Excimer laser refractive surgery : corneal wound healing and clinical results

Although the first procedure in a seeing human eye using excimer laser was reported in 1988 (McDonald et al. 1989, O'Connor et al. 2006) just three studies (Kymionis et al. 2007, O'Connor et al. 2006, Rajan et al. 2004) with a follow-up over ten years had been published when this thesis was started. The present thesis aims to investigate 1) the long-term outcomes of excimer laser refractive surgery performed for myopia and/or astigmatism by photorefractive keratectomy (PRK) and laser-in situ- keratomileusis (LASIK), 2) the possible differences in postoperative outcomes and complications when moderate-to-high astigmatism is treated with PRK or LASIK, 3) the presence of irregular astigmatism that depend exclusively on the corneal epithelium, and 4) the role of corneal nerve recovery in corneal wound healing in PRK enhancement. Our results revealed that in long-term the number of eyes that achieved uncorrected visual acuity (UCVA)≤0.0 and ≤0.5 (logMAR) was higher after PRK than after LASIK. Postoperative stability was slightly better after PRK than after LASIK. In LASIK treated eyes the incidence of myopic regression was more pronounced when the intended correction was over >6.0 D and in patients aged <30 years.Yet the intended corrections in our study were higher for LASIK than for PRK eyes. No differences were found in percentages of eyes with best corrected visual acuity (BCVA) or loss of two or more lines of visual acuity between PRK and LASIK in the long-term. The postoperative long-term outcomes of PRK with two different delivery systems broad beam and scanning laser were compared and revealed no differences. Postoperative outcomes of moderate-to-high astigmatism yielded better results in terms of UCVA and less compromise or loss of two more lines of BCVA after LASIK that after PRK.Similar stability for both procedures was revealed. Vector analysis showed that LASIK outcomes tended to be more accurate than PRK outcomes, yet no statistically differences were found. Irregular astigmatism secondary to recurrent corneal erosion due to map-dot-fingerprint was successfully treated with phototherapeutic keratectomy (PTK). Preoperative videokeratographies (VK) showed irregular astigmatism. However, postoperatively, all eyes showed a regular pattern. No correlation was found between pre- and postoperative VK patterns. Postoperative outcomes of late PRK in eyes originally subjected to LASIK showed that all (7/7) eyes achieved UCVA ≤0.5 at last follow-up (range 3 — 11 months), and no eye lost lines of BCVA. Postoperatively all eyes developed and initial mild haze (0.5 — 1) into the first month. Yet, at last follow-up 5/7 eyes showed a haze of 0.5 and this was no longer evident in 2/7 eyes. Based on these results, we demonstrated that the long-term outcomes after PRK and LASIK were safe and efficient, with similar stability for both procedures. The PRK outcomes were similar when treated by broad-beam or scanning slit laser. LASIK was better than PRK to correct moderate-to-high astigmatism, yet both procedures showed a tendency of undercorrection. Irregular astigmatism was proven to be able to depend exclusively from the corneal epithelium. If this kind of astigmatism is present in the cornea and a customized PRK/LASIK correction is done based on wavefront measurements an irregular astigmatism may be produced rather than treated. Corneal sensory nerve recovery should have an important role in the modulation of the corneal wound healing and post-operative anterior stromal scarring. PRK enhancement may be an option in eyes with previous LASIK after a sufficient time interval that in at least 2 years.

Role of Basement Membrane and Extracellular Matrix Proteins in the Adhesion and Spreading of Immortalized Human Corneal Epithelial Cells

The repair of corneal wounds requires both epithelial cell adhesion and migration. Basement membrane (BM) and extracellular matrix (ECM) proteins function in these processes via integrin and non-integrin receptors. We have studied the adhesion, spreading and migration of immortalized human corneal epithelial (HCE) cells and their interactions with the laminins (Lms), fibronectins and tenascins produced. Human corneal BM expresses Lms-332 and -511, while Lm-111 was not found in these experiments. HCE cells produced both processed and unprocessed Lm-332, whereas neither Lm-111 nor Lm-511 was produced. Because HCE cells did not produce Lm-511, although it was present in corneal BM, we suggest that Lm-511 is produced by stromal keratocytes. The adhesion of HCE cells to Lms-111, -332 and -511 was studied first by determining the receptor composition of HCE cells and then by using quantitative cell adhesion assays. Immunofluorescence studies revealed the presence of integrin α2, α3, α6, β1 and β4 subunits. Among the non-integrin receptors, Lutheran (Lu) was found on adhering HCE cells. The cells adhered via integrin α3β1 to both purified human Lms-332 and -511 as well as to endogenous Lm-332. However, only integrin β1 subunit functioned in HCE cell adhesion to mouse Lm-111. The adhesion of HCE cells to Lm-511 was also mediated by Lu. Since Lm-511 did not induce Lu into focal adhesions in HCE cells, we suggest that Lm-511 serves as an ECM ligand enabling cell motility. HCE cells produced extradomain-A fibronectin, oncofetal fibronectin and tenascin-C (Tn-C), which are also found during corneal wound healing. Monoclonal antibodies (MAbs) against integrins α5β1 and αvβ6 as well as the arginine-glycine-aspartic acid (RGD) peptide inhibited the adhesion of HCE cells to fibronectin. Although the cells did not adhere to Tn-C, they adhered to the fibronectin/Tn-C coat and were then more efficiently inhibited by the function-blocking MAbs and RGD peptide. During the early adhesion, HCE cells codeposited Lm-332 and the large subunit of tenascin-C (Tn-CL) beneath the cells via the Golgi apparatus and microtubules. Integrin β4 subunit, which is a hemidesmosomal component, did not mediate the early adhesion of HCE cells to Lm-332 or Lm-332/Tn-C. Based on these results, we suggest that the adhesion of HCE cells is initiated by Lm-332 and modulated by Tn-CL, as it has been reported to prevent the assembly of hemidesmosomes. Thereby, Tn-CL functions in the motility of HCE cells during wound healing. The different distribution of processed and unprocessed Lm-332 in adhering, spreading and migrating HCE cells suggests a distinct role for these isoforms. We conclude that the processed Lm-332 functions in cell adhesion, whereas the unprocessed Lm-332 participates in cell spreading and migration.

Associations among Emdogain®, human oral carcinoma cells and oral proteolytic enzymes

The Enamel matrix derivative Emdogain® (EMD) is a commercially available tissue extract preparation of porcine enamel origin. Studies have shown EMD to be clinically useful in promoting periodontal regeneration. EMD has been widely used in periodontal therapy for over ten years, but the mechanism of its action and the exact composition are not completely clear. EMD is predominantly amelogenin (>90%). However, unlike amelogenin, EMD has a number of growth factor-like effects and it has been shown to enhance the proliferation, migration and other cellular functions of periodontal ligament fibroblasts and osteoblasts. In contrast, the effects of EMD on epithelial cell lines and in particular on oral malignant cells have not been adequately studied. In addition, EMD has effects on the production of cytokines by several oral cell lines and the product is in constant interaction with different oral enzymes. Regardless of the various unknown properties of EMD, it is said to be clinically safe in regenerative procedures, also in medically compromised patients. The aim of the study was to examine whether gingival crevicular fluid (GCF), which contains several different proteolysis enzymes, could degrade EMD and alter its biological functions. In addition, the objective was to study the effects of EMD on carcinogenesis-related factors, in particular MMPs, using in vitro and in vivo models. This study also aimed to contribute to the understanding of the composition of EMD. GCF was capable of degrading EMD, depending on the periodontal status, with markedly more degradation in all states of periodontal disease compared to healthy controls. EMD was observed to stimulate the migration of periodontal ligament fibroblasts (PLF), whereas EMD together with GCF could not stimulate this proliferation. In addition, recombinant amelogenin, the main component of EMD, decreased the migration of PLFs. A comparison of changes induced by EMD and TGF-β1 in the gene profiles of carcinoma cells showed TGF-β1 to regulate a greater number of genes than EMD. However, both of the study reagents enhanced the expression of MMP-10 and MMP-9. Furthermore, EMD was found to induce several factors closely related to carcinogenesis on gene, protein, cell and in vivo levels. EMD enhanced the production of MMP-2, MMP-9 and MMP-10 proteins by cultured carcinoma cells. In addition, EMD stimulated the migration and in vitro wound closure of carcinoma cells. EMD was also capable of promoting metastasis formation in mice. In conclusion, the diseased GCF, containing various proteases, causes degradation of EMD and decreased proliferation of PLFs. Thus, this in vitro study suggests that the regenerative effect of EMD may decrease due to proteases present in periodontal tissues during the inflammation and healing of the tissues in vivo. Furthermore, EMD was observed to enhance several carcinoma-related factors and in particular the production of MMPs by benign and malignant cell lines. These findings suggest that the clinical safety of EMD with regard to dysplastic mucosal lesions should be further investigated.

Palladin, a novel microfilament protein

Palladin is a novel actin microfilament associated protein, which together with myotilin and myopalladin forms a novel cytoskeletal IgC2 domain protein family. Whereas the expression of myotilin and myopalladin is limited mainly to striated muscle, palladin is widely expressed in both epithelial and mesenchymal tissues, including heart and the nervous system. Palladin has a complex genetic structure and it is expressed as several different sized and structured splice variants, which also display differences in their expression pattern and interactions. In muscle cells, all the family members localize to the sarcomeric Z-disc, and in non-muscle cells palladin also localizes to the stress-fiber-dense regions, lamellipodia, podosomes and focal adhesions. A common feature of this protein family is the binding to α-actinin, but other interactions are mostly unique to each member. Palladin has been shown to interact with several proteins, including VASP, profilin, Eps8, LASP-1 and LPP. Its domain structure, lack of enzymatic activity and multiple interactions define it as a molecular scaffolding protein, which links together proteins with different functional modalities into large complexes. Palladin has an important role in cytoskeletal regulation, particularly in stress fiber formation and stabilization. This assumption is supported by several experimental results. First, over-expression of palladin in non-muscle cells results in rapid reorganization of the actin cytoskeleton and formation of thick actin bundles. Second, the knock-down of palladin with anti-sense and siRNA techniques or knock-out by genetic methods leads to defective stress fiber formation. Furthermore, palladin is usually up-regulated in situations requiring a highly organized cytoskeleton, such as differentiation of dendritic cells, trophoblasts and myofibroblasts, and activation of astrocytes during glial scar formation. The protein family members have also direct disease linkages; myotilin missense mutations are the cause of LGMD1A and myofibrillar myopathy. Palladin mutations and polymorphisms, on the other hand, have been linked to hereditary pancreatic cancer and myocardial infarction, respectively. In this study we set out to characterize human palladin. We identified several palladin isoforms, studied their tissue distribution and sub-cellular localization. Four novel interaction partners were identified; ezrin, ArgBP2, SPIN90 and Src-kinase.The previously identified interaction between palladin and α-actinin was also characterized in detail. All the identified new binding partners are actin cytoskeleton associated proteins; ezrin links the plasma membrane to the cytoskeleton, ArgBP2 and SPIN90 localize, among other structures, to the lamellipodia and in cardiomyocytes to the Z-disc. Src is a transforming tyrosine kinase, which besides its role in oncogenesis has also important cytoskeletal associations. We also studied palladin in myofibroblasts, which are specialized cells involved in diverse physiological and pathological processes, such as wound healing and tissue fibrosis. We demonstrated that palladin is up-regulated during the differentiation of myofibroblasts in an isoform specific manner, and that this up-regulation is induced by TGF-β via activation of both the SMAD and MAPK signalling cascades. In summary, the results presented here describe the initial characterization of human palladin and offer a basis for further studies.

Molecular biology and functions of epilysin (MMP-28)

Epilysin (MMP-28) is the most recently identified member of the matrix metalloproteinase (MMP) family of extracellular proteases. Together these enzymes are capable of degrading almost all components of the extracellular matrix (ECM) and are thus involved in important biological processes such as development, wound healing and immune functions, but also in pathological processes such as tumor invasion, metastasis and arthritis. MMPs do not act solely by degrading the ECM. They also regulate cell behavior by releasing growth factors and biologically active peptides from the ECM, by modulating cell surface receptors and adhesion molecules and by regulating the activity of many important mediators in inflammatory pathways. The aim of this study was to define the unique role of epilysin within the MMP-family, to elucidate how and when it is expressed and how its catalytic activity is regulated. To gain information on its essential functions and substrates, the specific aim was to characterize how epilysin affects the phenotype of epithelial cells, where it is biologically expressed. During the course of the study we found that the epilysin promoter contains a well conserved GT-box that is essential for the basic expression of this gene. Transcription factors Sp1 and Sp3 bind this sequence and could hence regulate both the basic and cell type and differentiation stage specific expression of epilysin. We cloned mouse epilysin cDNA and found that epilysin is well conserved between human and mouse genomes and that epilysin is glycosylated and activated by furin. Similarly to in human tissues, epilysin is normally expressed in a number of mouse tissues. The expression pattern differs from most other MMPs, which are expressed only in response to injury or inflammation and in pathological processes like cancer. These findings implicate that epilysin could be involved in tissue homeostasis, perhaps fine-tuning the phenotype of epithelial cells according to signals from the ECM. In view of these results, it was unexpected to find that epilysin can induce a stable epithelial to mesenchymal transition (EMT) when overexpressed in epithelial lung carcinoma cells. Transforming growth factor b (TGF-b) was recognized as a crucial mediator of this process, which was characterized by the loss of E-cadherin mediated cell-cell adhesion, elevated expression of gelatinase B and MT1-MMP and increased cell migration and invasion into collagen I gels. We also observed that epilysin is bound to the surface of epithelial cells and that this interaction is lost upon cell transformation and is susceptible to degradation by membrane type-1-MMP (MT1-MMP). The wide expression of epilysin under physiological conditions implicates that its effects on epithelial cell phenotype in vivo are not as dramatic as seen in our in vitro cell system. Nevertheless, current results indicate a possible interaction between epilysin and TGF-b also under physiological circumstances, where epilysin activity may not induce EMT but, instead, trigger less permanent changes in TGF-b signaling and cell motility. Epilysin may thus play an important role in TGF-b regulated events such as wound healing and inflammation, processes where involvement of epilysin has been indicated.

Microfiltration in cheese and whey processing

Milk microfiltration (0.05-0.2 um) is a membrane separation technique which divides milk components into casein-enriched and native whey fractions. Hitherto the effect of intensive microfiltration including a diafiltration step for both cheese and whey processing has not been studied. The microfiltration performance of skimmed milk was studied with polymeric and ceramic MF membranes. The changes caused by decreased concentration of milk lactose, whey protein and ash content for cheese milk quality and ripening were studied. The effects of cheese milk modification on the milk coagulation properties, cheese recovery yield, cheese composition, ripening and sensory quality as well as on the whey recovery yield and composition by microfiltration were studied. The functional properties of whey protein concentrate from native whey were studied and the detailed composition of whey protein concentrate powders made from cheese wheys after cheese milk pretreatments such as high temperature heat treatment (HH), microfiltration (MF) and ultrafiltration (UF) were compared. The studied polymeric spiral wound microfiltration membranes had 38.5% lower energy consumption, 30.1% higher retention of whey proteins to milk retentate and 81.9% lower permeate flux values compared to ceramic membranes. All studied microfiltration membranes were able to separate main whey proteins from skimmed milk. The optimal lactose content of Emmental cheese milk exceeded 3.2% and reduction of whey proteins and ash content of cheese milk with high concentration factor (CF) values increased the rate of cheese ripening. Reduction of whey protein content in cheese milk increased the concentration of caseinomacropeptide (CMP) of total proteins in cheese whey. Reduction of milk whey protein, lactose and ash content reduces milk rennet clotting time and increased the firmness of the coagulum. Cheese yield calculated from raw milk to cheese was lower with microfiltrated milks due to native whey production. Amounts of a-lactalbumin (a-LA) and b-lactoglobulin (b-LG) were significantly higher in the reference whey, indicating that HH, MF and UF milk pretreatments decrease the amounts of these valuable whey proteins in whey. Even low CF values in milk microfiltration (CF 1.4) reduced nutritional value of cheese whey. From the point of view of utilization of milk components it would be beneficial if the amount of native whey and the CMP content of cheese whey could be maximized. Whey protein concentrate powders made of native whey had excellent functional properties and their detailed amino acid composition differed from those of cheese whey protein concentrate powders.

VEGFR-2 and VEGFR-3 Specific Signaling in Lymphangiogenesis and Angiogenesis

The circulatory system consists of the blood and lymphatic vessels. While blood vessels transport oxygen, cells, and nutrients to tissues, the lymphatic vessels collect fluid, cells, and plasma proteins from tissues to return back to the blood circulation. Angiogenesis, the growth of new blood vessels from pre-existing ones, is an important process involved in several physiological conditions such as inflammation, wound healing, and embryonic development. Furthermore, angiogenesis is found in many pathological conditions such as atherosclerosis and the growth and differentiation of solid tumors. Many tumor types spread via lymphatic vessels to form lymph node metastasis. The elucidation of the molecular players coordinating development of the vascular system has provided an array of tools for further insight of the circulatory system. The discovery of the Vascular Endothelial Growth Factor (VEGF) family members and their tyrosine kinase receptors (VEGFRs) has facilitated the understanding of the vasculature in different physiological and pathological situations. The VEGFRs are expressed on endothelial cells and mediate the growth and maintenance of both the blood and lymphatic vasculatures. This study was undertaken to address the role of VEGFR-2 specific signaling in maturation of blood vessels during neoangiogenesis and in lymphangiogenesis. We also wanted to differentiate between VEGFR-2 and VEGFR-3 specific signaling in lymphangiogenesis. We found that specific VEGFR-2 stimulation alone by gene therapeutic methods is not sufficient for production of mature blood vessels. However, VEGFR-2 stimulation in combination with expression of platelet-derived growth factor D (PDGF-D), a recently identified member of the PDGF growth factor family, was capable of stabilizing these newly formed vessels. Signaling through VEGFR-3 is crucial during developmental lymphangiogenesis, but we showed that the lymphatic vasculature becomes independent of VEGFR-3 signaling after the postnatal period. We also found that VEGFR-2 specific stimulation cannot rescue the loss of lymphatic vessels when VEGFR-3 signaling is blocked and that VEGFR-2 specific signals promote lymphatic vessel enlargement, but are not involved in vessel sprouting to generate new lymphatic vessels in vivo, in contrast to the VEGFR-2 dependent sprouting observed in blood vessels. In addition, we compared the inhibitory effects of a small molecular tyrosine kinase inhibitor of VEGFR-2 vs. VEGFR-3 specific signaling in vitro and in vivo. Our results showed that the tyrosine kinase inhibitor could equally affect physiological and pathological processes dependent on VEGFR-2 and VEGFR-3 driven angiogenesis or lymphangiogenesis. These results provide new insights into the VEGFR specific pathways required for pre- and postnatal angiogenesis as well as lymphangiogenesis, which could provide important targets and therapies for treatment of diseases characterized by abnormal angiogenesis or lymphangiogenesis.

CADASIL: molecular studies on the most common hereditary vascular dementing disorder

Cerebral Autosomal Dominant Arteriopathy with Subcortical Infarcts and Leukoencephalopathy (CADASIL) is the most common hereditary vascular dementia. CADASIL is a systemic disease of small and medium-sized arteries although the symptoms are almost exclusively neurological, including migraineous headache, recurrent ischemic episodes, cognitive impairment and, finally, subcortical dementia. CADASIL is caused by over 170 different mutations in the NOTCH3 gene, which encodes a receptor expressed in adults predominantly in the vascular smooth muscle cells. The function of NOTCH3 is not crucial for embryonic development but is needed after birth. NOTCH3 directs postnatal arterial maturation and helps to maintain arterial integrity. It is involved in regulation of vascular tone and in the wound healing of a vascular injury. In addition, NOTCH3 promotes cell survival by inducing expression of anti-apoptotic proteins. NOTCH3 is a membrane-spanning protein with a large extracellular domain (N3ECD) containing 34 epidermal growth factor-like (EGF) repeats and a smaller intracellular domain with six ankyrin repeats. All CADASIL mutations are located in the EGF repeats and the majority of the mutations cause gain or loss of one cysteine residue in one of these repeats leading to an odd number of cysteine residues, which in turn leads to misfolding of N3ECD. This misfolding most likely alters the maturation, targetting, degradation and/or function of the NOTCH3 receptor. CADASIL mutations do not seem to affect the canonical NOTCH3 signalling pathway. The main pathological findings are the accumulation of the NOTCH3 extracellular domain on degenerating vascular smooth muscle cells (VSMCs), accumulation of granular osmiophilic material (GOM) in the close vicinity of VSMCs as well as fibrosis and thickening of arterial walls. Narrowing of the arterial lumen and local thrombosis cause insufficient blood flow, mainly in small arteries of the cerebral white matter, resulting in tissue damage and lacunar infarcts. CADASIL is suspected in patients with a suggestive family history and clinical picture as well as characteristic white matter alterations in magnetic resonance imaging. A definitive verification of the diagnosis can be achieved by identifying a pathogenic mutation in the NOTCH3 gene or through the detection of GOM by electron microscopy. To understand the pathology underlying CADASIL, we have generated a unique set of cultured vascular smooth muscle cell (VSMC) lines from umbilical cord, placental, systemic and cerebral arteries of CADASIL patients and controls. Analyses of these VSMCs suggest that mutated NOTCH3 is misfolded, thus causing endoplasmic reticulum stress, activation of the unfolded protein response and increased production of reactive oxygen species. In addition, mutation in NOTCH3 causes alterations in actin cytoskeletal structures and protein expression, increased branching and abnormal node formation. These changes correlate with NOTCH3 expression levels within different VSMCs lines, suggesting that the phenotypic differences of SMCs may affect the vulnerability of the VSMCs and, therefore, the pathogenic impact of mutated NOTCH3 appears to vary in the arteries of different locations. Furthermore, we identified PDGFR- as an immediate downstream target gene of NOTCH3 signalling. Activation of NOTCH induces up-regulation of the PDGFR- expression in control VSMCs, whereas this up-regulation is impaired in CADASIL VSMCs and might thus serve as an alternative molecular mechanism that contributes to CADASIL pathology. In addition, we have established the congruence between NOTCH3 mutations and electron microscopic detection of GOM with a view to constructing a strategy for CADASIL diagnostics. In cases where the genetic analysis is not available or the mutation is difficult to identify, a skin biopsy is an easy-to-perform and highly reliable diagnostic method. Importantly, it is invaluable in setting guidelines concerning how far one should proceed with the genetic analyses.

Bustle behind the scenes of action : nemosis as a model for fibroblast inflammatory activation

Wound healing is a complex process that requires an interplay between several cell types. Classically, fibroblasts have been viewed as producers of extracellular matrix, but more recently they have been recognized as orchestrators of the healing response, promoting and directing, inflammation and neovascularization processes. Compared to those from healthy tissue, inflammation-associated fibroblasts display a dramatically altered phenotype and have been described as sentinel cells, able to switch to an immunoregulatory profile on cue. However, the activation mechanism still remains largely uncharacterized. Nemosis is a model for stromal fibroblast activation. When normal human primary fibroblasts are deprived of growth support they cluster, forming multicellular spheroids. Clustering results in upregulation of proinflammatory markers such as cyclooxygenase-2 and secretion of prostaglandins, proteinases, cytokines, and growth factors. Fibroblasts in nemosis induce wound healing and tumorigenic responses in many cell types found in inflammatory and tumor microenvironments. This study investigated the effect of nemotic fibroblasts on two components of the vascular system, leukocytes and endothelium, and characterized the inflammation-promoting responses that arose in these cell types. Fibroblasts in nemosis were found to secrete an array of chemotactic cytokines and attract leukocytes, as well as promote their adhesion to the endothelium. Nuclear factor-kB, the master regulator of many inflammatory responses, is activated in nemotic fibroblasts. Nemotic fibroblasts are known to produce large amounts of hepatocyte growth factor, a motogenic and angiogenic factor. Also, as shown in this study, they produce vascular endothelial growth factor. These two factors induced migratory and sprouting responses in endothelial cells, both required for neovascularization. Nemotic fibroblasts also caused a decrease in the expression of adherens and tight junction components on the surface of endothelial cells. The results allow the conclusion that fibroblasts in nemosis share many similarities with inflammation-associated fibroblasts. Both inflammation and stromal fibroblasts are known to be involved in tumorigenesis and tumor progression. Nemosis may be viewed as a model for stromal fibroblast activation, or it may correlate with cell-cell interactions between adjacent fibroblasts in vivo. Nevertheless, due to nemosis-derived production of proinflammatory cytokines and growth factors, fibroblast nemosis may have therapeutic potential as an inducer of controlled tissue repair. Knowledge of stromal fibroblast activation gained through studies of nemosis, could provide new strategies to control unwanted inflammation and tumor progression.

Postoperative soft tissue defects in the ankle area : The etiology and methods of reconstruction

The risk is obvious for soft tissue complications after operative treatment of the Achilles tendon, calcaneal bone or after ankle arthroplasty. Such complications after malleolar fractures are, however, seldom seen. The reason behind these complications is that the soft tissue in this region is tight and does not allow much tension to the wound area after surgery. Furthermore the area of operation may be damaged by swelling after the injury, or can be affected by peripheral vascular disease. While complications in this area are unavoidable, they can be diminished. This study attempts to highlight the possible predisposing factors leading to complications in these operations and on the other hand, to determine the solutions to solve soft tissue problems in this region. The study consists of five papers. The first article is a reprint on the soft tissue reconstruction of 25 patients after their complicated Achilles tendon surgeries were analysed. The second study reviews a series of 126 patients after having undergone an operative treatment of calcaneal bone fractures and analyses the complications and possible reasons behind them. The third part analyses a series of corrections of 35 soft tissue complications after calcaneal fracture operations. The fourth part reviews a series of 7 patients who had undergone complicated ankle arthroplasties. The last article presents a series of post operative lateral defects of the ankle treated with a less frequently used distally based peroneus brevis muscle flap and analyses the results. What can be conducted from these studies is that in general, the results after the correction of even severe soft tissue complications in the ankle region are good. For the small defects around the Achilles tendon, the local flaps are useful, but the larger defects are best treated with a free flap. We found that a long delay from trauma to surgery and a long operating time were predisposing factors that lead to soft tissue complications after operatively treated calcaneal bone fractures. The more severe the injury, the greater the risk for wound complication. Surprisingly, the long-term results after infected calcaneal osteosyntheses were acceptable and the calcaneal bone seems to tolerate chronic infections very well if the soft tissue is reconstructed successfully. Behind the complicated ankle arthroplasties, unexpectedly high number of cases experiencing arteriosclerosis of the lower extremity was found. These complications lead to ankle fusion but can be solved with a free flap if the vascularity is intact or can be reconstructed. For this reason a vascular examination of the lower extremity arteries of the patients going to ankle arthroplasty is strongly recommended. Moreover postoperative lateral malleolar wound infections which typically create lateral ankle defects can successfully be treated with a peroneus brevis muscle flap covered with a free skin graft.

Applications of coralline hydroxyapatite with bioabsorbable containment and reinforcement as bone graft substitute : An experimental study

The aim of the present experimental study was to find out if the applications of coralline hydroxyapatite (HA) can be improved by using bioabsorbable containment or binding substance with particulate HA in mandibular contour augmentation and by using bioabsorbable fibre-reinforced HA blocks in filling bone defects and in anterior lumbar interbody fusion. The use of a separate curved polyglycolide (PGA) containment alone or together with a fast resorbing polyglycolide/polylactide (PGA/PLA) binding substance were compared to the conventional non-contained method in ridge augmentation in sheep. The contained methods decreased HA migration, but the augmentations did not differ significantly. The use of the containment caused a risk for wound dehiscence and infection. Histologically there was a rapid connective tissue ingrowth into the HA graft and it was more abundant with the PGA containment compared to the non-contained augmentation and even additionally rich when the HA particles were bound with PGA/PLA copolymer. However, the bone ingrowth was best in the non-contained augmentation exceeding 10-12 % of the total graft area at 24 weeks. Negligible or no bone ingrowth was seen in the cases where the polymer composite was added to the HA particles and, related to that, foreign-body type cells were seen at the interface between the HA and host bone. The PGA and poly-dl/l-lactide (PDLLA) fibre-reinforced coralline HA blocks were studied in the metaphyseal and in the diaphyseal defects in rabbits. A rapid bone ingrowth was seen inside the both types of implants. Both PGA and PDLLA fibres induced an inflammatory fibrous reaction around themselves but it did not hinder the bone ingrowth. The bone ingrowth pattern was directed according to the loading conditions so that the load-carrying cortical ends of the implants as well as the implants sited in the diaphyseal defects were the most ossified. The fibre-reinforced coralline HA implants were further studied as stand-alone grafts in the lumbar anterior interbody implantation in pigs. The strength of the HA implants proved not to be adequate, the implants fractured in six weeks and the disc space was gradually lost similarly to that of the discectomized spaces. Histologically, small quantities of bone ingrowth was seen in some of the PGA and PDLLA reinforced coralline implants while no bone formation was identified in any of the PDLLA reinforced synthetic porous HA implants. While fragmented, the inner structure of the implants was lost, the bone ingrowth was minimal, and the disc was replaced by the fibrous connective tissue. When evaluated radiologically the grade of ossification was assessed as better than histologically, and, when related to the histologic findings, CT was more dependable than the plain films to show ossification of the implanted disc space. Local kyphosis was a frequent finding along with anterior bone bridging and ligament ossification as a consequence of instability of the implanted segment.

Mediastinitis after cardiac surgery

Mediastinitis as a complication after cardiac surgery is rare but disastrous increasing the hospital stay, hospital costs, morbidity and mortality. It occurs in 1-3 % of patients after median sternotomy. The purpose of this study was to find out the risk factors and also to investigate new ways to prevent mediastinitis. First, we assessed operating room air contamination monitoring by comparing the bacteriological technique with continuous particle counting in low level contamination achieved by ultra clean garment options in 66 coronary artery bypass grafting operations. Second, we examined surgical glove perforations and the changes in bacterial flora of surgeons' fingertips in 116 open-heart operations. Third, the effect of gentamicin-collagen sponge on preventing surgical site infections (SSI) was studied in randomized controlled study with 557 participants. Finally, incidence, outcome, and risk factors of mediastinitis were studied in over 10,000 patients. With the alternative garment and textile system (cotton group and clean air suit group), the air counts fell from 25 to 7 colony-forming units/m3 (P<0.01). The contamination of the sternal wound was reduced by 46% and that of the leg wound by >90%. In only 17% operations both gloves were found unpunctured. Frequency of glove perforations and bacteria counts of hands were found to increase with operation time. With local gentamicin prophylaxis slightly less SSIs (4.0 vs. 5.9%) and mediastinitis (1.1 vs. 1.9%) occurred. We identified 120/10713 cases of postoperative mediastinitis (1.1%). During the study period, the patient population grew significantly older, the proportion of women and patients with ASA score >3 increased significantly. In multivariate logistic regression analysis, the only significant predictor for mediastinitis was obesity. Continuous particle monitoring is a good intraoperative method to control the air contamination related to the theatre staff behavior during individual operation. When a glove puncture is detected, both gloves are to be changed. Before donning a new pair of gloves, the renewed disinfection of hands will help to keep their bacterial counts lower even towards the end of long operation. Gentamicin-collagen sponge may have beneficial effects on the prevention of SSI, but further research is needed. Mediastinitis is not diminishing. Larger populations at risk, for example proportions of overweight patients, reinforce the importance of surveillance and pose a challenge in focusing preventive measures.