A novel non-toxic biodegradable lysine-di-isocyanate (LDI)-based urethane polymer was developed for use in tissue engineering applications. This matrix was synthesized with highly purified LDI made from the lysine diethylester. The ethyl ester of LDI was polymerized with glycerol to form a prepolymer. LDI-glycerol prepolymer when reacted with water foamed with the liberation of CO 2 to provide a pliable spongy urethane polymer. The LDI-glycerol matrix degraded in aqueous solutions at 100, 37, 22, and 4°C at a rate of 27.7, 1.8, 0.8, and 0.1 mM per 10 days, respectively. Its thermal stability in water allowed its sterilization by autoclaving. The degradation of the LDI-glycerol polymer yielded lysine, ethanol, and glycerol as breakdown products. The degradation products of LDI-glycerol polymer did not significantly affect the pH of the solution. The glass transition temperature (T g ) of this polymer was found to be 103.4°C. The physical properties of the polymer network were found to be adequate to support the cell growth in vitro, as evidenced by the fact that rabbit bone marrow stromal cells (BMSC) attached to the polymer matrix and remained viable on its surface. Culture of BMSC on LDI-glycerol matrix for long durations resulted in the formation of multilayered confluent cultures, a characteristic typical of bone cells. Furthermore, cells grown on LDI-glycerol matrix did not differ phenotypically from the cells grown on the tissue culture polystyrene plates as assessed by the cell growth, and expression of mRNA for collagen type I, and transforming growth factor-β1 (TGF-β1). The observations suggest that biodegradable peptide-based urethane polymers can be synthesized which may pave their way for possible use in tissue engineering applications.

Objective. The mechanisms by which chondrocytes convert biomechanical signals into intracellular biochemical events are not well understood. In this study, we sought to determine the intracellular mechanisms of the magnitude-dependent actions of mechanical signals.Methods. Chondrocytes isolated from rabbit articular cartilage were grown on flexible membranes. Cells were subjected to cyclic tensile strain (CTS) of various magnitudes in the presence or absence of interleukin-1␤ (IL-1␤), which was used as a proinflammatory signal for designated time intervals. The regulation of NF-B was measured by reverse transcriptasepolymerase chain reaction, electrophoretic mobility shift assay, and immunofluorescence.Results. CTS of low magnitudes (4-8% equibiaxial strain) was a potent inhibitor of IL-1␤-dependent NF-B nuclear translocation. Cytoplasmic retention of NF-B and reduction of its synthesis led to sustained suppression of proinflammatory gene induction. In contrast, proinflammatory signals generated by CTS of high magnitudes (15-18% equibiaxial strain) mimicked the actions of IL-1␤ and induced rapid nuclear translocation of NF-B subunits p65 and p50.Conclusion. Magnitude-dependent signals of mechanical strain utilize the NF-B transcription factors as common elements to abrogate or aggravate proinflammatory responses. Furthermore, the intracellular events induced by mechanical overload are similar to those that are initiated by proinflammatory cytokines in arthritis.

Macrophages respond to bacterial lipopolysaccharides (LPS) and activate several host defense functions through production of mediators. However, it is not clear whether the degree of macrophage responsiveness to different sources of LPS is equivalent to or varies with the source of LPS. Therefore, in this report, we examined the extent of the human monocyte response to LPS derived from two oral pathogens, Actinobacillus actinomycetemcomitans (Aa) and Porphyromonas gingivalis (Pg). Additionally, due to its well-established ability to activate monocytes, we used LPS from Escherichia coli (Ec). Human monocytes, when activated with a specific source of LPS, exhibited rapid expression of mRNA for IL-1 beta, TNF-alpha, and IL-8, which was followed by IL-6, as measured by RNA-PCR. Moreover, the expression of mRNA for these cytokines was followed by cytokine synthesis. Monocytes from the same subject, when activated with LPS from Pg, Aa, or Ec expressed quantitatively different levels of mRNA and proteins for all four cytokines. A given LPS induced either high or low expression of the battery of cytokines tested, indicating that the expression of these pro-inflammatory cytokines may be regulated by a single or a cluster of gene(s). However, no apparent differences in the time course of mRNA expression for these cytokines were observed in response to any of the LPS tested. Furthermore, the relative ability of the different sources of LPS to induce mRNA for cytokines varied throughout a wide range of LPS concentrations. This suggests that differences exist in the sensitivity of monocytes to a specific LPS, rather than in the kinetics of the secretory process itself.(ABSTRACT TRUNCATED AT 250 WORDS)

Mechanical signals play an integral role in bone homeostasis. These signals are observed at the interface of bone and teeth, where osteoblast-like periodontal ligament (PDL) cells constantly take part in bone formation and resorption in response to applied mechanical forces. Earlier, we reported that signals generated by tensile strain of low magnitude (TENS-L) are antiinflammatory, whereas tensile strain of high magnitude (TENS-H) is proinflammatory and catabolic. In this study, we examined the mechanisms of intracellular actions of the antiinflammatory and proinflammatory signals generated by TENS of various magnitudes. We show that both low and high magnitudes of mechanical strain exploit nuclear factor (NF)-kappaB as a common pathway for transcriptional inhibition/activation of proinflammatory genes and catabolic processes. TENS-L is a potent inhibitor of interleukin (IL)-1 beta-induced I-kappaBbeta degradation and prevents dissociation of NF-kB from cytoplasmic complexes and thus its nuclear translocation. This leads to sustained suppression of IL-1beta-induced NF-kappaB transcriptional regulation of proinflammatory genes. In contrast, TENS-H is a proinflammatory signal that induces I-kappaBbeta degradation, nuclear translocation of NF-kappaB, and transcriptional activation of proinflammatory genes. These findings are the first to describe the largely unknown intracellular mechanism of action of applied tensile forces in osteoblast-like cells and have critical implications in bone remodeling.

The Amelogenesis Imperfecta (AI) are a group of clinically and genetically heterogeneous disorders that affect enamel formation. To date, mutations in 4 genes have been reported in various types of AI. Mutations in the genes encoding the 2 enamel proteases, matrix metalloproteinase 20 (MMP20) and kallikrein 4 (KLK4), have each been reported in a single family segregating autosomal-recessive hypomaturation AI. To determine the frequency of mutations in these genes, we analyzed 15 Turkish probands with autosomal-recessive hypomaturation AI for MMP20 and KLK4 gene mutations. No KLK4 mutations were found. A novel MMP20 mutation (g.16250T>A) was found in one family. This missense mutation changed the conserved active-site His226 residue of the zinc catalytic domain to Gln (p.H226Q). Zymogram analysis demonstrated that this missense mutation abolished MMP20 proteolytic activity. No MMP20 mutations were found in the remaining 14 probands, underscoring the genetic heterogeneity of hypomaturation AI.

Applied mechanical loading induces inflammation in the periodontal ligament (PDL). However, the mechanisms involved in bone deposition at tension sites in an inflammatory environment are not clear. Here, in an in vitro model system, we show that equibiaxial tensile strain of low magnitude (TENS) provokes potent anti-inflammatory signals in PDL cells. TENS inhibits IL-1beta-induced synthesis of IL-1beta, IL-6, and IL-8 by inhibiting their mRNA expression, and thus significantly suppresses the amplification of IL-1beta-induced inflammatory responses in PDL cells. Additionally, as an anti-inflammatory signal, TENS induces IL-10 synthesis in the presence and absence of IL-1beta. These observations are the first to demonstrate that TENS antagonizes IL-1beta actions on PDL cells by (i) inhibiting IL-1beta-induced transcriptional regulation of proinflammatory cytokines, and (ii) inducing synthesis of IL-10, which may post-transcriptionally suppress the synthesis of pro-inflammatory cytokines.

Intracellular signals generated by mechanical strain profoundly affect the metabolic function of osteoblast-like periodontal ligament (PDL) cells, which reside between the tooth and alveolar bone. In response to applied mechanical forces, PDL cells synthesize bone-resorptive cytokines to induce bone resorption at sites exposed to compressive forces and deposit bone at sites exposed to tensile forces in an environment primed for catabolic processes. The intracellular mechanisms that regulate this bone remodeling remain unclear. Here, in an in vitro model system, we show that tensile strain is a critical determinant of PDL-cell metabolic functions. Equibiaxial tensile strain (TENS), when applied at low magnitudes, acts as a potent antagonist of interleukin (IL)-1β actions and suppresses transcriptional regulation of multiple proinflammatory genes. This is evidenced by the fact that TENS at low magnitude: (i) inhibits recombinant human (rh)IL-1β-dependent induction of cyclooxygenase-2 (COX-2) mRNA expression and production of prostaglandin estradiol (PGE 2 ); (ii) inhibits rhIL-1β-dependent induction matrix metalloproteinase-1 (MMP-1) and MMP-3 synthesis by suppressing their mRNA expression; (iii) abrogates rhIL-1β-induced suppression of tissue inhibitor of metalloprotease-II (TIMP-II) expression; and (iv) reverses IL-1β-dependent suppression of osteocalcin and alkaline phosphatase synthesis. Nevertheless, these actions of TENS were observed only in the presence of IL-1β, as TENS alone failed to affect any of the aforementioned responses. The present findings are the first to show that intra-cellular signals generated by low-magnitude mechanical strain interfere with one or more critical step(s) in the signal transduction cascade of rhIL-1β upstream of mRNA expression, while concurrently promoting the expression of osteogenic proteins in PDL cells.

We have examined the ability of gingival fibroblasts (GF) to participate in inflammatory response and function as accessory immune cells. The accessory immune function of GF cells was evaluated by their ability to elaborate proinflammatory cytokines following stimulation with lipopolysaccharides and interleukin-1 beta (IL-1 beta). Using three separate clonally derived and characterized human gingival fibroblast (GF) cell lines, we demonstrate that LPS from Actinobacillus actinomycetemcomitans (Aa) and Escherichia coli (Ec) induce mRNA and synthesis of proinflammatory cytokines, IL-1 beta, IL-6 and IL-8. IL-1 beta activation of GF cells showed that IL-1 beta non only induces the expression of IL-6, IL-8 and TNF-alpha, but also acts in an autocrine manner of GF cells and induces IL-1 beta expression. Furthermore, the continuous presence of IL-1 beta in GF cell cultures did not down regulate the response of GF cells to IL-1 beta. Pretreatment of GF cells with IL-1 beta resulted in the enhanced synthesis of TNF-alpha in response to additional IL-1 beta. These findings indicate that GF cells, in addition to providing structural support, may also function as accessory immune cells and play an important role in the initial inflammatory reaction as well as in the amplification of immune response.