Thermoplastic bioactive composite – with special reference to dissolution behaviour and tissue response

Bioactive glasses are surface-active ceramic materials which support and accelerate bone growth in the body. During the healing of a bone fracture or a large bone defect, fixation is often needed. The aim of this thesis was to determine the dissolution behaviour and biocompatibility of a composite consisting of poly(ε-caprolactone-co-DL-lactide) and bioactive glass (S53P4). In addition the applicability as an injectable material straight to a bone defect was assessed. In in vitro tests the dissolution behaviour of plain copolymer and composites containing bioactive glass granules was evaluated, as well as surface reactivity and the material’s capability to form apatite in simulated body fluid (SBF). The human fibroblast proliferation was tested on materials in cell culture. In in vivo experiments, toxicological tests, material degradation and tissue reactions were tested both in subcutaneous space and in experimental bone defects. The composites containing bioactive glass formed a unified layer of apatite on their surface in SBF. The size and amount of glass granules affected the degradation of polymer matrix, as well the material’s surface reactivity. In cell culture on the test materials the human gingival fibroblasts proliferated and matured faster compared with control materials. In in vitro tests a connective tissue capsule was formed around the specimens, and became thinner in the course of time. Foreign body cell reactions in toxicological tests were mild. In experimental bone defects the specimens with a high concentration of small bioactive glass granules (<45 μm) formed a dense apatite surface layer that restricted the bone ingrowth to material. The range of large glass granules (90-315 μm) with high concentrations formed the best bonding with bone, but slow degradation on the copolymer restricted the bone growth only in the superficial layers. In these studies, the handling properties of the material proved to be good and tissue reactions were mild. The reactivity of bioactive glass was retained inside the copolymer matrix, thus enabling bone conductivity with composites. However, the copolymer was noticed to degradate too slowly compared with the bone healing. Therefore, the porosity of the material should be increased in order to improve tissue healing.

Unidirectional fiber-reinforced composite as an oral implant abutment material: Experimental studies of E-glass fiber/BisGMA-TEGDMA polymer in vitro

Fiber-reinforced composites (FRCs) are a new group of non-metallic biomaterials showing a growing popularity in many dental and medical applications. As an oral implant material, FRC is biocompatible in bone tissue environment. Soft tissue integration to FRC polymer material is unclear. This series of in vitro studies aimed at evaluating unidirectional E-glass FRC polymer in terms of mechanical, chemical, and biological properties in an attempt to develop a new non-metallic oral implant abutment alternative. Two different types of substrates were investigated: (a) Plain polymer (BisGMA 50%–TEGDMA 50%) and (b) Unidirectional FRC. The mechanical behavior of high fiber-density FRCs was assessed using a three-point bending test. Surface characterization was performed using scanning electron and spinning disk confocal microscopes. The surface wettability/energy was determined using sessile drop method. The blood response, including blood-clotting ability and platelet morphology was evaluated. Human gingival fibroblast cell responses - adhesion kinetics, adhesion strength, and proliferation activity - were studied in cell culture environment using routine test conditions. A novel tissue culture method was developed and used to evaluate porcine gingival tissue graft attachment and growth on the experimental composite implants. The analysis of the mechanical properties showed that there is a direct proportionality in the relationship between E-glass fiber volume fraction and toughness, modulus of elasticity, and load bearing capacity; however, flexural strength did not show significant improvement when high fiber-density FRC is used. FRCs showed moderate hydrophilic properties owing to the presence of exposed glass fibers on the polymer surface. Blood-clotting time was shorter on FRC substrates than on plain polymer. The FRC substrates also showed higher platelet activation state than plain polymer substrates. Fibroblast cell adhesion strength and proliferation rate were highly pronounced on FRCs. A tissue culture study revealed that gingival epithelium and connective tissue established an immediate close contact with both plain polymer and FRC implants. However, FRC seemed to guide epithelial migration outwards from the tissue/implant interface. Due to the anisotropic and hydrophilic nature of FRC, it can be concluded that this material enhances biological events related with soft tissue integration on oral implant surface.

Pain-evoked alterations on gingival blood flow and gingival crevicular fluid (GCF) neuropeptide SP and collagenase-2 (MMP-8) levels

Previous studies have demonstrated that clinical pulpal pain can induce the expression of pro-inflammatory neuropeptides in the adjacent gingival crevice fluid (GCF). Vasoactive agents such as substance P (SP) are known to contribute to the inflammatory type of pain and are associated with increased blood flow. More recent animal studies have shown that application of capsaicin on alveolar mucosa provokes pain and neurogenic vasodilatation in the adjacent gingiva. Pain-associated inflammatory reactions may initiate expression of several pro- and anti-inflammatory mediators. Collagenase-2 (MMP-8) has been considered to be the major destructive protease, especially in the periodontitis-affected gingival crevice fluid (GCF). MMP-8 originates mostly from neutrophil leukocytes, the first line of defence cells that exist abundantly in GCF, especially in inflammation. With this background, we wished to clarify the spatial extensions and differences between tooth-pain stimulation and capsaicin-induced neurogenic vasodilatation in human gingiva. Experiments were carried out to study whether tooth stimulation and capsaicin stimulation of alveolar mucosa would induce changes in GCF MMP-8 levels and whether tooth stimulation would release neuropeptide SP in GCF. The experiments were carried out on healthy human volunteers. During the experiments, moderate and high intensity painful tooth stimulation was performed by a constant current tooth stimulator. Moderate tooth stimulation activates A-delta fibres, while high stimulation also activates C-fibres. Painful stimulation of the gingiva was achieved by topical application of capsaicin-moistened filter paper on the mucosal surface. Capsaicin is known to activate selectively nociceptive C-fibres of stimulated tissue. Pain-evoked vasoactive changes in gingivomucosal tissues were mapped by laser Doppler imaging (LDI), which is a sophisticated and non-invasive method for studying e.g. spatial and temporal characteristics of pain- and inflammation-evoked blood flow changes in gingivomucosal tissues. Pain-evoked release of MMP-8 in GCF samples was studied by immunofluorometric assay (IFMA) and Western immunoblotting. The SP levels in GCF were analysed by Enzyme immunoassay (EIA). During the experiments, subjective stimulus-evoked pain responses were determined by a visual analogue pain scale. Unilateral stimulation of alveolar mucosa and attached gingiva by capsaicin evoked a distinct neurogenic vasodilatation in the ipsilateral gingiva, which attenuated rapidly at the midline. Capsaicin stimulation of alveolar mucosa provoked clear inflammatory reactions. In contrast to capsaicin stimuli, tooth stimulation produced symmetrical vasodilatations bilaterally in the gingiva. The ipsilateral responses were significantly smaller during tooth stimulation than during capsaicin stimuli. The current finding – that tooth stimulation evokes bilateral vasodilatation while capsaicin stimulation of the gingiva mainly produces unilateral vasodilatation – emphasises the usefulness of LDI in clarifying spatial features of neurogenic vasoactive changes in the intra-oral tissues. Capsaicin stimulation of the alveolar mucosa induced significant elevations in MMP-8 levels and activation in GCF of the adjacent teeth. During the experiments, no marked changes occurred in MMP-8 levels in the GCF of distantly located teeth. Painful stimulation of the upper incisor provoked elevations in GCF MMP-8 and SP levels of the stimulated tooth. The GCF MMP-8 and SP levels of the non-stimulated teeth were not changed. These results suggest that capsaicin-induced inflammatory reactions in gingivomucosal tissues do not cross the midline in the anterior maxilla. The enhanced reaction found during stimulation of alveolar mucosa indicates that alveolar mucosa is more sensitive to chemical irritants than the attached gingiva. Analysis of these data suggests that capsaicin-evoked neurogenic inflammation in the gingiva can trigger the expression and activation of MMP-8 in GCF of the adjacent teeth. In this study, it is concluded that experimental tooth pain at C-fibre intensity can induce local elevations in MMP-8 and SP levels in GCF. Depending on the role of MMP-8 in inflammation, in addition to surrogated tissue destruction, the elevated MMP-8 in GCF may also reflect accelerated local defensive and anti-inflammatory reactions.

The regulation and function of collagenase-3 (MMP-13) in cutaneous wound healing and squamous cell carcinoma

Matrix metalloproteinase-13 (MMP-13) is a potent proteolytic enzyme, whose expression has been previously associated with fetal bone development and postnatal bone remodeling and with adult gingival wound healing. MMP-13 is also known to be involved in the growth and invasion of various cancers including squamous cell carcinoma (SCC) of the skin. The aim of this study was to further elucidate the function and regulation of MMP-13 in wound repair and cancer. In this study, it was shown that fetal skin fibroblasts express MMP-13 in response to transforming growth factor-β in a p38 MAP kinase dependent manner. In addition, MMP-13 was found to be expressed in vivo by wound fibroblasts in human fetal skin grafted on SCID mice. Adenovirally delivered expression of MMP-13 enhanced collagen matrix contraction by fibroblasts in vitro in association with altered cytoskeletal structure, enhanced proliferation and survival. These results indicate that MMP-13 is involved in cell-mediated collagen matrix remodeling and suggest a role for MMP-13 in superior matrix remodeling and scarless healing of fetal skin wounds. Using an MMP-13 deficient mouse strain, it was shown that MMP-13 is essential for the normal development of experimental granulation tissue in mice. MMP-13 was implicated in the regulation of myofibroblast function and angiogenesis and the expression of genes involved in cellular proliferation and movement, immune response, angiogenesis and proteolysis. Finally, epidermal mitogen, keratinocyte growth factor (KGF) was shown to suppress the malignant properties of skin SCC cells by downregulating the expression of several target genes with potential cancer promoting properties, including MMP-13, and by reducing SCC cell invasion. These results provide evidence that MMP-13 potently regulates cell viability, myofibroblast function and angiogenesis associated with wound healing and cancer. In addition, fibroblasts expressing MMP-13 show high collagen reorganization capacity. Moreover, the results suggest that KGF mediates the anti-cancer effects on skin SCC