The "HAT-TRICK" technique: A modification of soft tissue grafting using volume stable collagen matrix and cross-linked hyaluronic acid. Part A: The pontic site.

Vertical soft tissue augmentation between implants can be clinically challenging and burdensome for patients when employing conventional techniques. Recently, with the introduction of xenogenic collagen matrices, the principle of single-site surgery has become more common. However, some issues persist regarding graft stability and tissue integration. In the present technical note, the authors introduce the "HAT-TRICK" technique to address these observed difficulties. As the name suggests, this technique is believed to provide improved stability, volumetric gain, and histological integration of the implanted matrix by shaping it appropriately resembling a hat over the crest with apical bevels, stabilized with fixation pins and infused with cross-linked hyaluronic acid (xHya). A two-month observation of a bi-maxillary case is presented with detailed description of the technique and digitalized comparison methods for an easier explanation of the introduced technique.

Effects of hindlimb unloading and subsequent reloading on the structure and mechanical properties of Achilles tendon-to-bone attachment.

While muscle and bone adaptations to deconditioning have been widely described, few studies have focused on the tendon enthesis. Our study examined the effects of mechanical loading on the structure and mechanical properties of the Achilles tendon enthesis. We assessed the fibrocartilage surface area, the organization of collagen, the expression of collagen II, the presence of osteoclasts, and the tensile properties of the mouse enthesis both after 14 days of hindlimb suspension (HU) and after a subsequent 6 days of reloading. Although soleus atrophy was severe after HU, calcified fibrocartilage (CFc) was a little affected. In contrast, we observed a decrease in non-calcified fibrocartilage (UFc) surface area, collagen fiber disorganization, modification of morphological characteristics of the fibrocartilage cells, and altered collagen II distribution. Compared to the control group, restoring normal loads increased both UFc surface area and expression of collagen II, and led to a crimp pattern in collagen. Reloading induced an increase in CFc surface area, probably due to the mineralization front advancing toward the tendon. Functionally, unloading resulted in decreased enthesis stiffness and a shift in site of failure from the osteochondral interface to the bone, whereas 6 days of reloading restored the original elastic properties and site of failure. In the context of spaceflight, our results suggest that care must be taken when performing countermeasure exercises both during missions and during the return to Earth.

Antibacterial Activity Evaluation of ZnO, CuO, and TiO2 Nanoparticles in Solution and Thin Films.

This chapter introduces unique methodology of antibacterial activity evaluation of nanoparticles in both solution and thin films. Nanoparticles of ZnO, TiO2, and CuO are synthesized via the sol-gel method. Antibacterial tests are carried out against Gram-positive Staphylococcus aureus and Gram-negative Escherichia coli bacteria using disk diffusion and bioluminescence. To perform antibacterial tests on thin films and to overcome bacterial strains recuperation on the supports, a new method of bacterial detaching from the slides is developed based on French standard NF EN 14561.

Inherited connective tissue diseases highlight macromolecular network interdependences in skin extracellular matrix: a histomorphometric study.

Mutation of just a single extracellular matrix protein, a receptor or enzyme involved in connective tissue metabolism is sufficient to cause systemic pathologies and failure of tissues that are subjected to strong mechanical stresses. Skin histological and computerized image analyses can provide a good qualitative and quantitative indication of these inherited connective tissue diseases. In this study, skin biopsies from young (10 to 25 years) and middle-aged patients (26 to 50 years) suffering from Ehlers-Danlos syndromes (EDS), Marfan syndrome (MS) or pseudoxanthoma elasticum (PXE) were studied after specific staining of both the collagen and elastic networks. Findings from the histomorphometric analyses conducted on skin sections of the patients with EDS, MS and PXE were compared to skin sections of healthy subjects from the same age groups. Our results show that both the collagen and the elastic networks were affected in all the studied pathological cases, but that the adverse changes to the elastic network in older patients were distinct from the physiological changes observed during aging process for healthy subjects. This degenerative process may be explained by an added phenomenon involving a general connective tissue proteolysis.

Oral manifestations associated with inherited hyperhomocysteinemia: A first case description.

Hyperhomocysteinemia is a rare disease caused by nutritional deficiencies or genetic impairment of cysteine metabolism. To date, no oral manifestations of hyperhomocysteinemia have been described in humans. Therefore, to our knowledge, the present case report is the first description of a hyperhomocysteinemic patient showing oral tissue alterations leading to both early tooth loss and failed implant osseointegration. The patient presented with a methylenetetrahydrofolate reductase gene mutation (677T polymorphism) leading to mild hyperhomocysteinemia. The radiologic analysis showed hyperdense lesions scattered in the maxillae. The histologic observations indicated alterations in both collagen and elastic networks in the gingiva and dermis. Interestingly, the presence of ectopic mineralized inclusions was noted in both periodontal ligament and gingiva. Strong osteoclastic activity was associated with abnormal calcification of trabecular spaces. Uneven oral tissue remodeling due to high tissue levels of homocysteine could explain the pathologic manifestations observed in this case.

Poor Biointegration of Porcine Acellular Dermal Matrix Associated with Unfavorable Gingival Healing: A Report of Three Cases.

AIM: The aim of the present work was to explain the poor biointegration of acellular dermal xenogeneic matrix, leading to an unfavorable gingival healing following a grafting procedure for the treatment of soft tissue deficiencies. BACKGROUND: Numerous works have demonstrated the successful use of acellular dermal matrix (ADM) in soft tissue augmentation procedures. However, spare human investigations reported adverse healing outcomes at microscopic level. CASE DESCRIPTION: Three patients showing various soft tissue deficiencies (recession, gingival thickening) requiring a gingival augmentation were grafted using an ADM porcine acellular dermal matrices (pADM) as a soft tissue substitute. For this purpose, appropriate soft tissue augmentation surgeries were performed and the grafted pADM was left for proper healing. Biopsies were harvested from two out of the three patients, respectively, at 11 and 27 weeks in order to conduct a histological evaluation of the pADM's doubtful biointegration. Moreover, the ultrastructural analysis of pADM was performed using scanning electron microscopy, and additional histological procedures were used to assess its ability to support human gingival fibroblast cultures. Signs of gingival inflammation persisted several months postoperatively. Histologically, numerous inflammatory cells characterized the grafted site. Indeed, the high number of foreign body giant cell granulomas and the very densified newly formed collagen fibers highlighted a fibrotic process within gingival connective tissue. The ultrastructural and histological analysis showed that pADM was characterized by very thick and dense collagen bundles demonstrating a nonphysiological collagen network organization. Cell culture experiments showed fibroblasts proliferating on the matrix surface, sparing its deeper part, even though the collagen matrix degradation seemed to occur following a gradient from the pADM surface inward. CONCLUSION: The unfavorable clinical results may be caused by the poor colonization of matrix cells and poor angiogenesis leading to the inadequate biointegration of pADM. Hence, the pADM structure in terms of porosity and degradability should be further investigated. CLINICAL SIGNIFICANCE: The present cases highlighted a poor integration of pADM following soft tissue grafting procedures, which was caused by the inadequate ultrastructure of the used pADM. Therefore, despite the utility of such tissue substitutes, their manufacturing improvement could be required to obtain a better biointegration.

The Use of Platelet-Rich Plasma to Promote Cell Recruitment into Low-Molecular-Weight Fucoidan-Functionalized Poly(Ester-Urea-Urethane) Scaffolds for Soft-Tissue Engineering.

Due to their elastomeric behavior, polyurethane-based scaffolds can find various applications in soft-tissue engineering. However, their relatively inert surface has to be modified in order to improve cell colonization and control cell fate. The present study focuses on porous biodegradable scaffolds based on poly(ester-urea-urethane), functionalized concomitantly to the scaffold elaboration with low-molecular-weight (LMW) fucoidan; and their bio-activation with platelet rich plasma (PRP) formulations with the aim to promote cell response. The LMW fucoidan-functionalization was obtained in a very homogeneous way, and was stable after the scaffold sterilization and incubation in phosphate-buffered saline. Biomolecules from PRP readily penetrated into the functionalized scaffold, leading to a biological frame on the pore walls. Preliminary in vitro assays were assessed to demonstrate the improvement of scaffold behavior towards cell response. The scaffold bio-activation drastically improved cell migration. Moreover, cells interacted with all pore sides into the bio-activated scaffold forming cell bridges across pores. Our work brought out an easy and versatile way of developing functionalized and bio-activated elastomeric poly(ester-urea-urethane) scaffolds with a better cell response.

Picrosirius red staining: a useful tool to appraise collagen networks in normal and pathological tissues.

Specific staining of the extracellular matrix components is especially helpful in studying tissue remodeling, particularly in the case of connective tissue pathologies. As developed by Junqueira and colleagues in 1979, specific staining by Picrosirius red is one of the most important stains to study collagen networks in different tissues. Under polarized light, collagen bundles appear green, red or yellow, and are easily differentiated from the black background, thus allowing for quantitative morphometric analysis. As Junqueira and colleagues point out, many studies use color staining to differentiate collagen bundles and to specify collagen types, yet other studies report that polarized colors only reflect fiber thickness and packing. Using a simple histological example, our study illustrates the inability of Picrosirius red staining to differentiate collagen types, since the absorbed amount of polarized light by this dye strictly depends on the orientation of the collagen bundles.

Unusual glycosaminoglycans from a deep sea hydrothermal bacterium improve fibrillar collagen structuring and fibroblast activities in engineered connective tissues.

Biopolymers produced by marine organisms can offer useful tools for regenerative medicine. Particularly, HE800 exopolysaccharide (HE800 EPS) secreted by a deep-sea hydrothermal bacterium displays an interesting glycosaminoglycan-like feature resembling hyaluronan. Previous studies demonstrated its effectiveness to enhance in vivo bone regeneration and to support osteoblastic cell metabolism in culture. Thus, in order to assess the usefulness of this high-molecular weight polymer in tissue engineering and tissue repair, in vitro reconstructed connective tissues containing HE800 EPS were performed. We showed that this polysaccharide promotes both collagen structuring and extracellular matrix settle by dermal fibroblasts. Furthermore, from the native HE800 EPS, a low-molecular weight sulfated derivative (HE800 DROS) displaying chemical analogy with heparan-sulfate, was designed. Thus, it was demonstrated that HE800 DROS mimics some properties of heparan-sulfate, such as promotion of fibroblast proliferation and inhibition of matrix metalloproteinase (MMP) secretion. Therefore, we suggest that the HE800EPS family can be considered as an innovative biotechnological source of glycosaminoglycan-like compounds useful to design biomaterials and drugs for tissue engineering and repair.

Clinical, histological, and histomorphometrical analysis of maxillary sinus augmentation using inorganic bovine in humans: preliminary results.

The aim of the present study was to evaluate bone formation after maxillary sinus augmentation using bovine bone substitute material Bio-Oss alone by means of clinical, histological, and histomorphometrical examination of human biopsies. Deproteinized bovine bone (DPBB, Bio-Oss) was used to fill cavities after elevation of the sinus mucosa following major sinus pneumatization. Twenty patients with edentulous posterior maxillae were treated with 20 sinus augmentation procedures using a 2-stage technique. Residual lateral maxillary bone height was less than 3 mm. Forty-nine Straumann endosseous implants were used to complete the implant-prosthetic rehabilitation. Forty cylinder-shaped bone biopsies were taken from the augmented maxillary region 8 months after grafting during the second-stage surgery before implant placement. All implants were loaded 3 months after insertion, and no failures were recorded. Histomorphometrical analysis showed an average percentage of newly formed bone of 17.6% (± 2.8%) and a proportion of residual bone substitute material of 29.9% (± 4.9%) of the total biopsy area. Intimate contact between newly formed bone and Bio-Oss was detected along 28.2% (± 6.8%) of the particle surfaces. The results also showed that in all cases, the DPBB granules had been interconnected by bridges of vital newly formed bone. Inorganic bovine bone appears to be biocompatible and osteoconductive, and it can be used with success as a bone substitute in maxillary sinus augmentation procedures.

Marine polysaccharides: a source of bioactive molecules for cell therapy and tissue engineering.

The therapeutic potential of natural bioactive compounds such as polysaccharides, especially glycosaminoglycans, is now well documented, and this activity combined with natural biodiversity will allow the development of a new generation of therapeutics. Advances in our understanding of the biosynthesis, structure and function of complex glycans from mammalian origin have shown the crucial role of this class of molecules to modulate disease processes and the importance of a deeper knowledge of structure-activity relationships. Marine environment offers a tremendous biodiversity and original polysaccharides have been discovered presenting a great chemical diversity that is largely species specific. The study of the biological properties of the polysaccharides from marine eukaryotes and marine prokaryotes revealed that the polysaccharides from the marine environment could provide a valid alternative to traditional polysaccharides such as glycosaminoglycans. Marine polysaccharides present a real potential for natural product drug discovery and for the delivery of new marine derived products for therapeutic applications.

Human bone marrow-derived cells: an attractive source to populate dermal substitutes.

We have previously shown the importance of dermal fibroblasts within skin substitutes for promoting the emergence of a functional neodermis after grafting in humans. However, the use of fibroblasts from sources other than the dermis needs to be evaluated for patients with extensive skin loss. Here we examined the capacity of human bone marrow-derived cells (BMDCs), selected for their ability to adhere to plastic culture dishes, to behave like human dermal fibroblasts when incorporated within a 3D in vitro reconstructed tissue that promotes dermal fibroblast differentiation. Like dermal fibroblasts, BMDCs contracted a collagen matrix and were growth regulated by the matrix environment. They had the same shape and their nuclei had the same form factor as dermal fibroblasts. In addition, both cell types expressed desmin and vimentin but not alpha-smooth muscle actin. BMDCs deposited collagen types I and III, and fibrillin-1 with similar efficiency to dermal fibroblasts. In addition, BMDCs have the potential to regulate this deposition, as they produced metalloproteinases (MMP1, MMP2, and MMP9) and metalloproteinase inhibitors (TIMP1) very similarly to dermal fibroblasts. BMDCs can thus be induced to express functions resembling those of dermal fibroblasts, including those involved in the wound healing process.

Epigallocatechin gallate's protective effect against MMP7 in recessive dystrophic epidermolysis bullosa patients.

The analysis of phenotype-genotype correlations of patients suffering from recessive dystrophic epidermolysis bullosa (RDEB) evidenced intrafamilial and interfamilial phenotype variability occurring for the same mutation of COL7A1; this underscores the role of other genetics environmental factors in the expressivity of the disease. In this work, we checked whether matrilysin 1 (matrix metalloproteinase (MMP)7) could take part in the epidermal detachment in RDEB. Furthermore, we investigated epigallocatechin 3 gallate (EGCG) to determine whether it could inhibit matrilysin activities on collagen type VII and fibrillin 1 known to be associated with the dermo-epidermal junction. In this work, matrilysin 1 was detected in affected and unaffected skins of the three RDEB patients; furthermore, MMP7 was shown to degrade ex vivo on healthy normal skin collagen VII and fibrillin 1. Thus, we suspect that MMP7 could take an active part in the epidermal detachment occurring during RDEB. We evidenced that EGCG in in vitro as well as in ex vivo experiments was a good inhibitor of MMP7 and developed a good protection of collagen type VII and fibrillin 1 susceptible of being degraded by MMP7. We therefore propose that EGCG could be used beneficially in patients suffering from RDEB.

Comparison of extracellular matrix in skin and saphenous veins from patients with varicose veins: does the skin reflect venous matrix changes?

Varicose vein disease is a frequently occurring pathology with multifactorial causes and a genetic component. An intense remodelling of the varicose vein wall has been described and could be at the origin of its weakness and altered elasticity. We have described previously a dysregulation of collagen synthesis in cultured smooth muscle cells from saphenous veins and in dermal fibroblasts from the skin of patients with varicose veins, suggesting a systemic defect in their connective tissue. The present study describes comparative morphological and immunohistochemical data in both the skin and saphenous veins of eight control subjects (undergoing coronary bypass surgery) and eight patients with varicose veins. Histological staining of glycoproteins, the elastic fibre network and collagen bundles showed that the remodelling and fragmentation of elastic fibres observed in varicose veins were also present in the skin of the patients. When compared with control subjects, we observed in both the veins and skin of patients with varicose veins (i) an increase in the elastic network, as quantified by image analysis; (ii) an accumulation of collagen type I, fibrillin-1 and laminin; and (iii) an overproduction of MMP (matrix metalloproteinase)-1, MMP-2 and MMP-3, analysed by immunohistochemistry, but normal levels of other MMPs (MMP-7 and MMP-9) and their inhibitors (TIMP-1, TIMP-2 and TIMP-3). An imbalance of extracellular matrix production/degradation was thus observed in veins as well as in the skin of the patients with varicose veins and, taken together, these findings show that remodelling is present in different organs, confirming systemic alterations of connective tissues.

Fucoidan a sulfated polysaccharide from brown algae is a potent modulator of connective tissue proteolysis.

Fucoidans are sulfated fucosylated polymers from brown algae cell wall that exhibit some heparin/heparan sulfate properties. We previously demonstrated that these polysaccharides were able in vitro to stimulate dermal fibroblast proliferation and extracellular matrix deposition. Here, we investigated the action of a 16kDa fucoidan fraction on parameters involved in connective tissue breakdown. This fucoidan is able to inhibit gelatinase A secretion and stromelysin 1 induction by interleukin-1beta on dermal fibroblasts in culture. Furthermore, we observed that fucoidan increases the rate of association of MMPs with their specific inhibitors namely TIMPs. Using tissue sections of human skin in ex vivo experiments, we evidenced that this polysaccharide was able to minimize human leukocyte elastase activity resulting in the protection of human skin elastic fiber network against the enzymatic proteolysis due to this serine proteinase. These results suggested that fucoidan could be used for treating some inflammatory pathologies in which uncontrolled extracellular matrix degradation takes place.

Magnesium and connective tissue.

Magnesium (Mg2+) is the fourth most abundant cation and the second most abundant intracellular cation in vertebrates. Magnesium plays a critical role in cellular functions such as: cell adhesion via integrins on various macromolecular substrates, cell migration, DNA transcription and protein synthesis.

Are cytokines linked to collagen breakdown during periodontal disease progression?

BACKGROUND: Evidence of the role of cytokines produced by resident and inflammatory cells during inflammation is well established. The aim of this study was to quantify in healthy and diseased human gingiva the area fraction (AA%) occupied by collagen fibers and the amount of cytokines such as interleukin (IL)-1beta, IL-4, IL-6, tumor necrosis factor (TNF)-alpha, transforming growth factor (TGF)-beta, and epidermal growth factor (EGF) to investigate a possible correlation between such cytokines, collagen degradation, and the gingival index. METHODS: Gingival tissue specimens from 6 healthy controls (group 1), 6 patients with mild gingival inflammation (group 2), 6 patients with moderate gingival inflammation (group 3), and 6 patients with severe gingival inflammation (group 4) were cultured for 72 hours, and the cytokines present in the culture media were quantified using an enzyme-linked immunosorbent assay (ELISA). Paraffin gingival sections from the 24 subjects were stained with sirius red F3Ba for visualization of collagen fibers, then the area fraction (AA%) occupied by the gingival fibers was determined by automated image analysis. RESULTS: The present study revealed significant differences (P < 0.05) between means of AA% in group 1 (53%), group 2 (41%), group 3 (39.5%), and group 4 (35%) for collagen fibers. Compared to controls, there were significant increases of IL-1beta (groups 3 and 4), IL-6, and TNF-alpha (group 3); a significant decrease of IL-4 (groups 2, 3, and 4) and TGF-beta (groups-2 and, 3); and no change of EGF. The collagen AA% was significantly correlated with the amounts of IL-4 and TGF-beta, and significantly inversely correlated with the amounts of IL-1beta for all 3 inflamed groups and IL-6 and TNF-alpha for groups 2 and 3. CONCLUSION: The present study showed that EGF was not changed in inflamed gingival tissue and that IL-1beta and IL-4 were particularly and intensively correlated with collagen loss. These 2 cytokines could be markers of clinical severity during active periodontitis.