Antonios G. Mikos

Biomimetic materials for tissue engineering

The development of biomaterials for tissue engineering applications has recently focused on the design of biomimetic materials that are capable of eliciting specific cellular responses and directing new tissue formation mediated by biomolecular recognition, which can be manipulated by altering design parameters of the material. Biomolecular recognition of materials by cells has been achieved by surface and bulk modification of biomaterials via chemical or physical methods with bioactive molecules such as a native long chain of extracellular matrix (ECM) proteins as well as short peptide sequences derived from intact ECM proteins that can incur specific interactions with cell receptors. The biomimetic materials potentially mimic many roles of ECM in tissues. For example, biomimetic scaffolds can provide biological cues for cell-matrix interactions to promote tissue growth, and the incorporation of peptide sequences into materials can also make the material degradable by specific protease enzymes. This review discusses the surface and bulk modification of biomaterials with cell recognition molecules to design biomimetic materials for tissue engineering. The criteria to design biomimetic materials such as the concentration and spatial distribution of modified bioactive molecules are addressed. Recent advances for the development of biomimetic materials in bone, nerve, and cardiovascular tissue engineering are also summarized.

Preparation and characterization of poly(l-lactic acid) foams

Abstract A particulate-leaching method was developed to prepare highly porous biodegradable polymer membranes. It involves the casting of polymer/salt composite membranes followed by the dissolution of the salt. Poly( l -lactic acid) porous membranes of controlled porosity, surface/volume ratio, and crystallinity were prepared with sodium chloride, sodium tartrate or sodium citrate sieved particles. For salt weight fractions of 50 and 60 wt%, asymmetric membranes were formed, independent of salt particle size. When 70–90 wt% salt was used, the membranes were homogeneous with interconnected pores. The membrane properties were independent of the salt type and were only related to the salt weight fraction and particle size. The porosity increased with the salt weight fraction, and the median pore diameter increased as the salt particle size increased. The polymer/salt composite membranes could be quenched or annealed to yield amorphous or semicrystalline foams with desired crystallinity. All foams were 99.9 wt% salt free and had porosities as high as 0.93 and median pore diameters up to 150 μm.

Poly(ethylenimine) and its role in gene delivery.

Since the first published examination of poly(ethylenimine) (PEI) as a gene delivery vehicle, there has been a flurry of research aimed at this polycation and its role in gene therapy. Here we will briefly review PEI chemistry and the characterization of PEI/DNA complexes used for gene delivery. Additionally, we will note various PEI transfection considerations and examine findings involving other polycationic gene delivery vehicles used with cellular targeting ligands. The current state of our knowledge regarding the mechanism of PEI/DNA transfection will also be discussed. Finally, we will survey toxicity issues related to PEI transfection.

Review: tissue engineering for regeneration of articular cartilage.

Joint pain due to cartilage degeneration is a serious problem, affecting people of all ages. Although many techniques, often surgical, are currently employed to treat this affliction, none have had complete success. Recent advances in biology and materials science have pushed tissue engineering to the forefront of new cartilage repair techniques. This review seeks to condense information for the biomaterialist interested in developing materials for this application. Articular cartilage anatomy, types of injury, and current repair methods are explained. The need for biomaterials, current commonly used materials for tissue-engineered cartilage, and considerations in scale-up of cell-biomaterial constructs are summarized.

Bone formation by three-dimensional stromal osteoblast culture in biodegradable polymer scaffolds

Bone formation was investigated in vitro by culturing stromal osteoblasts in three-dimensional (3-D), biodegradable poly(DL-lactic-co-glycolic acid) foams. Three polymer foam pore sizes, ranging from 150-300, 300-500, and 500-710 microns, and two different cell seeding densities, 6.83 x 10(5) cells/cm2 and 22.1 x 10(5) cells/cm2, were examined over a 56-day culture period. The polymer foams supported the proliferation of seeded osteoblasts as well as their differentiated function, as demonstrated by high alkaline phosphatase activity and deposition of a mineralized matrix by the cells. Cell number, alkaline phosphatase activity, and mineral deposition increased significantly over time for all the polymer foams. Osteoblast foam constructs created by seeding 6.83 x 10(5) cells/cm2 on foams with 300-500 microns pores resulted in a cell density of 4.63 x 10(5) cells/cm2 after 1 day in culture; they had alkaline phosphatase activities of 4.28 x 10(-7) and 2.91 x 10(-6) mumol/cell/min on Days 7 and 28, respectively; and they had a cell density that increased to 18.7 x 10(5) cells/cm2 by Day 56. For the same constructs, the mineralized matrix reached a maximum penetration depth of 240 microns from the top surface of the foam and a value of 0.083 mm for mineralized tissue volume per unit of cross sectional area. Seeding density was an important parameter for the constructs, but pore size over the range tested did not affect cell proliferation or function. This study suggests the feasibility of using poly(alpha-hydroxy ester) foams as scaffolding materials for the transplantation of autogenous osteoblasts to regenerate bone tissue.

Size matters: molecular weight affects the efficiency of poly(ethylenimine) as a gene delivery vehicle.

Poly(ethylenimine) (PEI) samples of various molecular weights and pHs were used to transfect endothelial cells to achieve levels of gene expression for comparison. PEIs with nominal molecular weights of 600, 1200, 1800, 10,000, and 70,000 Da were examined at pHs of 5. 0, 6.0, 7.0, and 8.0, and the results were recorded in terms of transfection efficiencies at 24, 48, 68, 92, and 120 h post-transfection. Trials were performed on the human endothelial cell-derived cell line EA.hy 926. We found that, for the polymers tested, transfection efficiency increased as the molecular weight of PEI increased. Representative values of PEIs at pH 6 and molecular weight 70,000 produced average transfection efficiencies of 25.6 +/- 7.9% (n = 8) at the greatest average expression levels, while PEI of molecular weight 10,000 yielded efficiencies of only 11.4 +/- 1.7% (n = 6). Transfection efficiencies for molecular weight 1,800 PEI were essentially zero, and PEIs of lower molecular weights produced no transfection at all. In contrast, the pH of the PEI solutions had no discernible effect on transfection. Optimal expression of the green fluorescent protein reporter occurred between 2 and 3 days post-transfection. The amount of reporter expression also was noted, as determined by the brightness of fluorescing cells under UV. The data obtained demonstrate that the molecular weight of the PEI carrier has an effect on transfection efficiency while the pH of the PEI solutions prior to DNA complexation has no such effect.

Evolution of bone transplantation: molecular, cellular and tissue strategies to engineer human bone *

Bone defects occur in a wide variety of clinical situations, and their reconstruction to provide mechanical integrity to the skeleton is a necessary step in the patients rehabilitation. The current gold standard for bone reconstruction, the autogenous bone graft, works well in many circumstances. However, autograft reconstruction, along with the available alternatives of allogenous bone graft or poly(methylmethacrylate) bone cement, do not solve all instances of bone deficiency. Novel materials, cellular transplantation and bioactive molecule delivery are being explored alone and in various combinations to address the problem of bone deficiency. The goal of these strategies is to exploit the bodys natural ability to repair injured bone with new bone tissue, and to then remodel that new bone in response to the local stresses it experiences. In general, the strategies discussed in this paper attempt to provide the reconstructed region with appropriate initial mechanical properties, encourage new bone to form in the region, and then gradually degrade to allow the new bone to remodel and assume the mechanical support function. Several of the concepts presented below are already finding clinical applications in early patient trials.

Laminated three-dimensional biodegradable foams for use in tissue engineering

A novel processing technique is reported to construct three-dimensional biodegradable polymer foams with precise anatomical shapes. The technique involved the lamination of highly-porous membranes of porosities up to 90%. Implants with specific shapes were prepared made of poly(L-lactic acid) and copolymers of poly(DL-lactic-co-glycolic acid) to evaluate feasibility. The biomaterials produced have pore morphologies similar to those of the constituent membranes. The pores of adjacent layers of laminated devices are interconnected, resulting in continuous pore structures. The compressive creep behaviour of multilayered devices is also similar to that of the individual layers. Recent discoveries from our group and others that organs and tissues can be regenerated and reconstructed, using cells cultured on synthetic biodegradable polymers, renders this method useful in creating polymer-cell graft for use in cell transplantation.

Fluid flow increases mineralized matrix deposition in 3D perfusion culture of marrow stromal osteoblasts in a dose-dependent manner

Bone is a complex highly structured mechanically active 3D tissue composed of cellular and matrix elements. The true biological environment of a bone cell is thus derived from a dynamic interaction between responsively active cells experiencing mechanical forces and a continuously changing 3D matrix architecture. To investigate this phenomenon in vitro, marrow stromal osteoblasts were cultured on 3D scaffolds under flow perfusion with different rates of flow for an extended period to permit osteoblast differentiation and significant matrix production and mineralization. With all flow conditions, mineralized matrix production was dramatically increased over statically cultured constructs with the total calcium content of the cultured scaffolds increasing with increasing flow rate. Flow perfusion induced de novo tissue modeling with the formation of pore-like structures in the scaffolds and enhanced the distribution of cells and matrix throughout the scaffolds. These results represent reporting of the long-term effects of fluid flow on primary differentiating osteoblasts and indicate that fluid flow has far-reaching effects on osteoblast differentiation and phenotypic expression in vitro. Flow perfusion culture permits the generation and study of a 3D, actively modeled, mineralized matrix and can therefore be a valuable tool for both bone biology and tissue engineering.

In vitro and in vivo degradation of porous poly(DL-lactic-co-glycolic acid) foams.

This study investigated the in vitro degradation of porous poly(DL-lactic-co-glycolic acid) (PLGA) foams during a 20-week period in pH 7.4 phosphate-buffered saline (PBS) at 37 degrees C and their in vivo degradation following implantation in rat mesentery for up to 8 weeks. Three types of PLGA 85 : 15 and three types of 50 : 50 foams were fabricated using a solvent-casting, particulate-leaching technique. The two types had initial salt weight fraction of 80 and 90%, and a salt particle size of 106-150 microm, while the third type had 90% initial weight fraction of salt in the size range 0-53 microm. The porosities of the resulting foams were 0.82, 0.89, and 0.85 for PLGA 85 : 15, and 0.73, 0.87, and 0.84 for PLGA 50 : 50 foams, respectively. The corresponding median pore diameters were 30, 50, and 17 microm for PLGA 85: 15, and 19, 17, and 17 microm for PLGA 50 : 50. The in vitro and in vivo degradation kinetics of PLGA 85: 15 foams were independent of pore morphology with insignificant variation in foam weight, thickness, pore distribution, compressive creep behavior, and morphology during degradation. The in vitro foam half-lives based on the weight average molecular weight were 11.1 +/- 1.8 (80%, 106-150 microm), 12.0 +/- 2.0 (90%, 106-150 microm), and 11.6 +/- 1.3 (90%, 0-53 microm) weeks, similar to the corresponding values of 9.4 +/- 2.2, 14.3 +/- 1.5, and 13.7 +/- 3.3 weeks for in vivo degradation. In contrast, all PLGA 50 : 50 foams exhibited significant change in foam weight, water absorption, and pore distribution after 6-8 weeks of incubation with PBS. The in vitro foam half-lives were 3.3 +/- 0.3 (80%, 106-150 microm), 3.0 +/- 0.3 (90%, 106-150 microm), and 3.2 +/- 0.1 (90%, 0-53 microm) weeks, and the corresponding in vivo half-lives were 1.9 micro 0.1, 2.2 +/- 0.2, and 2.4 +/- 0.2 weeks. The significantly shorter half-lives of PLGA 50: 50 compared to 85: 15 foams indicated their faster degradation both in vitro and in vivo. In addition, PLGA 50: 50 foams exhibited significantly faster degradation in vivo as compared to in vitro conditions due to an autocatalytic effect of the accumulated acidic degradation products in the medium surrounding the implants. These results suggest that the polymer composition and environmental conditions have significant effects on the degradation rate of porous PLGA foams.