Bone morphogenetic proteins (BMPs) are considered to have important regulatory roles in skeletal embryogenesis and bone healing. Recombinant human BMPs (rhBMPs) have been shown to heal critical size defects and promote spinal fusion. We studied the effects of rhBMP-2 in an absorbable collagen sponge (ACS) on bone healing in a large animal tibial fracture model. Bilateral closed tibial fractures were created in 16 skeletally mature goats and reduced and stabilized using external fixation. In each animal, one tibia received the study device (0.86 mg of rhBMP-2/ACS or buffer/ACS), and the contralateral fracture served as control. The device was implanted as a folded onlay or wrapped circumferentially around the fracture. Six weeks following fracture, the animals were sacrificed and the tibiae harvested for torsional testing and histomorphologic evaluation. Radiographs indicated increased callus at 3 weeks in the rhBMP-2/ACS treated tibiae. At 6 weeks, the rhBMP-2/ACS wrapped fractures had superior radiographic healing scores compared with buffer groups and controls. The rhBMP-2/ACS produced a significant increase in torsional toughness (p ‫؍‬ 0.02), and trends of increased torsional strength and stiffness ( p ‫؍‬ 0.09) compared with fracture controls. The device placed in a wrapped fashion around the fracture produced significantly tougher callus ( p ‫؍‬ 0.02) compared with the onlay application. Total callus new bone volume was significantly increased ( p ‫؍‬ 0.02) in the rhBMP-2/ACS fractures compared with buffer groups and controls regardless of the method of device application. The rhBMP-2/ACS did not alter the timing of onset of periosteal/ endosteal callus formation compared with controls. Neither the mineral apposition rates nor bone formation rates were affected by rhBMP-2/ACS treatment. The increased callus volume associated with rhBMP-2 treatment produced only moderate increases in strength and

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Porous matrices of biodegradable polymers are extensively used as scaffolds in tissue engineering and as drug delivery devices. A critical component of the design, processing, and utility of such polymeric systems concerns the local void microarchitecture. In this study, a novel approach based on confocal fluorescence imaging was employed to visualize and quantify in 3 dimensions (3-D) the individual and population-level void morphology within porous polymeric matrices. Poly(lactic acid-glycolic acid) copolymer matrices were cast to yield void configurations of variable void sizes but constant cumulative voidage. Using confocal microscopy, fluorescently saturated polymer matrices were optically sectioned into serial 2-D images, and 3-D void contours were reconstructed via object discrimination and connectivity analysis. The resultant data was used to quantitate the matrix microstructure and map its evolution following polymer degradation. Under conditions of accelerated degradation, matrix erosion was found to cause a significant change in the disposition of voids; this involves two processes (void formation and void enlargement), the extent of which was influenced by the initial void size and the duration of erosion. By virtue of providing both static and dynamic descriptions of the void morphology in poly(lactic acid-glycolic acid) matrices, this is the first spatiotemporal study of the 3-D microarchitecture of porous, bioerodible tissue analog matrices.

In vivo, motile epidermal tissues frequently encounter ligand microinterfaces that are dynamic, owing to rapid cell-mediated substrate phagocytosis. In this study, we have examined cell motility phenomena in response to the adhesion ligand, collagen, which was presented on cell-internalizable 400-nm colloidal gold microcarriers. Normal human keratinocytes were seeded onto collagen-adsorbed poly(lactide-co-glycolide) (PLGA) films that were predeposited with varying densities of the ligand-associated microcarriers (LAMs), such that the overall ligand density and the ligand loading per microcarrier were invariant. Cells seeded on LAMs exhibited rapid and distinct cytoskeletal redistribution resulting in numerous filopodial extensions, indicative of activation of cell motility processes. We report that the population-averaged cell migration rate of cells, mu, was increased from 2 microm2/min on collagen substrates lacking the microcarriers, to approximately 50 microm2/min on collagen presenting LAMs. An increase in LAM density to 1.2 LAMs per microm2 was found to maximize mu, as well as the directional persistence and speed of cell migration, whereas very high LAM densities saturated cell internalization and diminished migration rates. The cell-LAM interactions essential to enhanced migration were ligand-activated, as mu; values were reduced 400% on internalizable microcarriers lacking ligands; moreover, cooperative ligand elicitation was possible, for example, when 10 microg/mL soluble fibronectin was introduced as a costimulant of keratinocytes, yielding the highest reported mu values (80 microm2/min) on collagen LAM-PLGA substrates. Notably, cell migration rates were severely repressed when cell internalization processes were challenged through covalent conjugation of ligand carriers to the substrate, indicating that signals from cell-LAM binding alone were inadequate for elevated levels of cell migration. Further analysis indicated that the presence of LAMs did not alter the protease-resistant adhesivity between the cell and the underlying substrate, suggesting that the activation governing ligand interactions likely arose at the cell-LAM interface rather than the cell-substrate interface. This study highlights the novel use of secondary ligand-presenting microscale/nanoscale depots at polymer substrates to elicit, via dynamic cell internalization processes, significantly enhanced levels of cell migration over traditional interfaces with ligand-bearing substrates.

Currently, synthetic degradable polymers are frequently employed as culture substrates prior to cell transplantation and as implantable scaffolds for cellular infiltration during soft and hard tissue repair. The surface microstructure of matrices based on such polymers may be important in controlling cellular anchorage, spreading, and growth on the external surface, as well as infiltration into the voids of porous polymer scaffolds. While the chemistry, bulk structure, and mechanical properties of such polymers have been extensively studied, the surface microstructure has not yet been systematically examined, particularly following polymer degradation. In this study, we present the first account of the use of confocal laser-scanning reflection microscopy (CLSM) to visualize and quantitate the microtopography of the surface of porous matrices of poly(lactic acid)/poly(glycolic acid) (PLAGA) copolymers following polymer degradation. Utilizing this technique, we report that the surface morphology of PLAGA matrices changes significantly upon degradation, with increased local clustering of textured regions. Our quantitative analysis suggests that polymer degradation results in a lower spatially-averaged surface roughness, with significant cyclical variations observed at later time points. The computed surface correlation function was observed to increase upon degradation, confirming the results from our morphological studies. Finally, we demonstrate the efficacy of CLSM to concomitantly image both the polymer surface and locally attached cells, in real time.