membranes. Increasing the radiation dose increases the value of tensile strength, while elongation and absorption of the membrane decrease. The PVA-collagen-chitosan membrane has the potential to develop as an alternative membrane for guided tissue regeneration.

Effects of bone types, particle sizes, and gamma irradiation doses in feline demineralized freeze-dried bone allograft.

BACKGROUND AND AIM: Fracture cases significantly increase recently, demanding high quality of bone graft materials. This research aimed to evaluate the effects of bone types, particle sizes, and gamma irradiation doses on morphological performance and cell viability of feline demineralized freeze-dried bone allograft (DFDBA) through an in vitro study. MATERIALS AND METHODS: Feline DFDBA derived from feline cortical and cancellous long bones was processed into four different sizes: Group A (larger than 1000 µm), B (841-1000 µm), C (420-840 µm), and D (250-419 µm) for each type of bones. The materials were then irradiated with two doses of gamma rays, 15 and 25 kGy, resulting in 16 variants of feline DFDBA. The surfaces of each material were then observed with the scanning electron microscope (SEM). The in vitro evaluation of feline DFDBA was then performed using 3-(4,5-dimethythiazol-2)-2,5-diphenyltetrazolium bromide (MTT) assay with calf pulmonary artery endothelial cells. RESULTS: The MTT assay results showed that the lowest inhibition rate (14.67±9.17 %) achieved by feline DFDBA in Group A derived from cortical bones irradiated with 15 kGy. Group D generally showed high inhibition rate in both cancellous and cortical bones, irradiated with either 15 or 25 kGy. The SEM results showed that cancellous and cortical bones have numerous macropores and micropores structure in 170× and 3000×, respectively. CONCLUSION: The material derived from cortical bones in Group A (larger than 1000 µm in particle size) irradiated with 15 kGy is the best candidate for further development due to its abundance of micropores structure and ability in preserving the living cells.

Periodontal Ligament Cell Sheets and RGD-Modified Chitosan Improved Regeneration in the Horizontal Periodontal Defect Model.

OBJECTIVE: The aim of this study was to examine the potential of periodontal ligament (PDL) cells sheet and arginine-glycyl-aspartic acid (RGD)-modified chitosan scaffold for periodontal tissue regeneration in horizontal periodontal defect model. MATERIALS AND METHODS: PDL cell cytotoxicity was tested with 3-[4,5- dimethylthiazol-2yl]-2,5-diphenyl-2H-tetrazolium bromide assay. Cell migration toward the chitosan-based materials was analyzed with trans-well migration assay. Horizontal periodontal defect model was created in four maxillary and mandibular lateral incisors of Macaque nemestrina. Following periodontal therapy, the sites were transplanted with various regenerative materials: (1) chitosan, (2) RGD-modified chitosan, (3) PDL cell sheet with chitosan, (4) PDL cell sheet with RGD-modified chitosan. The periodontal tissue regeneration was evaluated clinically and radiographically. Gingival crevicular fluids were collected each week to evaluate cementum protein-1 (CEMP-1) expression with enzyme-linked immunosorbent assay, while the biopsies were retrieved after 4 weeks for histological and microcomputed tomography evaluation. STATISTICAL ANALYSIS: Data was statistically analyzed using GraphPad Prism 6 for MacOS X. Normality was tested using the Shapiro-Wilk normality test. The Kruskal-Wallis test was used to compare the groups. Significance was accepted when p < 0.05. RESULTS: Clinical examination revealed more epithelial attachment was formed in the group with PDL cell sheet with RGD-modified chitosan. Similarly, digital subtraction radiography analysis showed higher gray scale, an indication of higher alveolar bone density surrounded the transplanted area, as well as higher CEMP-1 protein expression in this group. The incorporation of RGD peptide to chitosan scaffold in the group with or without PDL cells sheet reduced the distance of cement-enamel junction to the alveolar bone crest; hence, more periodontal tissue formed. CONCLUSIONS: Horizontal periodontal defect model could be successfully created in M. nemestrina model. Combination of PDL cell sheet and RGD-modified chitosan resulted in the higher potential for periodontal tissue regeneration. The results of this study highlight the PDL cell sheet and RGD-modified chitosan as a promising approach for future clinical use in periodontal regeneration.

The impact of the International Atomic Energy Agency (IAEA) program on radiation and tissue banking in Indonesia.

In 1986, the National Nuclear Energy Agency (Batan) in Jakarta started the research and development for the setting up of a tissue bank (Batan Research Tissue Bank/BRTB) by preserving fresh amnion or fetal membranes by lyophilisation and then sterilising by gamma irradiation. During the period of 1990 and 2000, three more tissue banks were set up, i.e., Biomaterial Centre in Surabaya, Jamil Tissue Bank in Padang, and Sitanala Tissue Bank in Tangerang. In 1994, BRTB produced bone allografts. The banks established under the IAEA program concentrated its work on the production of amnion, bone and soft tissues allografts, as well as bone xenografts. These tissues (allografts and xenografts) were sterilised using gamma irradiation (about 90%) and the rest were sterilized by ETO and those products have been used in the treatment of patients at more than 50 hospitals in Indonesia. In 2004, those tissue banks produced 8,500 grafts and 5,000 of them were amnion grafts for eye treatment and wound dressing. All of those grafts were used for patients as well as for research. In 2006, the production increased to 9,000 grafts. Although the capacity of those banks can produce more grafts, we are facing problems on getting raw materials from suitable donors. To fulfill the demand of bone grafts we also produced bone xenografts. The impact of the IAEA program in tissue banking activities in Indonesia can be summarised as follows: to support the national program on importing substitutes for medical devices. The price of imported tissues are between US$ 50 and US$ 6,000 per graft. Local tissue bank can produce tissues with the same quality with the price for about 10-30% of the imported tissues.

Fabrication and Characterization of Chitosan-Collagen Membrane from Barramundi (Lates Calcarifer) Scales for Guided Tissue Regeneration.

OBJECTIVES: The aim of this study was to evaluate the synthesis, mechanical strength, and morphology of chitosan-collagen membranes from barramundi scales for guided tissue regeneration technique. MATERIALS AND METHODS: Collagen was extracted from barramundi scales by immersion in acetic acid. The resulting wet collagen was later dried. The membrane was fabricated by mixing chitosan with collagen from barramundi scales. Membrane characterization parameters were measured using Fourier transform infrared (FTIR), scanning electron microscopy (SEM) and mechanical property. RESULTS: The FTIR spectrum showed the typical peak of the mixture of chitosan and collagen. The tensile strength and elongation at break of the membrane in dry condition were 0.28 MPa and 8.53%, respectively, while in the wet condition these were 0.12 MPa and 25.6%. The membrane porosity test result was 38.85%; SEM result showed a porous membrane surface with size varying around 16 to 100 µm. CONCLUSION: The chitosan-collagen membrane from the barramundi scale showed the fibrous membrane surface that has ideal porous size as guided tissue regeneration membrane and the lower mechanical strength.