Microgrooved silicone subcutaneous implants in guinea pigs

Cell-substratum interactions are of fundamental importance for the reaction of body tissues to surgically implanted foreign materials. In our study we investigated the influence of 2 microm wide microgrooves, with various depths (0.5-6 microm), on capsule formation around subcutaneous silicone implants, in an animal experiment. Silicone sheets with microtexture were glued around silicone tubes. These implants were placed subcutaneously in eight guinea pigs for 10 weeks. The implanted tubes were removed including all surrounding tissues, and processed for light microscopy and subsequent histomorphometrical evaluation. All removed implants were surrounded by a thin fibrous capsule, and it was observed that this capsule was separated from the implants by a thin, single layer of mono- and multinucleated phagocytotic cells. In histomorphometry no significant differences were seen in relation to the reaction towards the various textures. We conclude that microtextures do not have an effect on the morphological characteristics of capsule formation around silicone implants in soft tissue.     

Morphological study of capsules formed around various breast endoprostheses

Silicone implants used in mammaplasty are often associated with constrictive capsule fibrosis, leading to induration and deformation of the prosthetic organs. In connection with this, there has been developed and is currently successfully applied in the clinical practice of the A. V. Vishnevsky Institute of Surgery an original method of mammary gland endoprosthesis with oily implants. The objective of this work was to make a comparative study of the late results of mammaplasty based upon the morphologic study of capsules formed around silicone and oily implants, removed during secondary surgical interventions. The dynamics of capsule formation around temporary organic glass endoprostheses has also been studied. The results showed that implantation of silicone prostheses results in a formation of a rough thick capsule, an active inflammatory reaction in the surrounding tissues and the development of constrictive fibrosis. Implantation of a temporary organic glass endoprosthesis that is filled with oil at the second stage of the operation, is followed by a thin capsule formation without signs of inflammation. The stability of the capsule structure up to 3 years has been noted. On the whole, this prosthesis provides a good cosmetic effect during long time after mammaplasty.     

Alleviation of capsular formations on silicone implants in rats using biomembrane-mimicking coatings

Despite their popular use in breast augmentation and reconstruction surgeries, the limited biocompatibility of silicone implants can induce severe side effects, including capsular contracture – an excessive foreign body reaction that forms a tight and hard fibrous capsule around the implant. This study examines the effects of using biomembrane-mimicking surface coatings to prevent capsular formations on silicone implants. The covalently attached biomembrane-mimicking polymer, poly(2-methacryloyloxyethyl phosphorylcholine) (PMPC), prevented nonspecific protein adsorption and fibroblast adhesion on the silicone surface. More importantly, in vivo capsule formations around PMPC-grafted silicone implants in rats were significantly thinner and exhibited lower collagen densities and more regular collagen alignments than bare silicone implants. The observed decrease in α-smooth muscle actin also supported the alleviation of capsular formations by the biomembrane-mimicking coating. Decreases in inflammation-related cells, myeloperoxidase and transforming growth factor-β resulted in reduced inflammation in the capsular tissue. The biomembrane-mimicking coatings used on these silicone implants demonstrate great potential for preventing capsular contracture and developing biocompatible materials for various biomedical applications.     

In vitro and in vivo characterization of synthetic polymer/biopolymer composites

Collagen, extracted from rat tail tendons using dilute acetic acid, was fabricated into films for subsequent characterization and biocompatibility testing. The reconstituted collagen was characterized with infrared spectroscopy, solution viscosity, contact angle, and tensile testing techniques and was found to be pure with molecular and physical properties consistent with findings of previous researchers. Composites composed of collagen coated on urethane and Silastic Rubber films were fabricated to give improved tear resistance. The biocompatibility of the composites and individual polymers was evaluated by discs implanted in the paravertebral muscle of rabbits. After four weeks none of the materials induced any gross changes in the muscle. Histopathological evaluation revealed a fibrous capsule around all of the materials. Collagen and collagen composites exhibited a stronger reaction as evidenced by a larger fibroblast layer and a variety of inflammatory cells, lymphocytes, eosinophils, and macrophages. The urethane was rated with a response index of 1.5 versus 3.25 for the urethane/collagen composite; Silastic Rubber rated a response index of 1.67 versus 3.12 for the Silastic Rubber/collagen composite; collagen rated a response index of 3.3. The polyester sutures also induced a reaction with a larger fibrous capsule but fewer inflammatory cells as compared to collagen and collagen composites.     

Silica coatings formed on noble dental casting alloy by the sol-gel dipping process.

The sol-gel dipping process, in which liquid silicon alkoxide is transformed into the solid silicon-oxygen network, can produce a thin film coating of silica (SiO2). The features of this method are high homogeneity and purity of the thin SiO2 film and a low sinter temperature, which are important in preparation of coating films that can protect from metallic ion release from the metal substrate and prevent attachment of dental plaque. We evaluated the surface characteristics of the dental casting silver-palladium-copper-gold (Ag-Pd-Cu-Au) alloy coated with a thin SiO2 film by the sol-gel dipping process. The SiO2 film bonded strongly (over 40 MPa) to Ti-implanted Ag-Pd-Cu-Au alloy substrate as demonstrated by a pull test. Hydrophobilization of Ti-implanted/SiO2-coated surfaces resulted in a significant increase of the contact angle of water (80.5 degrees) compared with that of the noncoated alloy specimens (59.3 degrees). Ti-implanted/SiO2-coated specimens showed the release of many fewer metallic ions (192 ppb/cm2) from the substrate than did noncoated specimens (2,089 ppb/cm2). The formation of a thin SiO2 film by the sol-gel dipping process on the surface of Ti-implanted Ag-Pd-Cu-Au alloy after casting clinically may be useful for minimizing the possibilities of the accumulation of dental plaque and metal allergies caused by intraoral metal restorations.     

Influence of local complications on capsule formation around model implants in a rat model

We studied the capsule formation around various filled breast implants and other related changes in distant organs (e.g., liver, spleen, lymph nodes) in a rat model 3 and 6 months after implantation. Model implants, one per rat, (filled with saline, n = 19; silicone, n = 14; cohesive silicone, n = 17; and hydrogel, n = 19) were implanted subcutaneous in the lower back of rats. The animals were sacrificed regularly after 3 and 6 months of implantation or when wound defects occurred. The capsules and organs were examined histologically, and immunohistology of the capsules was performed. A monoclonal antibody specific for AIF-1 (allograft inflammatory factor 1) was used to detect activated macrophages. Wound defects occurred most frequently after implantation of saline and hydrogel implants. Increased capsule thickness was associated with increased grade of inflammation, fibrosis, and the type of implant filling. There was a significant positive correlation between capsule thickness, presence of chronic inflammation, and AIF-1-positive macrophages (p < 0.0001), indicating that inflammation plays an important role in capsule formation. Remarkably, saline and silicone implants (in absence of local complications) cause only a blande slight fibrosis in capsules after 6 months of implantation, whereas capsules around cohesive silicone implants exhibited a more severe fibrosis with an increased capsule thickness. Most importantly, hydrogel seems to be most potent to induce an inflammatory infiltrate with AIF-1 expressing macrophages at the implantation site, independent of implantation time, and capsules also produced a significant increase in thickness after 6 months.     

The effect of microgeometry,implant thickness and polyurethane chemistry on the foreign body response to subcutaneous implants

We addressed the effect of implant thickness, implant porosity, and polyurethane (PU) chemistry on angiogenesis and on the foreign body response in rats. The following materials were implanted subcutaneously for 7 weeks then excised for histologic analysis: a solid PU; a solid polyurethane with silicone and polyethylene oxide (PU-S-PEO); porous expanded polytetrafluoroethylene (ePTFE); and porous polyvinyl alcohol sponge (PVA). Two thicknesses of PU-S-PEO were compared: 300 microns (thin) and 2000 microns (thick). Foreign body capsule (FBC) thickness was much less in PU-S-PEO implants than in PU implants. In addition, FBC were thinner in thin implants than in thick implants. FBC was much more dense in solid implants than in porous implants. As compared with solid implants, porous implants (PVA and ePTFE) led to a marked increase in the number of microvessels that developed adjacent to the implant, as observed both with hematoxylin/eosin staining and with an immunohistochemical anti-endothelial stain. We conclude that the polyethylene oxide and silicone moieties in PU reduce the thickness of the subsequent FBC. In addition, thin implants lead to a thin FBC. Porous implants (PVA and ePTFE) cause more angiogenesis than solid implants. These results may have implications for the measurement of blood-derived analytes by biosensors.     

Role of toll-like receptor 4 in the inflammation reaction surrounding silicone prosthesis

The inflammation which occurs around the silicone prosthesis is a complex process that can provoke the failure of the device and compromise the health of the implanted patient. Toll-like receptors (TLRs), which are transmembrane proteins, are now known to act in the innate immune response and in endogenous inflammation. The aim of our study was to assess the role of TLR4 in the foreign body reaction to a silicone shell prosthesis. Disks of shell silicone prosthesis were implanted in the subcutaneous tissue of C57BL6-TLR4-/- and C57BL6-WT mice. At day 14, inflammatory cell infiltrate and vessel sections around the prosthesis were less numerous in TLR4-/- than in WT mice. A histomorphometric analysis showed that the capsule around the implant was 1.96-fold less thick in depleted TLR4 than in wild-type mice. In addition, vascular endothelial growth factor and transforming growth factor 1 were underexpressed in the surrounding tissue of the prosthesis in TLR4-/- mice. Our study suggests, from this foreign body response model against silicone in mice, that TLR4 plays a key role in the reaction process around silicone implants.     

The subcutaneous capsules for foreign body in fetal rabbits: preliminary report.

In terms of wound healing, there are fundamental intrinsic and extrinsic differences between fetuses (scar-free healing) and adults. The fetus exhibits less typical inflammatory response (significantly neutropenic) with an underdeveloped self-nonself immunologic identity and a lack of cellular immunity. The recruitment of inflammatory cells to a wound may play an important role in the resulting cellular processes and ultimately affect the quality of the healing response. Foreign bodies can act as a source of infection and immunologic reactions. In contrast, there have been few studies of the wound healing of fetus with foreign bodies, where in adults, wounds are healed by tissue regeneration rather than capsule formation and a foreign body reaction. In this study, the wound healing process in an adult rabbit and fetus group, in which either silicone or a sponge was inserted in the uterus, were compared. All specimens showed capsule formation with fibroblast, collagen deposition, neovascularization, and infiltration of inflammatory cells. However, the fetal specimen exhibited mainly acute inflammatory responses and the capsule contained less fibroblasts and collagen deposition. In addition, myofibroblast expression, which mediates wound contracture, was lower in the fetal specimen. These findings were common with cotton implants, which were expected to induce a severe inflammatory response. The inflammatory response induced by foreign materials in fetal tissue showed similar response with that of incisional wound healing. This study may provide a basis for the use of implants such as silicone in future fetal surgery.     

In vivo evaluation and comparison of collagen, acetylated collagen and collagen/glycosaminoglycan composite films and sponges as candidate biomaterials

Native collagen, acetylated collagen, collagen/10% chondroitin sulphate, collagen/2.5% hyaluronic acid and collagen/20% hyaluronic acid were implanted both as film and as sponge into rat lumbar muscle for 7 and 14 d. After 7 d implantation, all materials elicited an acute inflammatory cell response characterized by numerous polymorphs and histocytes. The cell population after 14 d was principally mononuclear, i.e. leucocytes, neutrophils, macrophages, lymphocytes and fibroblasts. Both films and sponges followed a similar pattern. Native collagen elicited a subacute inflammatory response after 7 d. However, 14 d after implantation, a marked infiltration by neutrophils was apparent with subsequent degradation of existing collagen material. Acetylated collagen film evoked a much greater inflammatory cell response than native collagen. Both collagen/hyaluronic acid composites elicited a similar response. The collagen/10% chondroitin sulphate composite elicited the least inflammatory cell response at 7 d, whereas infiltration by host fibroblasts after 14 d implantation was clearly seen.     

A trial to prepare biodegradable collagen-hydroxyapatite composites for bone repair

This paper is a trial to prepare collagen-hydroxyapatite composites in vitro by an alternate immersion method. Collagen sponges of different biodegradabilities were prepared through chemical cross-linking of Type I collagen with glutaraldehyde (GA) at concentrations of 0.2, 1.0, and 2.0 wt%. The sponges were immersed at 37 degrees C in Tris-HCl-buffered solution containing 200 mM CaCl2 (pH 7.4) for 2 h and then in an aqueous solution of 120 mM Na2HPO4 (pH 9.3) for a 2 h further (one immersion cycle). The alternate immersion cycle was repeated for different times to obtain collagen-hydroxyapatite composites. The characterization of the resulting composites was performed by Fourier transform infrared spectroscopy (FT-IR). X-ray diffraction (XRD), and scanning electron microscopy (SEM). The weight of composites increased with an increase in immersion cycles and the rate of increase became greater with higher GA cross-linking levels for collagen sponge preparation. The pH of the phosphate solution decreased with the immersion cycle, which suggests H+ generation accompanied hydroxyapatite formation. Irrespective of the GA concentration and immersion cycle, every composite showed IR absorption bands attributable to phosphate and hydroxyl groups at 950-1100 or 550-650 and 3000-3500 cm(-1) and broad peaks specific to hydroxyapatite on the XRD charts. SEM study revealed small white clusters of hydroxyapatite interspersed uniformly on/in the collagen framework without any preferential orientation. The composite prepared from 0.2 wt% GA cross-linked collagen sponge which showed favourable characteristics was applied to a rat skull defect to evaluate its osteoconductivity as well as biodegradability. The formation of new bone tissue was histologically observed at the defect 12 weeks after application in marked contrast to the collagen sponge alone. The composite degraded without any inflammation reaction. It is concluded that the collagen-hydroxyapatite composite prepared by the present method is a biodegradable biomaterial of osteoconductivity applicable to bone repair.     

Thin films of oriented collagen fibrils for cell motility studies

Collagen films with oriented fibrils mimic tissues that have been remodeled by fibroblasts, which naturally tend to orient collagen fibrils in vivo. We have prepared thin films of ordered fibrils of collagen I, a major component of the extracellular matrix. The films were prepared by modifying a technique previously used to produce collagen I films for studies of cell morphology and intracellular signaling. By modifying the drying step, we were able to produce thin monolayers of collagen fibrils with consistent orientations over macroscopic (>100 microm) distances. We quantified the degree of orientation of the collagen fibrils using Fourier analysis of optical microscopy images. We also conducted experiments with vascular endothelial cells, and found that cell orientation and migration are well-correlated with fibril orientation. Using polarized cells, we showed oriented thin collagen film induces natural migration along the fibrils without using any sort of attractor. Taken together, these results demonstrate additional functionality and physiological relevance for a class of films being successfully applied in a variety of cell biology experiments.     

Porous Silk Fibroin Film as a Transparent Carrier for Cultivated Corneal Epithelial Sheets

Biological carriers, such as the amniotic membrane and serum-derived fibrin, are currently used to deliver cultivated corneal epithelial sheets to the ocular surface. Such carriers require being transparent and allowing the diffusion of metabolites in order to maintain a healthy ocular surface. However, safety issues concerning biological agents encouraged the development of safer, biocompatible materials as cell carriers. We examined the application of porous silk fibroin films with high molecular permeability prepared by mixing silk fibroin and poly(ethylene glycol) (PEG), and then removal of PEG from the silk-PEG films. Molecular permeability of porous silk fibroin film is higher than untreated silk fibroin film. Epithelial cells were isolated from rabbit limbal epithelium, and seeded onto silk fibroin coated wells and co-cultured with mitomycin C-treated 3T3 fibroblasts. Stratified epithelial sheets successfully engineered on porous silk fibroin film expressed the cornea-specific cytokeratins K3 and K12, as well as the corneal epithelial marker pax6. Basement membrane components such as type-IV collagen and integrin β1 were expressed in the stratified epithelial sheets. Further more, colony-forming efficiency of dissociated cells was similar to primary corneal epithelial cells showing that progenitor cells were preserved. The biocompatibility of fibroin films was confirmed in rabbit corneas for up to 6 months. Porous silk fibroin film is a highly transparent, biocompatible material that may be useful as a carrier of cultivated epithelial sheets in the regeneration of corneal epithelium.     

A collagen-phosphophoryn sponge as a scaffold for bone tissue engineering

Non-collagenous phosphoproteins that interact with a type-I collagen are thought to nucleate bone mineral into collagen networks of mineralized tissues. Previously, phosphophoryn cross-linked to type-I collagen was reported to be an effective nucleator of appatite. However, free phosphophoryn molecules inhibit the formation of apatite in vitro. On the basis of the above study, we expected a collagen-phosphophoryn sponge to be a good scaffold for bone-tissue engineering and examined the formation of bone in orthotopically transplanted composites of the sponge and bone marrow osteoblasts in vivo in Fischer rats. Osteoblastic primary cells were obtained from the bone shaft of femorae of Fisher rats, according to the method of Maniatopoulous et al. A suspension of marrow cells was distributed through a flask with standard culture medium and incubated at 37 degrees C. When cultures were nearly confluent after 10 days, they were concentrated by centrifugation to 10(6) cells/ml and subcultured onto the synthesized collagen-phosphophoryn sponge and a collagen sponge (control). After 14 days, the composites of collagen-phosphophoryn and osteoblastic cells as well as control composites were transplanted into bone-defect sites of Fisher rats (holes 2 mm in diameter) and then the wounds were sutured. The composites were harvested at 1-8 weeks after implantation, and stained with hematoxylin and eosin. It was found that more bone was formed in the composites of collagen-phosphophoryn sponge and osteoblasts than control composites from 1 week to 8 weeks, suggesting that the collagen-phosphophoryn sponge is a good candidate as a scaffold for bone-tissue engineering.     

A collagen–phosphophoryn sponge as a scaffold for bone tissue engineering

Non-collagenous phosphoproteins that interact with a type-I collagen are thought to nucleate bone mineral into collagen networks of mineralized tissues. Previously, phosphophoryn cross-linked to type-I collagen was reported to be an effective nucleator of appatite. However, free phosphophoryn molecules inhibit the formation of apatite in vitro. On the basis of the above study, we expected a collagen-phosphophoryn sponge to be a good scaffold for bone-tissue engineering and examined the formation of bone in orthotopically transplanted composites of the sponge and bone marrow osteoblasts in vivo in Fischer rats. Osteoblastic primary cells were obtained from the bone shaft of femorae of Fisher rats, according to the method of Maniatopoulous et al. A suspension of marrow cells was distributed through a flask with standard culture medium and incubated at 37°C. When cultures were nearly confluent after 10 days, they were concentrated by centrifugation to 10⁶ cells/ml and subcultured onto the synthesized collagen-phosphophoryn sponge and a collagen sponge (control). After 14 days, the composites of collagen-phosphophoryn and osteoblastic cells as well as control composites were transplanted into bone-defect sites of Fisher rats (holes 2 mm in diameter) and then the wounds were sutured. The composites were harvested at 1-8 weeks after implantation, and stained with hematoxylin and eosin. It was found that more bone was formed in the composites of collagen-phosphophoryn sponge and osteoblasts than control composites from 1 week to 8 weeks, suggesting that the collagen-phosphophoryn sponge is a good candidate as a scaffold for bone-tissue engineering.     

In vivo biocompatibility of collagen-poly(hydroxyethyl methacrylate) hydrogels

Collagen-p(HEMA) hydrogels were subcutaneously implanted in rats for up to 6 month and the immediate short- and long-term tissue response to these implants was studied. Histopathological data indicated that the tissue reaction at the implant site progressed from an initial acute inflammatory response characterized by the presence of eosinophils and polymorphs to a chronic response marked by few macrophages, foreign body giant cells and fibroblasts. After one month a very thin fibrous capsule (approximately 11 microns thick) was observed around the implant. Even 6 month post-implantation, the capsule thickness was maintained at about 11-12 microns. No necrosis, calcification, tumorigenesis or infection was observed at the implant site up to 6 month. Fibrous capsule analysis showed that the collagen content and the capsule thickness were well within the threshold limits. The collagen-p(HEMA) hydrogels were found to be well-tolerated, non-toxic and highly biocompatible.     

A trial to prepare biodegradable collagen–hydroxyapatite composites for bone repair

This paper is a trial to prepare collagen-hydroxyapatite composites in vitro by an alternate immersion method. Collagen sponges of different biodegradabilities were prepared through chemical cross-linking of Type I collagen with glutaraldehyde (GA) at concentrations of 0.2, 1.0, and 2.0 wt%. The sponges were immersed at 37°C in Tris-HCl-buffered solution containing 200 mM CaCl₂ (pH 7.4) for 2 h and then in an aqueous solution of 120 mM Na₂HPO₄ (pH 9.3) for a 2 h further (one immersion cycle). The alternate immersion cycle was repeated for different times to obtain collagen-hydroxyapatite composites. The characterization of the resulting composites was performed by Fourier transform infrared spectroscopy (FT-IR), X-ray diffraction (XRD), and scanning electron microscopy (SEM). The weight of composites increased with an increase in immersion cycles and the rate of increase became greater with higher GA cross-linking levels for collagen sponge preparation. The pH of the phosphate solution decreased with the immersion cycle, which suggests H⁺ generation accompanied hydroxyapatite formation. Irrespective of the GA concentration and immersion cycle, every composite showed IR absorption bands attributable to phosphate and hydroxyl groups at 950-1100 or 550-650 and 3000-3500 cm^-1 and broad peaks specific to hydroxyapatite on the XRD charts. SEM study revealed small white clusters of hydroxyapatite interspersed uniformly on/in the collagen framework without any preferential orientation. The composite prepared from 0.2 wt% GA cross-linked collagen sponge which showed favourable characteristics was applied to a rat skull defect to evaluate its osteoconductivity as well as biodegradability. The formation of new bone tissue was histologically observed at the defect 12 weeks after application in marked contrast to the collagen sponge alone. The composite degraded without any inflammation reaction. It is concluded that the collagen-hydroxyapatite composite prepared by the present method is a biodegradable biomaterial of osteoconductivity applicable to bone repair.     

Effects of fibroblasts and basic fibroblast growth factor on facilitation of dermal wound healing by type I collagen matrices

Healing of large open dermal wounds is associated with decreased values of the tensile strength even up to 6 months post-wounding. Results of previous studies have shown that healing is facilitated in the presence of a type I collagen sponge by promoting deposition of newly synthesized large-diameter collagen fibers parallel to the fibers of the sponge. In this study healing is evaluated in dermal wounds treated with a collagen sponge seeded with fibroblasts or coated with basic fibroblast growth factor (bFGF). Experimental results indicate that the presence of a collagen sponge results in increased wound tensile strength and increased collagen fiber diameters in the upper dermis 15 days post-wounding in an excisional guinea pig dermal wound model. In comparison, dermal wounds treated with collagen sponges seeded with fibroblasts or coated with bFGF showed increased tensile strengths 15 days postimplantation and increased degree of reepithelialization. These results indicate that fibroblast seeding and bFGF coating in conjunction with a type I collagen sponge matrix facilitate early dermal and epidermal wound healing.     

Novel collagen sponge reinforced with polyglycolic acid fiber produces robust, normal hair in murine hair reconstitution model

The hair reconstitution assay is a useful system for studying cell-cell and epithelial-mesenchymal interaction. The current method consists of transplantation of both epidermal and dermal cells, using a silicone chamber placed on an athymic nude mouse. However, because of leakage and tilting of the grafted cells, the rate and area of hair growth vary depending on the chamber. We modified this method by using a collagen sponge as a scaffold and compared two types of collagen sponges, each having different tensile strengths. A conventional collagen sponge disturbed normal hair follicle formation; in contrast, a collagen sponge containing polyglycolic acid (PGA) fiber supported proper restructuring of skin and hair follicles. These data suggested the usefulness of PGA fiber-containing collagen sponges for hair reconstitution in research and clinical applications.     

Thin collagen film scaffolds for retinal epithelial cell culture

Collagen films have been used in biological implantation and surgical grafts. The development of thin collagen films on the order of 10 microm thick that ensure a planar distribution of implanted cells is a necessary step towards surgical grafts for treatment of age-related macular degeneration (AMD). Here, collagen films were manufactured on a Teflon support to a thickness of 2.4+/-0.2 microm, comparable to that of native Bruch's membrane. Because one important function of Bruch's membrane is allowing the flow of nutrients and waste to and from the retinal pigment epithelium the diffusion properties of the collagen films were studied using blind-well chambers. The diffusion coefficient of the collagen film was determined to be 4.1 x 10(-10)cm(2)/s for 71,200 Da dextran molecules. Viability studies utilizing the blind-well chambers also confirmed that nutrient transport through the films was sufficient to sustain retinal pigment epithelial (RPE) cells. The films were bioassayed in a RPE cell culture model to confirm cell attachment and viability. RPE cells were shown to form an epithelial phenotype and were able to phagocytize photoreceptor outer segments.