Vascularization and improved in vivo survival of VEGF-secreting cells microencapsulated in HEMA-MMA

Vascularization caused by encapsulated cells engineered to secrete vascular endothelial growth factor (VEGF) improved the in vivo survival of the encapsulated cells in a syngeneic mouse Matrigel plug model. Murine fibroblast cells (L929) were engineered to secrete recombinant human vascular endothelial growth factor (rhVEGF(165)). Transfected and nontransfected L929 cells were microencapsulated in a 75:25 hydroxyethyl methacrylate-methyl methacrylate (HEMA-MMA) copolymer. Capsules containing transfected cells induced vascularization in vivo at 1 and 3 weeks postimplantation. In histological sections, a significant positive correlation was seen between the number of capsules and blood vessel density for VEGF-secreting cell capsule implants. New vessels, many positively stained for smooth muscle cells and pericytes, were seen surrounding these VEGF-secreting cell capsule explants. Few vessels were seen in nontransfected L929 capsule implants. The viability of transfected and nontransfected encapsulated cells was assessed on explantation. Although the viability of all encapsulated cells decreased at both 1 and 3 weeks, encapsulated VEGF-secreting cells retained more of the viability than did encapsulated nontransfected control cells. Genetically modified cells promoted vascularization in this context and appeared to enhance the viability of the encapsulated cells, although the extent of the functional benefit was less than expected. Additional effort is required to enhance the benefit, to quantify it, and to understand further the host response to HEMA-MMA microencapsulated cells and tissue constructs, more generally.     

Implantation of porous acrylic cement in soft tissues: an animal and human biopsy histological study

Long-term (8 and 24 month) reactions of the (hypo) dermis of the guinea pig to solid and porous (50 vol%) acrylic implants and four human biopsies from porous subcutaneous acrylic implants were studied light microscopically. The solid implants were encapsulated by dense connective tissue. Mobility was evidenced by the loss of 4 out of 36 after 2 yr and was considered the explanation for the occurrence of ectopic cartilage and mineralized material at some solid implants' surfaces after 2 yr. A dense capsule was not evident with the porous implants, instead vascularized collagenous connective tissues penetrated into and filled the pores, thus anchoring the implant to the body. None of these implants, was lost. Notwithstanding the presence of some multinucleated giant cells, scattered inflammatory cells and loosely packed inflammatory foci with both implant materials, the materials were considered well-tolerated by the body. The histology of the human biopsy did not differ significantly from the porous animal implants.     

Mast cells and tissue reaction to intraperitoneally implanted polymer capsules.

The inflammatory reaction to implanted biomaterials often compromises the clinical usefulness of implantable devices. Dexamethasone, an anti-inflammatory agent, acts on macrophages to decrease production of inflammatory mediators, and on mast cells to prevent degranulation. Systemic administration of dexamethasone (dms) in rats decreases the tissue reaction to intraperitoneally implanted vinyl chloride-acrylic copolymer capsules. Local release of even smaller amounts of dms from a polymeric substrate placed inside an acrylic copolymer capsule may control the tissue reaction while avoiding the undesirable side effects of systemic treatment. Such a system also allows investigation of the local effect of soluble molecules on tissue-material interactions without altering the surface properties of the implant or adding the effect of a releasing material. In the present study, we investigated the effect of dms released from ethylene vinyl acetate (EVAc) rods placed in acrylic copolymer capsules and implanted in the peritoneal cavity of rats. In vitro the release of dms from EVAc rods was quasilinear for 5 weeks. When implanted intraperitoneally into rats, polymer capsules containing EVAc/dms rods generated a tissue reaction that was significantly thinner and featured fewer fibroblast and collagen layers than that around capsules containing pure EVAc rods at all time points studied. The tissue reaction layer was also thinner than that previously described in rats treated systemically with dms. The trabeculae of implants with dms-loaded EVAc rods contained significantly more intact mast cells than implants with EVAc alone, suggesting that degranulation of mast cells is involved in the tissue reaction to intraperitoneal polymer implants.     

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.     

Effects of environmental temperature, relative humidity and vaccination on Pasteurella haemolytica in lungs of mice

Groups of mice were kept in four combinations of environmental temperature (8 and 20 degrees C) and humidity [50 per cent and 90 per cent relative humidity (RH)]. The mice were anaesthetized and inoculated intranasally with Pasteurella haemolytica growing logarithmically. Groups of 11 mice were killed at 0.5 h intervals for 6 h after inoculation and the number of colony-forming-units (CFU) of Pasteurella haemolytica in the lungs was determined. The CFU in the lungs of mice were affected by a significant interaction of temperature, relative humidity and time after inoculation (P less than 0.01). From 0.05 to 5.0 h after inoculation, fewer CFU were found in the lungs of mice kept in 8 degrees C 50 per cent RH than in mice kept in 8 degrees C 90 per cent RH, 20 degrees C 50 per cent RH and 20 degrees C 90 per cent RH. In each environment, CFU in the lungs of mice increased from inoculation to approximately 3.5 h after inoculation and then decreased (P less than 0.01). In a similar experiment in an environment of approximately 20 degrees C and 50 per cent RH, there were significantly fewer CFU in the lungs of mice vaccinated intranasally 21 days earlier with live P. haemolytica than in the lungs of mice either vaccinated intranasally with formalized P. haemolytica or not vaccinated with P. haemolytica (P less than 0.01).     

Effect of an immobilization matrix and capsule membrane permeability on the viability of encapsulated HEK cells

The effect of inclusion of an immobilization matrix and the capsule membrane permeability on the viability, metabolic activity, and proliferation of encapsulated HEK cells was investigated in vitro. In the absence of a matrix, a particular transfected HEK cell line formed a single aggregate in the core of the poly(hydroxyethyl methacrylate-co-methyl methacrylate) (HEMA-MMA) capsule, and the number of live cells decreased significantly with the passage of time. In contrast, co-encapsulation with a 1% (w/v) ultralow gelling temperature agarose matrix promoted the proliferation of the encapsulated cells. The initial number of approximately 200 live cells/capsule doubled 14 d after encapsulation and reached a plateau of approximately 500 live cells/capsule 28 d after encapsulation. The agarose matrix provided uniform distribution of the cells within the capsule core giving rise to multiple aggregates upon proliferation. Reduction of the polymer solution concentration, and hence the increase of the permeability of the capsule membrane, did not have an effect on the extent or rate of proliferation of cells co-encapsulated with agarose, and did not improve the viability of cells that were encapsulated without a matrix. These cells (transfected with the cDNA for human hepatic lipase) served as a model as part of a program evaluating the use of encapsulated cells for gene therapy.     

Myofibroblasts in cotton-induced granulation tissue and the bovine adrenal capsule: morphological aspects

Ultrastructural studies of one week old cotton-induced granulation tissue revealed the presence of polymorphonuclear neutrophils (PMN), macrophages and mast cells, whilst the granulation tissue from two week old cotton implants contained fibroblasts, myofibroblasts and abundant collagen fibres. Similarly, the bovine adrenal capsule contained both fibroblasts and myofibroblasts. It is proposed that the bovine adrenal capsule and two week cotton-induced granulation tissue could be utilized as in-vitro models to study the mechanism(s) involved in myofibroblast contraction.     

Modification of inflammatory response to implanted biomedical materials in vivo by surface bound superoxide dismutase mimics

The healing response to implanted biomedical materials involves varying degrees and stages of inflammation and healing which in some cases leads to device failure. In this article, we describe synthetic methods and in vivo results of a novel surface treatment for biomedical materials involving covalent conjugation of a low molecular weight superoxide dismutase mimic (SODm), which imparts anti-inflammatory character to the material. SODm investigated in this study are a new class of anti-inflammatory drugs consisting of a Mn(II) complex of a macrocyclic polyamine ring that catalyze the dismutation of superoxide at rates equivalent to that of native enzyme. The SODms were covalently linked to small disks of ultra-high molecular weight polyethylene, poly(etherurethane urea), and tantalum metal at two concentrations and implanted in a subcutaneous rat implant model for 3, 7, 14, and 28 days. Histological examination of the implant tissue performed at 3 and 28 days revealed striking anti-inflammatory effects on both acute and chronic inflammatory responses. At 3 days, the formation of a neutrophil-rich acute inflammatory infiltrate seen in control implants was inhibited for all three materials treated with SODm. At 28 days, foreign body giant cell formation (number of FBGCs per field) and fibrous capsule formation (mean thickness of implant capsule) were also significantly inhibited over untreated control implants. A mechanism based on our current understanding of superoxide as an inflammatory mediator at implanted biomedical materials is proposed.     

Formation of granulation tissue in subcutaneously implanted sponges in rats. A comparison between granulation tissue developed in viscose cellulose sponges (Visella) and in polyvinyl alcohol sponges (Ivalon).

A comparison was made between the granulation tissue formation in two different synthetic sponge types. Visella and Ivalon, of different sizes. The granulation tissue formed in the two sponge types did not differ qualitatively, and had the character of wound tissue and inflammatory tissue in man. The rate of tissue formation in the Visella sponges was faster and the tissue was more homogenous than in the Ivalon sponges. Fourteen-day-old Visella implants of either size contained more granulation tissue than Ivalon sponges, probably owing to the smaller pore size of the former material. This may also account for the more frequent occurrence of giant cells in the Visella implants. In contrast to the Visella sponges, the trabeculae of the Ivalon polymer showed calcification and positive staining properties with histological staining procedures, and deformation was frequent among the Ivalon implants. Thin sponges of either type closed in about 21 days, thick ones after about 42 days of implantation. Calculated per 2 cm3 of implant, thin sponges produced more tissue after 14 days of implantation than thick noes. It is concluded that the Visella sponge type is best suitable for this experimental model of inflammation.     

Immunohistochemical and ultrastructural study of subacute thyroiditis, with special reference to multinucleated giant cells

Twenty-four cases of subacute thyroiditis were examined immunohistochemically and ultrastructurally, with special attention being directed to the multinucleated giant cells that characterize the lesions of this condition. Immunohistochemical study revealed that 99 per cent of the giant cells contained lysozyme, 82 per cent contained vimentin, 66 per cent contained alpha 1-antitrypsin, and 61 per cent contained thyroxine. Only 17 per cent contained thyroglobulin, 5 per cent contained keratin, 16 per cent contained epithelial membrane antigen, and 4 per cent contained leukocyte common antigen. Mononuclear cells infiltrating the follicular spaces showed a staining pattern similar to that of the multinucleated giant cells. Desquamated follicular epithelial cells were strongly positive for thyroglobulin and thyroxine, while regenerated epithelia that formed small follicles were positive for thyroglobulin but negative for thyroxine. Electron microscopic examination of cell ultrastructure revealed that most multinucleated giant cells had structures identical with those of infiltrating mononuclear cells, particularly with histiocytes, and that giant cells were formed by mononuclear cell fusion. These findings indicate the majority of multinucleated giant cells in subacute thyroiditis are derived from mononuclear-histiocyte cells.     

In vivo delivery of recombinant human growth hormone from genetically engineered human fibroblasts implanted within Baxter immunoisolation devices

Continuous delivery of therapeutic peptide to the systemic circulation would be the optimal treatment for a variety of diseases. The Baxter TheraCyte^® system is a membrane encapsulation system developed for implantation of tissues, cells such as endocrine cells or cell lines genetically engineered for therapeutic peptide delivery in vivo. To demonstrate the utility of this system, cell lines were developed which expressed human growth hormone (hGH) at levels exceeding 1 microgram per million cells per day. These were loaded into devices which were then implanted into juvenile nude rats. Significant levels of hGH of up to 2.5 ng/ml were detected in plasma throughout the six month duration of the study. In contrast, animals implanted with free cells showed peak plasma levels of 0.5 to 1.2 ng four days after implantation with no detectable hGH beyond 10 days. Histological examination of explanted devices showed they were vascularized and contained cells that were viable and morphologically healthy. After removal of the implants, no hGH could be detected which confirmed that the source of hGH was from cells contained within the device. The long term expression of human growth hormone as a model peptide has implications for the peptide therapies for a variety of human diseases using membrane encapsulated cells.     

High molecular weight caldesmon positive stromal cells in the capsule of thyroid follicular tumours and tumour-like lesions

AIMS: To investigate the smooth muscle nature of the spindle stromal cells in the capsule of thyroid tumours and tumour-like lesions. METHODS: Immunostaining for high molecular weight caldesmon (HCD), a highly specific marker for smooth muscle differentiation, was performed in 70 primary thyroid tumours and tumour-like lesions (21 hyperplastic nodules, 29 follicular adenomas, five minimally invasive follicular carcinomas, six widely invasive follicular carcinomas, and nine encapsulated papillary carcinomas). RESULTS: HCD positive stromal cells (HCD+ cells) were detected in the capsule of 20 of the 21 hyperplastic nodules, and all of the 29 follicular adenomas and five minimally invasive follicular carcinomas, whereas HCD+ cells were seen in the capsule of only four of the six widely invasive follicular carcinomas and no HCD+ cells were seen in the capsule of the nine encapsulated papillary carcinomas examined. CONCLUSIONS: The presence of HCD+ cells in the capsule is characteristic of thyroid follicular tumours and tumour-like lesions. The stromal cells in the capsule of thyroid follicular tumours and tumour-like lesions are different from those of encapsulated papillary carcinoma.     

Cellular reactions and bone apposition to titanium surfaces with different surface roughness and oxide thickness cleaned by oxidation

Titanium surfaces with three different surface characteristics were exposed to an intraperitoneal milieu in mouse or rat, or inserted into rabbit bone. The cleaning regimen of the TiO2 surfaces in this study included oxidation by heat or acid and a final rinsing and storage in water. Intraperitoneal exposure ranged from 1 to 64 min and the healing period in bone was 6 weeks. Cell recruitment to the surfaces was quantified by acridine orange staining and specific antibodies directed against cell membrane antigens. Removal torque, bone-to-metal contact, total bone area and histological evaluations were used to evaluate fixture stability and the healing-in of the implants. After the healing period of 6 weeks only a transient significant difference was seen in the total number of cells adherent on the surfaces. No significant differences were observed between any of the surfaces for removal torque, bone-to-metal contact, or bone area. The areas lacking bone-to-metal contact were filled with normal vascularised connective tissue with no signs of fibrous capsule formation or giant cells. These findings differ from findings published earlier of Ti implants that underwent a cleaning regimen with alcohol as the final rinsing step. The tissues around the implants were richly vascularised and there was continued bone growth toward the surfaces. The bone-to-metal contact in this study was lower than that seen with alcohol-cleaned TiO2.     

Quantitative in-vivo studies on angiogenesis in a rat sponge model.

A method for quantitative in-vivo studies on angiogenesis is described in this article. It is based on subcutaneous implantation of sterile polyester sponges in the rat and subsequent measurement of blood flow in the implants as they become vascularized. The blood flow in an implant was measured in terms of per cent 133Xe-saline clearance 6 min after the radio-isotope was injected into the sponge via a cannula attached to it. Since originally the sponge contained no blood vessels, the development of blood flow would represent a neovascularization. Histological examination of implants removed at fixed time intervals confirmed that the sponges were initially encapsulated by granulation tissue and gradually infiltrated by host blood vessels. Under standard conditions, the 133Xe clearance from sponges 16 days post-implantation approached the clearance obtained in normal skin. The new blood vessels in the sponges were reactive to vasodilator prostaglandin-E2 and vasoconstrictor noradrenaline applied topically. Furthermore, we have shown that local administration of endothelial cell growth supplement accelerated angiogenesis while protamine delayed its onset. Thus the model offers a new means for objective, continuous and reproducible studies on the controlling mechanisms of angiogenesis.     

Quantitative in-vivo studies on angiogenesis in a rat sponge model

A method for quantitative in-vivo studies on angiogenesis is described in this article. It is based on subcutaneous implantation of sterile polyester sponges in the rat and subsequent measurement of blood flow in the implants as they become vascularized. The blood flow in an implant was measured in terms of per cent 133Xe-saline clearance 6 min after the radio-isotope was injected into the sponge via a cannula attached to it. Since originally the sponge contained no blood vessels, the development of blood flow would represent a neovascularization. Histological examination of implants removed at fixed time intervals confirmed that the sponges were initially encapsulated by granulation tissue and gradually infiltrated by host blood vessels. Under standard conditions, the 133Xe clearance from sponges 16 days post-implantation approached the clearance obtained in normal skin. The new blood vessels in the sponges were reactive to vasodilator prostaglandin-E2 and vasoconstrictor noradrenaline applied topically. Furthermore, we have shown that local administration of endothelial cell growth supplement accelerated angiogenesis while protamine delayed its onset. Thus the model offers a new means for objective, continuous and reproducible studies on the controlling mechanisms of angiogenesis.     

Formation of Multinucleate Giant Cells in Organized Epithelioid Cell Granulomas

Organized epithelioid cell granulomas were produced experimentally by injecting intradermally dilute suspensions of zirconium and beryllium salts into individuals who had been previously sensitized to these metals. Biopsies were obtained at intervals of between 5 days and 13 months later, and the specimens were fixed and prepared for light and electron microscopy. Tritiated thymidine was injected into a number of the granulomas; the biopsy specimens were secured from 40 minutes to 2 or more weeks later, and the tissues were processed for light microscopic autoradiography. Giant cells occurred commonly, both within organized tubercles and in relation to areas of necrosis, and had markedly different cytoplasmic features from typical secretory epithelioid cells which enabled them to be readily recognized at scanning magnifications. The characteristic hallmark of these giant cells was the presence of myriads of small mitochondria adjacent to nuclei with numerous membrane-lined vesicles in the center of the cell. Giant cells occurred mainly in edematous disrupted tubercles. In these tubercles, epithelioid cells contained cytoplasmic components more like giant cells. Direct evidence of cell fusion was not seen, although fusion of membranes seemed to occur between cells having similar cytoplasmic features. The failure to find labeled nuclei in any giant cells 40 minutes after injection of tritiated thymidine indicates that normal nuclear division does not occur within giant cells. We postulate that epithelioid cells containing vesicles develop in damaged and necrotic areas, and that mainly this type of epithelioid cell fuses to form giant cells.     

Vascularization and tissue infiltration of a biodegradable polyurethane matrix

Urethanes are frequently used in biomedical applications because of their excellent biocompatibility. However, their use has been limited to bioresistant polyurethanes. The aim of this study was to develop a nontoxic biodegradable polyurethane and to test its potential for tissue compatibility. A matrix was synthesized with pentane diisocyanate (PDI) as a hard segment and sucrose as a hydroxyl group donor to obtain a microtextured spongy urethane matrix. The matrix was biodegradable in an aqueous solution at 37 degrees C in vitro as well as in vivo. The polymer was mechanically stable at body temperatures and exhibited a glass transition temperature (Tg) of 67 degrees C. The porosity of the polymer network was between 10 and 2000 microm, with the majority of pores between 100 and 300 microm in diameter. This porosity was found to be adequate to support the adherence and proliferation of bone-marrow stromal cells (BMSC) and chondrocytes in vitro. The degradation products of the polymer were nontoxic to cells in vitro. Subdermal implants of the PDI-sucrose matrix did not exhibit toxicity in vivo and did not induce an acute inflammatory response in the host. However, some foreign-body giant cells did accumulate around the polymer and in its pores, suggesting its degradation is facilitated by hydrolysis as well as by giant cells. More important, subdermal implants of the polymer allowed marked infiltration of vascular and connective tissue, suggesting the free flow of fluids and nutrients in the implants. Because of the flexibility of the mechanical strength that can be obtained in urethanes and because of the ease with which a porous microtexture can be achieved, this matrix may be useful in many tissue-engineering applications.     

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.     

Normal wound healing compared to healing within porous Dacron implants

This study examined the hypothesis that healing within porous implants differs from that in normal connective tissue. Special attention was given to extracellular components including collagen, reticular fibers, and ground substance, and to enzymes associated with activated macrophages. Using Dacron velour and the rabbit as host, the healing of normal connective tissue and that of the tissue/implant interface were histologically compared 10 and 28 days postimplantation. The results exhibited significant differences between connective tissue healing, implant capsule formation, and granulation tissue generation. The healing of connective tissue and implant capsule formation were essentially complete at 28 days. However, tissue inside the implant was qualitatively different and did not significantly change between 10 and 28 days. It was characterized by macrophages and giant cells, a predominantly acid mucopolysaccharide ground substance, and qualitatively fewer and less well defined collagen and reticular fibers were observed than in normal wound healing. Thus we conclude that the connective tissue inside Dacron velour does not resemble normal connective tissue after 10 or 28 days of healing. Furthermore, the collagen never fully matures into orderly bundles, a phenomenon which may be related to an altered mucopolysaccharide composition and a diminished reticular network. The lysosomal enzymatic activity of the macrophages and perhaps the giant cells at the tissue/implant interface may be linked to these differences.     

Reaction of skeletal muscle to small implants of titanium or stainless steel: a quantitative histological and autoradiographic study.

Small wire implants of titanium or stainless steel were inserted into mouse leg muscles to test the reaction of regenerating skeletal muscle. Muscle fibres regenerated rapidly, starting at 3 d; by 2 wk all implants were encapsulated in thin (10 microns) fibrous tissue capsule surrounded by myotubes for 15-20 microns. Quantitatively there were no detectable differences in muscle regeneration between the two metals. The initiation of myoblast precursor cell replication was determined in regenerating muscle next to the implants. Tritiated thymidine was injected 18-156 h after implant insertion and labelled myotube nuclei in the regenerated muscle indicated that their precursors had started DNA synthesis 24 h after implant insertion. This is similar to myogenesis in many other muscle lesions and indicated that neither titanium nor stainless steel retarded muscle regeneration.