Mark Borden

Tissue engineered microsphere-based matrices for bone repair: design and evaluation.

The need for synthetic alternatives to conventional bone grafts is due to the limitations of current grafting materials. Our approach has been to design polymer-based graft substitutes using microsphere technology. The gel microsphere matrix and the sintered microsphere matrix were designed using the random packing of poly(lactide-co-glycolide) microspheres to create a three-dimensional porous structure. The evaluation of these methods dealt with analysis of effects of matrix composition and processing. Matrices were evaluated structurally by scanning electron microscopy and porosimetry, and biomechanically by compression testing. The evaluation revealed the high modulus of the gel microsphere matrix and the versatility of the sintered microsphere matrix. The gel microsphere matrix incorporated hydroxyapatite particles and had a Youngs modulus of 1651 MPa, but structural analysis through SEM revealed a pore system less optimal for bone in-growth. The sintered microsphere matrices were fabricated without hydroxyapatite particles by thermally fusing polymeric microspheres into a three-dimensional array, possessing interconnectivity and a modulus range of 241 (+/-82)-349 (+/-89) MPa. The sintered microsphere matrix demonstrated a connected pore system and mechanical properties in the mid-range of cancellous bone. Porosimetry data indicated that matrix pore diameter varied directly with microsphere diameter, while pore volume was independent of microsphere diameter in the range of diameters examined. The microsphere-based matrices show promise as polymeric substitutes for bone repair.

Structural and human cellular assessment of a novel microsphere-based tissue engineered scaffold for bone repair.

The limitations of current grafting materials have driven the search for synthetic alternatives for the regeneration of trabecular bone. A variety of biodegradable polymer foams composed of 85/15 poly(lactide-co-glycolide) (PLAGA) have been evaluated for such uses. However, structural limitations may restrict the clinical use of these scaffolds. We have developed a novel sintered microsphere scaffold with a biomimetic pore system equivalent to the structure of trabecular bone. By modifying processing parameters, several different sintered microsphere structures were fabricated. Optimization of the structure dealt with modifications to sphere diameter and heating time. Compressive testing illustrated a trend between microsphere diameter and modulus, where increased microsphere diameter resulted in decreased modulus. In addition, evaluation of the pore system showed a positive correlation between sphere diameter and pore diameter. Mercury porosimetry showed increased median pore size with an increased microsphere diameter. Heating time modifications showed that compressive modulus was dependent on the period of heating with longer heating times resulting in higher moduli. It was also shown that heating time did not affect the pore structure. Analysis of the structural data indicated that the microsphere matrix sintered for 4h at a temperature of 160 degrees C with a microsphere diameter of 600-710 microm resulted in an optimal, biomimetic structure with range in pore diameter of 83-300 microm, a median pore size of 210 microm, 35% porosity, and a compressive modulus of 232 MPa. An in vitro evaluation of human osteoblasts seeded onto the sintered matrix indicated that the structure was capable of supporting the attachment and proliferation of cells throughout its pore system. Immunofluorescent staining of actin showed that the cells were proliferating three-dimensionally through the pore system. The stain for osteocalcin was used and showed that cells maintained phenotypic expression for this bone specific protein. Through this work, it was shown that an osteoconductive PLAGA scaffold with a pore system used as a reverse template to the structure of trabecular bone could be fabricated through the sintered microsphere method.

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.

Tissue-engineered bone formation in vivo using a novel sintered polymeric microsphere matrix

We have evaluated in vivo a novel, polymer-based, matrix for tissue engineering of bone. A segmental defect of 15 mm was created in the ulna of New Zealand white rabbits to determine the regenerative properties of a porous polylactide-co-glycolide matrix alone and in combination with autogenous marrow and/or the osteoinductive protein, BMP-7. In this study four implant groups were used: 1) matrix alone; 2) matrix with autogenous marrow; 3) matrix with 20 microg of BMP-7; and 4) matrix with 20 microg of BMP-7 and autogenous marrow. The results showed that the degree of bone formation was dependent on the properties of the graft material. The osteoconductive sintered matrix structure showed significant formation of bone at the implant-bone interface. The addition of autogenous marrow increased the penetration of new bone further into the central area of the matrix and also increased the degree of revascularisation. The osteoinductive growth factor BMP-7 induced penetration of new bone throughout the entire structure of the implant. The most effective treatment was with the combination of marrow cells and osteoinductive BMP-7.

Advancements in tissue engineered bone substitutes

The limitations of autogenous and allogenic bone grafts have driven the search for synthetic alternatives. Using a tissue engineering approach, researchers have developed a series of scaffolds for bone repair applications. In the past year, implants with a variety of structures and compositions have

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 Ewings 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. Taxols bioactivity and radiosensitizing ability were measured using flow cytometry. Co-culture of Ewings 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 Ewings 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.

Structural assessment of a tissue engineered scaffold for bone repair

The limitations of current grafting materials have driven the search for synthetic alternatives to cancellous bone. A variety of biodegradable polymer foams composed of poly(lactide-co-glycolide) [PLAGA] have been evaluated for such uses. However, structural limitations may restrict the clinical use of these scaffolds. We have developed a sintered microsphere scaffold composed of 85:15 poly(lactide-co-glycolide) with a biomimetic pore system equivalent to the structure of cancellous bone. Analysis of the structural data, indicated that the microsphere matrix sintered at a temperature of 160/spl deg/C with a microsphere diameter of 355-425 /spl mu/m resulted in a optimal, biomimetic structure with an approximate pore diameter of 75 to 275 /spl mu/m, 35% porosity, and a compressive modulus of 272 MPa. The in vitro evaluation of human osteoblasts on the sintered matrix indicated that the structure was capable of supporting the attachment and proliferation of the cells throughout its pore system. Immunofluorescent staining of actin showed that the cells were proliferating 3-dimensionally through the pore system. The stain for osteocalcin showed that the cells had maintained the phenotypic expression for this bone specific protein. Through this work, it was shown that an osteoconductive PLAGA scaffold with a pore system equivalent to the structure of cancellous bone could be fabricated through the sintered microsphere method.