Poly(lactide-co-glycolide)/hydroxyapatite delivery of BMP-2-producing cells: a regional gene therapy approach to bone regeneration.

Currently, functional treatment of fracture non-unions and bone loss remains a significant challenge in the field of orthopaedic surgery. Tissue engineering of bone has emerged as a new treatment alternative in bone repair and regeneration. Our approach is to combine a polymeric matrix with a cellular vehicle for delivery of bone morphogenetic protein-2 (BMP-2), constructed through retroviral gene transfer. The objective of this study is to develop an osteoinductive, tissue-engineered bone replacement system by culturing BMP-2-producing cells on an osteoconductive, biodegradable, polymeric-ceramic matrix. The hypothesis is that retroviral gene transfer can be used effectively in combination with a biodegradable matrix to promote bone formation. First, we examined the in vitro attachment and growth of transfected BMP-producing cells on a PLAGA-HA scaffold. Second, the bioactivity of the produced BMP in vitro was evaluated using a mouse model. It was found that the polymer-ceramic scaffold supported BMP-2 production, allowing the attachment and growth of retroviral transfected, BMP-2-producing cells. In vivo, the scaffold successfully functioned as a delivery vehicle for bioactive BMP-2, as it induced heterotopic bone formation in a SCID mouse model.

Regional drug delivery with radiation for the treatment of Ewing's sarcoma. In vitro development of a taxol release system.

Recently, several studies have suggested the radiosensitizing effect of taxol, a microtubular inhibitor. Our overall hypothesis is that a combination of radiation and taxol may demonstrate therapeutic efficacy over doses of either individually. Studies examining taxol use have mostly focused on systemic administration, which can lead to undesired effects. To circumvent these side effects, we propose a locally administered polymeric microsphere delivery system combined with radiation therapy for the treatment of Ewing's sarcoma. The present study focuses on the in vitro ability of taxol when present as a microencapsulated drug delivery system, and delivered locally at the site of the sarcoma/tumor, to block cells in the G2/M phase of the cell cycle and potentially enhance the radiation sensitivity of cells. Using the bioresorbable poly(anhydride-co-imide), poly[pyromellityl-imidoalanine-1,6-bis(carboxy-phenoxy)hexane] (PMA-CPH), and the radiosensitizing agent taxol, a microsphere based delivery system was fabricated. A solvent evaporation technique was used to encapsulate taxol at doses of 1%, 5%, and 10% in PMA-CPH microspheres. Release kinetics studies demonstrated that the total amount of taxol released and the release rate were directly dependent on loading percentage. Taxol's bioactivity and radiosensitizing ability were measured using flow cytometry. Co-culture of Ewing's sarcoma cells with and without taxol-loaded microspheres demonstrated that released taxol retained its bioactivity and effectively blocked cells in the radiosensitive G2/M phase of mitosis. The taxol-radiation delivery system studied achieved an 83% decrease in tumor cell count compared to control. Taxol effectively sensitized Ewing's sarcoma cells to radiation with radiosensitivity shown to be independent of radiation dose at levels of dosages studied. This work has demonstrated that taxol can be effectively released from a biodegradable PMA-CPH microsphere delivery system while maintaining potent combined cytotoxic and radiosensitizing abilities.

Circulating antipericyte autoantibodies in diabetic retinopathy.

PURPOSE: A number of reports suggest that autoimmune mechanisms may play a role in diabetic microangiopathy, and the existence of circulating antiendothelial cell autoantibodies in diabetic retinopathy has been reported. Because capillary pericyte injury/dysfunction is considered to be an early event in the pathogenesis of diabetic retinopathy and pericyte "drop out" or loss is considered pathognomonic in diabetic retinopathy, we screened diabetic sera for the presence of circulating antipericyte autoantibodies. METHODS: Diabetic sera were screened for the presence of antipericyte autoantibodies by indirect immunofluorescence on tissue cultured bovine retinal pericytes. Analysis of autoantibody prevalence data was performed with 2x2 contingency tables analyzed using Fisher's exact test. RESULTS: Diabetic subjects were found to have autoantibodies to microvascular pericytes in their circulation. The prevalence of these antibodies declines with increasing severity of retinopathy. A peak of antibody prevalence was seen at 5-10 years' duration of diabetes. These autoantibodies were found in both Type I and Type II diabetics and in pred iabetics. CONCLUSIONS: The finding of antipericyte autoantibodies in the circulation of a subpopulation of diabetic subjects suggests that the immune system may play a role in the early pathophysiology of diabetic retinopathy in some patients. These results may contribute to understanding why retinopathy progresses in some patients despite consistent reduction of blood sugar.

Proliferation, morphology, and protein expression by osteoblasts cultured on poly(anhydride-co-imides).

In vitro cell biocompatibility models are crucial in the study of any newly synthesized material. Our focus has been on the development of a new class of biocompatible, degradable, high-strength polymeric materials, the poly(anhydride-co-imides), for use in bone regeneration. This study examined osteoblast cell adherence, proliferation, viability, and phenotypic preservation on the surface of the poly(anhydride-co-imide) poly[pyromellitylimidoalanine (PMA-ala):1,6-bis(carboxyphenoxy) hexane (CPH)] over a period of time. Cell proliferation on PMA-ala:CPH degradable matrices over 21 days was examined. Throughout the 21-day period of study, osteoblast proliferation was similar on PMA-ala:CPH and on tissue culture polystyrene controls. Osteoblasts maintained their characteristic morphology as demonstrated by both scanning electron microscopy and immunofluorescence studies. Alkaline phosphatase activity for cells grown on PMA-ala:CPH was confirmed. Retention of the osteoblastic phenotype was demonstrated using immunofluorescence techniques and staining with antibodies against osteocalcin (an extracellular matrix protein of bone) and osteopontin (a marker of cell adhesion). Radioimmunoassay results provided evidence that levels of osteocalcin production by osteoblasts were similar when cells were cultured on PMA-ala:CPH and on tissue culture polystyrene controls. The present study provided evidence of normal osteoblast function on PMA-ala:CPH surfaces. PMA-ala:CPH may therefore be useful as a synthetic material for orthopedic applications.

Coculture system to assess biocompatibility of candidate orthopaedic materials.

When examining the biocompatibility of materials for orthopaedic use, it is important to examine the in vitro response of osteoblasts and macrophages to the material. The purpose of the current study was to develop an in vitro system that more accurately models the dynamic macrophage and osteoblast interactive response to biomaterials. A filter coculture system was designed that enables two cell types to be separated, while permitting cellular mediators to diffuse through its porous membrane. Using the filter coculture system, macrophage and osteoblast interaction in response to polymethylmethacrylate particle exposure was examined. As a marker of bone resorption potential, the level of prostaglandin E2 released from the macrophage and osteoblast coculture was compared with traditionally used in vitro culture systems. Cells interacting in the filter coculture system were found to produce prostaglandin E2 levels significantly greater than those of traditional conditioned media transfer systems. This filter coculture system offers an in vitro model that accounts for the continual cell to cell mediator interaction of two cell types simultaneously exposed to an implant material. This system may be useful in examining the biocompatibility of candidate materials at the bone interface, and thus elucidating the mechanisms of material induced bone resorption.

Tissue engineered bone-regeneration using degradable polymers: the formation of mineralized matrices.

In the development of 3-dimensional cell-polymer matrices for tissue engineering, the ability of osteoblast cells to maintain their phenotypic properties and form a mineralized matrix while seeded on the polymer surface is very important. Osteoblast cell differentiation and bone formation using rat calvaria cells were studied on the surface of a porous poly(lactide/glycolide)/hydroxyapatite (PLAGA/HA) 3-dimensional polymer matrix. Cell adhesion and proliferation were determined at 24 hr, 3, 7, 14, and 21 days. Cell attachment and proliferation were observed to increase throughout the first two weeks of the study, followed by a period of gradual plateauing of cell numbers. Environmental scanning electron microscopy demonstrated that cells grown on the surface of the 3-dimensional porous PLAGA/HA matrix retained their characteristic morphology and grew in a multi-layer fashion. Light microscopy observations of experiment cultures revealed active osteoblastic cells forming a characteristic mineralized matrix in the presence of beta-glycerophosphate as a phosphate donor. Mineralization did not occur in media either not supplemented with beta-glycerophosphate or when the matrix without cells was incubated with the reagents, indicating that the mineralization was due to the cells and not the HA in the matrix. These results suggest that the 3-dimensional PLAGA/HA matrix could provide a matrix for bone cell differentiation and mineralization in vitro and, therefore, may be a candidate as a synthetic implant for bone regeneration.

In vitro bone biocompatibility of poly (anhydride-co-imides) containing pyromellitylimidoalanine.

Poly(anhydride-co-imides) are currently under study for applications involving bone. The cytotoxicity of a series of poly(anhydride-co-imides) with osteoblast-like cells (MC3T3-E1) was evaluated. The imide component of the copolymers was based on pyromellitylimidoalanine and the anhydride component was based on either sebacic acid or 1,6-bis(carboxyphenoxy)hexane. Cell adhesion and proliferation on the surfaces of the polymer discs were observed by environmental scanning electron microscopy. During the first 24 hours of attachment, the cells showed normal morphology when cultured on copolymers containing 1,6-bis(carboxyphenoxy)hexane. The cells did not adhere to the polymers containing sebacic acid, probably due to the rapid degradation of the polymer surfaces. Concurrently, the effects of polymer breakdown products on osteoblast-like cells were evaluated by studying their proliferation (cell numbers), viability (dye exclusion), morphology (light microscopy), and phenotypic expression. The morphology of osteoblast-like cells cultured in the presence of the polymer breakdown products pyromellitylimidoalanine and pyromellitic acid was found to be similar to that of the same cells grown on tissue culture polystyrene and consisted of a characteristic polygonal shape. With use of a monoclonal antibody to osteocalcin, these cells were shown to demonstrate preserved osteoblast phenotype with growth over a 21-day period. In addition, the cells reached confluency after 3-4 days, similar to cells grown on tissue culture polystyrene. This in vitro evaluation showed that the poly(anhydride-co-imides) evaluated are non-cytotoxic and may be viable biomaterials for orthopaedic applications.

Three-dimensional degradable porous polymer-ceramic matrices for use in bone repair.

A degradable polymer-ceramic matrix for use as a bone graft material is described. The fabrication method used produces 3-dimensional macroporous matrices which are structurally similar to cancellous bone in their porosity, mechanically similar to cancellous bone in compressive elastic modulus and chemically comparable to the mineral matrix of bone in that they contain hydroxyapatite (HA). A 50:50 copolymer of poly(lactide/glycolide) (PLAGA) reinforced by a particulate calcium phosphate ceramic, HA, was used to create a matrix composed of polymeric microspheres. The channels between these spheres were pores approximately 100 microns in diameter. Four polymer/ceramic ratios were used in matrix fabrication: 1:0, 1:1, 2.5:1, and 5:1. The mechanical behavior of the material was found to vary with ceramic content. Increased levels of HA resulted in increased compressive elastic moduli. Prior to polymer degradation, moduli ranged from a high of 1459 MPa (50% HA) to a low of 293 MPa (0% HA). Degradation studies over a 6-week period showed that 0 and 16.7% HA-containing matrices lost up to 50% of their original weight, while the 28.6 and 50% IIA-containing matrices lost up to 20% of their original weight. Increased HA matrix content translated into decreased rates of matrix degradation. Environmental scanning electron microscopy (ESEM) confirmed that the polymer matrix contained pores that were interconnected during degradation. Viewed via ESEM, 10% HA containing matrices completely degraded by 6 weeks, while 50% HA matrices remained relatively stable. These studies indicate that the porous 3-dimensional polymer/ceramic matrix may potentially be useful as a synthetic material for bone repair.

Cytotoxicity testing of poly(anhydride-co-imides) for orthopedic applications.

The cytotoxicity of a series of poly(anhydride-co-imides) with osteoblast-like cells (MC3T3-E1) was evaluated. The imide component of the copolymers was based on trimellitylimidoglycine (TMA-gly), and the anhydride component was based on either sebacic acid (SA) or 1,6-bis(carboxyphenoxy)hexane (CPH). Cell adhesion and proliferation on surfaces of the polymer discs were observed by environmental scanning electron microscopy (ESEM). During the first 24 h of attachment, cells showed normal morphology when cultured on the various copolymers of CPH. Concurrently, the effects of polymer breakdown products on osteoblast-like cells were evaluated by studying their proliferation (cell numbers), viability (dye exclusion), and morphology (light microscopy). Cell cultures in the presence of these breakdown products resulted in normal morphologies and reached confluency after 7 days. This initial in vitro evaluation with osteoblast-like cells suggests that the poly(anhydride-co-imides) may be viable carriers for osteoblasts.

Osteoblast-like cell adherance and migration through 3-dimensional porous polymer matrices.

Osteoblast cells collected from rat calvaria were plated onto a biodegradable 3-dimensional porous composite of poly(lactide-co-glycolide) and hydroxyapatite to quantitatively assess the matrix's ability to support living cell adhesion throughout an initial 24 hour period. Numbers of cells adhering to the polymer exceeded the plating number of cells, demonstrating that during the 24 hour period, proliferation of cells began. Using immunofluorescent staining for anti-osteocalcin, an exclusive marker for bone cells, osteoblasts were seen to adhere to both the exterior surface of the polymer and to have migrated to the interior surface of the matrix. It is proposed that this biodegradable cell/polymer composite may be useful in bone grafting applications. These studies demonstrated early cellular attachment, proliferation and ingrowth of osteoblast cells can occur within the matrix, with preservation of bone cell phenotypes.

Immunofluorescence and confocal laser scanning microscopy studies of osteoblast growth and phenotypic expression in three-dimensional degradable synthetic matrices.

In the development of three-dimensional cell-polymer synthetic matrices for tissue regeneration, visualization of cells growing in these porous structures can be difficult. The focus of this study was the development and use of a novel method that would allow for visualization of osteoblasts inside opaque matrices. The morphologic responses and phenotypic characterization of osteoblasts as they attach, spread, and migrate through a porous three-dimensional biodegradable polymer-ceramic matrix in vitro were studied using immunofluorescence and confocal laser scanning microscopy (CLSM). CLSM offers several advantages over the most commonly used imaging methods [traditional light microscopy and scanning electron microscopy (SEM)]. CLSM filters out-of-focus background and provides more structural details of cells. In addition, CLSM does not require extensive sample preparation as does SEM. When used in conjunction with fluorescence-labeled antibodies to identify cells and their products, it can characterize morphology of growing cells and successfully determine phenotypic function. Using monoclonal antibody to osteocalcin, a bone cell-specific protein, cells throughout the matrix were found to have preserved osteoblast-like phenotype with growth. The morphology of cells throughout the matrix was found to be similar to osteoblast cells grown on tissue culture polystyrene and consisted of spread polygonal forms. Using the technique of CLSM with immunofluorescent antibodies, we have demonstrated for the first time that these three-dimensional degradable polymer matrices can support osteoblast growth and phenotypic expression throughout its structure.

Role of epicardial mesothelial cells in the modification of phenotype and function of adult rat ventricular myocytes in primary coculture.

Adult rat ventricular myocytes undergo a well-documented sequence of phenotypic changes during adaptation to primary culture. However, we observed that coculture of myocytes with a specific subset of nonmyocyte cardiac cells could slow and even reverse the process of adaptation. These nonmyocyte cells were isolated and identified by immunohistochemical and ultrastructural criteria as being of epicardial mesothelial origin. When added to long-term primary cultures of adult ventricular myocytes, epicardial mesothelial cells appeared to induce myofibrillar arrays that were more organized than those seen in noncocultured myocytes; these changes that occurred were concurrent with the appearance of large amplitude contractions and multicellular synchronous beating that was facilitated by gap junctions between myocytes and epicardial mesothelial cells. The changes in morphology and function were accompanied by a marked increase in beta-myosin heavy chain isoform transcription in cocultured myocytes, a return to the ratio of cardiac to skeletal alpha-actin expected in adult rat myocardium, and a much reduced expression of smooth muscle alpha-actin. These changes in myocyte phenotype and function appeared to require epicardial cell-myocyte contact, or close apposition, because media conditioned by epicardial mesothelial cells alone or in coculture had no effect. Thus, these rapid and reversible changes in myocyte ultrastructure, function, and gene expression may provide a useful in vitro model with which to study the mechanism responsible for regulating the plasticity of ventricular myocyte phenotype and the role of specific cell-cell interactions.

Expression of a monoclonal antibody (3G5) defined ganglioside antigen in the renal cortex.

The monoclonal antibody (mAb) 3G5 was found, by indirect immunofluorescence, to bind to renal cortical structures in frozen sections of human, rat and calf kidneys. Double indirect immunofluorescence studies on frozen sections of rat kidneys showed that 3G5 stained only the glomerulus and the distribution of the 3G5 antigen on the glomerulus was more extensive than the staining observed with antibodies to Factor VIII antigen. 3G5 stained the proximal convoluted tubules and collecting tubules in bovine renal sections but glomeruli did not stain with 3G5. The 3G5 mAb did not stain tissue cultured bovine glomerular endothelial cells or mesangial cells, but did stain bovine glomerular epithelial cell cultures. 3G5 did not stain MDCK cell cultures. The binding of mAb 3G5 to glomeruli was investigated by immunoelectron microscopy of rat renal tissue. In contrast to the podocyte specificity on bovine glomerular cells in vitro, it was found that the specificity of 3G5 expression on rat glomerular cells in vivo was broader. No binding of mAb 3G5 was found outside the glomerulus in the rat renal cortex. Podocytes, endothelial cells and capsular epithelial cells expressed the 3G5 antigen most strongly. A lesser amount of binding was found in the glomerular basement membrane. The mesangium showed a little binding of mAb 3G5 and no binding at all was found to other cortical structures. The 3G5 antigen in rat renal tissue was found to be a glycolipid that migrated between the ganglioside markers GM2 and GM1 by immunostaining of thin layer chromatograms.(ABSTRACT TRUNCATED AT 250 WORDS)