OBJECTIVES: This study investigated the indirect effect of calcium-enriched mixture (CEM) cement and mineral trioxide aggregate (MTA), as 2 calcium silicate-based hydraulic cements, on human dental pulp stem cells (hDPSCs) through different dentin thicknesses.MATERIALS AND METHODS: Two-chamber setups were designed to simulate indirect pulp capping (IPC). Human molars were sectioned to obtain 0.1-, 0.3-, and 0.5-mm-thick dentin discs, which were placed between the 2 chambers to simulate an IPC procedure. Then, MTA and CEM were applied on one side of the discs, while hDPSCs were cultured on the other side. After 2 weeks of incubation, the cells were removed, and cell proliferation, morphology, and attachment to the discs were evaluated under scanning electron microscopy (SEM). Energy-dispersive X-ray (EDXA) spectroscopy was performed for elemental analysis. Alkaline phosphatase (ALP) activity was assessed quantitatively. The data were analyzed using the Kruskal-Wallis and Mann-Whitney tests.RESULTS: SEM micrographs revealed elongated cells, collagen fibers, and calcified nucleations in all samples. EDXA verified that the calcified nucleations consisted of calcium phosphate. The largest calcifications were seen in the 0.1-mm-thick dentin subgroups. There was no significant difference in ALP activity across the CEM subgroups; however, ALP activity was significantly lower in the 0.1-mm-thick dentin subgroup than in the other MTA subgroups (p < 0.05).CONCLUSIONS: The employed capping biomaterials exerted biological activity on hDPSCs, as shown by cell proliferation, morphology, and attachment and calcific precipitations, through 0.1- to 0.5-mm-thick layers of dentin. In IPC, the bioactivity of these endodontic biomaterials is probably beneficial.
Alkaline Phosphatase ; Biocompatible Materials ; Calcium ; Cell Proliferation ; Collagen ; Dental Pulp Capping ; Dental Pulp ; Dentin ; Endodontics ; Humans ; Microscopy, Electron, Scanning ; Miners ; Molar ; Pemetrexed ; Spectrum Analysis ; Stem Cells

This study aimed to obtain a recombinant human-source collagen for industrialization. First, based on the Gly-X-Y sequence of human type I collagen, we optimized the hydrophilic Gly-X-Y collagen peptide, designed the human collagen amino acid sequence and the corresponding nucleotide sequence. Next, the expression vector pPIC9K-COL was constructed via endonuclease digestion technology. We obtained an engineering strain of human-source collagen by electrotransforming Pichia pastoris, and then it was fermented, purified and identified. As a result, the expression level reached 4.5 g/L and the purity was over 95%. After amino acid N-terminal sequencing, molecular weight analysis, amino acid analysis and collagenase degradation test, we confirmed that the obtained collagen was consistent with designed primary structure of human-source collagen. After freeze-drying, we analyzed the collagen by scanning electron microscope and cell cytotoxicity, confirming that the collagen has porous fiber reticular structure and superior cytocompatibility. This indicates that human-source collagen has potential to be applied as biomedical material. In conclusion, we successfully obtained the expected human-source collagen and laid a foundation to its further application.
Amino Acid Sequence ; Biocompatible Materials ; Collagen ; analysis ; Freeze Drying ; Humans ; Pichia ; Recombinant Proteins

OBJECTIVE: Graft rejection, with the possibility of a violent immune response, may be severe and life threatening. Our aim was to thoroughly investigate the biocompatibility and immunotoxicology of collagen-based dermal matrix (DM) before assessment in clinical trials. METHODS: DM was subcutaneously implanted in BALB/c mice in two doses to induce a potential immune response. The spleen and lymph nodes were assessed for shape, cell number, cell phenotype via flow cytometry, cell activation via CCK8 kit, Annexin V kit, and Ki67 immunostaining. Serum samples were used to measure antibody concentration by enzyme-linked immunosorbent assay. Local inflammation was analyzed by histology and immunohistochemistry staining. Data analysis was performed by one-way ANOVA and non-parametric tests. RESULTS: Our data illustrate that the spleen and lymph node sizes were similar between the negative control mice and mice implanted with DM. However, in the high-dose DM (DM-H) group, the total cell populations in the spleen and lymph nodes, T cells and B cells in the spleen had slight increases in prophase, and the low-dose DM (DM-L) group did not display gross abnormities. Moreover, DM-H initiated moderate cell activation and proliferation in the early phase post-immunization, whereas DM-L did not. Neither DM-H nor DM-L implantation noticeably increased IgM and IgG serum concentrations. Examination of the local cellular response revealed only benign cell infiltration and TNF-α expression in slides of DM in the early phase. CONCLUSION Overall, DM-H may have induced a benign temporary acute immune response post-implantation, whereas DM-L had quite low immunogenicity. Thus, this DM can be regarded as a safe product.
Animals ; Biocompatible Materials ; adverse effects ; analysis ; Collagen ; adverse effects ; immunology ; Dermis ; immunology ; surgery ; Female ; Flow Cytometry ; Immunity, Cellular ; Lymph Nodes ; immunology ; Mice ; Mice, Inbred BALB C ; Prostheses and Implants ; adverse effects ; Spleen ; immunology

PURPOSE: We aimed to investigate the gene expression of human gingival fibroblasts on microgroove surface using DNA microarray. MATERIALS AND METHODS: Microgrooves were applied on grade II titanium discs to have 0/0 µm (NE0, control group), 60/10 µm (E60/10, experimental group) of respective width/depth by photolithography. The entire surface of the microgrooved Ti substrata was further acid etched and used as the two experimental groups in this study. Human gingival fibroblasts were cultured in the experimental group and the control group, and total RNA was extracted. The oligonucleotide microarray was performed to confirm the changes of various gene expression levels between experimental group and control group. Changes of gene expression level were determined at the pathway level by mapping the expression results of DNA chips, using the KEGG (Kyoto Encyclopedia of Genes and Genomes) pathway analysis. RESULTS: Gene expression levels on E60/10 and NE0 were analyzed, there were 123 genes showing significant differences in expression more than 1.5 times on E60/10 microgrooved surface compared to NE0 surface, and 19 genes showing significant differences in expression more than 2 times. The KEGG pathway analysis confirmed the changes in gene expression levels under experimental conditions. Cell signaling, proliferation, and activity among the various gene expression results were identified. CONCLUSION: Microgrooved surfaces induce gene expression changes and related cell signaling. According to the results of this study, microgrooves can be used as the surface of various biomaterials which need to improve cell activity through gene expression changes and activation of cell signaling.
Biocompatible Materials ; DNA* ; Fibroblasts* ; Gene Expression* ; Humans* ; Oligonucleotide Array Sequence Analysis* ; RNA ; Titanium

OBJECTIVE: In addition to bone bridging inside a cage or graft (intragraft bone bridging, InGBB), extragraft bone bridging (ExGBB) is commonly observed after anterior cervical discectomy and fusion (ACDF) with a stand-alone cage. However, solid bony fusion without the formation of ExGBB might be a desirable condition. We hypothesized that an insufficient contact area for InGBB might be a causative factor for ExGBB. The objective was to determine the minimal area of InGBB by finite element analysis. METHODS: A validated 3-dimensional, nonlinear ligamentous cervical segment (C3–7) finite element model was used. This study simulated a single-level ACDF at C5–6 with a cylindroid interbody graft. The variables were the properties of the incorporated interbody graft (cancellous bone [Young’s modulus of 100 or 300 MPa] to cortical bone [10000 MPa]) and the contact area between the vertebra and interbody graft (Graft-area, from 10 to 200 mm²). Interspinous motion between the flexion and extension models of less than 2 mm was considered solid fusion. RESULTS: The minimal Graft-areas for solid fusion were 190 mm², 140 mm², and 100 mm² with graft properties of 100, 300, and 10000 MPa, respectively. The minimal Graft-areas were generally unobtainable with only the formation of InGBB after the use of a commercial stand-alone cage. CONCLUSION: ExGBB may be formed to compensate for insufficient InGBB. Although various factors may be involved, solid fusion with less formation of ExGBB may be achieved with refinements in biomaterials, such as the use of osteoinductive cage materials; changes in cage design, such as increasing the area of polyetheretherketone or the inside cage area for bone grafts; or surgical techniques, such as the use of plate/screw systems.
Biocompatible Materials ; Diskectomy* ; Finite Element Analysis* ; Ligaments ; Spinal Fusion ; Spine ; Transplants

Deformities in tissues and organs can be treated by using tissue engineering approach offering the development of biologically functionalized scaffolds from a variety of polymer blends which mimic the extracellular matrix and allow adjusting the material properties to meet the defect architecture. In recent years, research interest has been shown towards the development of chitosan (CS) based biomaterials for tissue engineering applications, because of its minimal foreign body reactions, intrinsic antibacterial property, biocompatibility, biodegradability and ability to be molded into various geometries and forms thereby making it suitable for cell ingrowth and conduction. The present work involves the fabrication of nanofibrous scaffold from CS and poly(vinyl alcohol) blends by free-surface electrospinning method. The morphology and functional characteristics of the developed scaffolds were assessed by field emission scanning electron microscopy and fourier transformed infra-red spectra analysis. The morphological analysis showed the average fiber diameter was 269 nm and thickness of the mat was 200–300 µm. X-ray diffraction study confirmed the crystalline nature of the prepared scaffolds, whereas hydrophilic characteristic of the prepared scaffolds was confirmed by measured contact angle. The scaffolds possess an adequate biodegradable, swelling and mechanical property that is found desirable for tissue engineering applications. The cell study using umbilical cord blood-derived mesenchymal stem cells has confirmed the in vitro biocompatibility and cell supportive property of the scaffold thereby depicting their potentiality for future clinical applications.
Biocompatible Materials ; Chitosan ; Congenital Abnormalities ; Crystallins ; Extracellular Matrix ; Foreign Bodies ; Fourier Analysis ; Fungi ; In Vitro Techniques ; Mesenchymal Stromal Cells ; Methods ; Microscopy, Electron, Scanning ; Nanofibers* ; Polymers ; Tissue Engineering* ; Umbilical Cord ; X-Ray Diffraction

The aim of this study was to establish an assessment method for determining α-Gal (α-1, 3-galactosyle) epitopes contained in animal tissue or animal tissue-derived biological materials with ELISA inhibition assay. Firstly, a 96 well plate was coated with Gal α-1, 3-Gal/bovine serum albumin (BSA) as a solid phase antigen and meanwhile, the anti-α-Gal M86 was used to react with α-Gal antigens which contained in the test materials. Then, the residual antibodies (M86) in the supernatant of M86-Gal reaction mixture were measured using ELISA inhibition assay by the α-Gal coating plate. The inhibition curve of the ELISA inhibition assay, the R2 = 0.999, was well established. Checking using both α-Gal positive materials (rat liver tissues) and α-Gal negative materials (human placenta tissues) showed a good sensitivity and specificity. Based on the presently established method, the α-Gal expression profile of rat tissues, decellular animal tissue-derived biological materials and porcine dermal before and after decellular treatment were determined. The M86 ELISA inhibition assay method, which can quantitatively determine the α-Gal antigens contained in animal tissues or animal tissue-derived biomaterials, was refined. This M86 specific antibody based-ELISA inhibition assay established in the present study has good sensitivity and specificity, and could be a useful method for determining remnant α-1, 3Gal antigens in animal tissue-derived biomaterials.
Animals ; Antibodies ; Biocompatible Materials ; Enzyme-Linked Immunosorbent Assay ; methods ; Epitopes ; analysis ; Humans ; Rats ; Sensitivity and Specificity ; Serum Albumin, Bovine ; Trisaccharides ; analysis

BACKGROUND: There has been controversy whether methylmethacrylate precoating of the cemented femoral stem is a solution for aseptic loosening or rather contributes to increased failure rates in cemented total hip arthroplasties. METHODS: On a retrospective basis, we analyzed 76 primary hybrid total hip arthroplasties from 63 patients with precoated, cemented femoral stems between October 1990 and December 1995. The mean age of the patients was 46.8 years (range, 22 to 77 years) with a minimum follow-up of 14 years (mean, 15.5 years; range, 14 to 19.5 years). Third generation cementing techniques were employed in all cases. RESULTS: Twenty-four out of 76 cases (31.6%) showed aseptic loosening of the femoral stems, of which 23 stems were revised at an average revision time of 8 years (range, 3 to 14.8 years). The main mode of loosening was cement-stem interface failure in 22 hips (91.7%). Twenty-one out of 24 failed hips (87.5%) demonstrated C2 cementing grades (p < 0.001). Kaplan-Meier survivorship analysis using radiographic aseptic loosening of the femoral stem as the endpoint for failure showed survival rates of 76.5% at 10 years (95% confidence interval [CI], 71.4 to 81.6) and 63.2% at 19 years (95% CI, 57.3 to 69.1). CONCLUSIONS: An early failure of the precoated femoral stem in this study was mainly due to an insufficient cementing technique. Achievement of good cement mantle may improve the survival rates.
Adult ; Aged ; Aged, 80 and over ; Arthroplasty, Replacement, Hip/*instrumentation ; *Coated Materials, Biocompatible ; Equipment Failure Analysis ; Female ; Femur Head Necrosis/diagnosis/surgery ; *Hip Prosthesis ; Humans ; Kaplan-Meier Estimate ; Male ; Methylmethacrylate ; Middle Aged ; Prosthesis Failure ; Retrospective Studies ; Treatment Outcome ; Young Adult

Quantification of residual DNA in animal-derived biological scaffold materials is one of technical specifications for evaluating decellularization process and immunotoxicity risk. Up to now, there have been no standard methods available for quantification of residues DNA in animal-derived biological scaffold materials. In this study, a three-step method, including proteinase K digestion, DNA purification and determination of DNA using fluorescence assay, was designed for residual DNA quantification. A parallel recovery experiment of standard DNA using the same protocol to test article determination was used for adjusting final results of residuul DNA amount. DNA purification based on magnetic beads enabled the experiments to get high accuracy and repeatability. The validation experiment showed that the three-step method had high sensitivity up to 6.25ng of DNA per sample with good linearity (recovery curve R2 > 0.99) in the concentration range of 3. 125-100ng, and 25-400ng per sample. This method is useful for determining micro or trace amount DNA remained in the biomaterials.
Animals ; Biocompatible Materials ; chemistry ; DNA ; analysis ; isolation & purification ; Materials Testing ; methods ; Tissue Scaffolds ; chemistry

OBJECTIVETo investigate whether multiple coatings can improve the bond durability of one-step self-etching adhesive to primary dentin.

METHODSTwelve caries-free human primary molars were randomly divided into 2 groups. In group 1, each tooth was hemisected into 2 halves. One half was assigned to the control subgroup 1, which was bonded with a commercially available one-step self-etching adhesive according to the manufacturer's instructions; the other half was assigned to experimental subgroup 1, in which the adhesive was applied three times before light curing. In group 2, one split half tooth was bonded with a commercially available one-step self-etching adhesive according to the manufacturer's instructions; for the other half, three layers of adhesive were applied with each successive layer of light curing. Specimens were stored in 0.9% NaCl containing 0.02% sodium azide at 37℃ for 18 months and then were subjected to microtensile bond strength test and the fracture mode analysis.

RESULTSWhen the adhesive was applied three times before light curing, the bond strength of the experimental subgroup 1 was significantly higher than that of the control subgroup 1 (47.46∓13.91 vs. 38.12∓11.21 MPa, P<0.05). When using the technique of applying multiple layers of adhesive with each successive layer of light curing, no difference was observed in bond strength between the control subgroup and the experimental subgroup (39.40±8.87 vs. 40.87±9.33 MPa, P>0.05).

CONCLUSIONMultiple coatings can improve the bond durability of one-step self-etching adhesive to primary dentin when using the technique of light-curing after applying 3 layers of adhesive.

Acid Etching, Dental ; methods ; Adhesiveness ; Child ; Coated Materials, Biocompatible ; chemical synthesis ; chemistry ; pharmacology ; Dental Cements ; chemical synthesis ; chemistry ; pharmacology ; Dental Prosthesis ; Dental Restoration Failure ; Dentin ; chemistry ; drug effects ; Dentin-Bonding Agents ; pharmacology ; Electroplating ; methods ; Equipment Failure Analysis ; Humans ; Materials Testing ; Tensile Strength ; drug effects