A control model for tibial cortex neovascularization in the bone chamber.

Neovascularization across a gap defect in a rabbit tibial cortex was monitored using the optical bone chamber implant (BCI). Cortical bone growing by apposition as trabeculae was observed weekly as it penetrated a slit into a tissue space in vivo and in situ. Each rabbit was viewed weekly with an intravital microscope from 3 to 8 weeks postimplantation. The constant field of view was the slit-gap tissue space, which was 100 microns thick and 2 mm in diameter. Vessels were imaged with epi-illuminated fluorescence microscopy as they carried FITC-dextran 70 that had been injected into an aural vein. Observations were videotaped and photographed. Videotape frames were analyzed with a digital image processing system to obtain measures of vessel length per unit volume (L/V) of fibroblastic granular tissue and trabeculae, caliber C, and flow velocity u, all as functions of time. Observations supported the conclusions that (1) neovascularization precedes neo-osteogenesis, (2) major vessels tend to align with the tibial axis, (3) bone apposition-generated destruction of fibrous granular tissue vessels stimulates fibrous granular tissue angiogenesis, which keeps its L/V constant, (4) L/V in trabeculae increases with time, and (5) blood supply (Q) and nutrient exchange in healing trabeculae are not positively correlated. Thus, O2 supply to the trabeculum cannot be predicted from Q alone because the nutrient exchange area is not constant. It was noted that an increase in the potential nutrient exchange area occurred in both fibrous granular tissue and osseous vessels and the volume fraction of blood decreased in the fibrous granular tissue and remained constant in the trabeculae.

The role of microvasculature in normal and perturbed bone healing as revealed by intravital microscopy.

Bone chamber intravital microscopy brings to the study of bone circulation a combination of the control volume of in vitro models and the chemical complexity of in vivo models. As an optical tool it provides a window to circulatory events at the tissue level of magnification. In particular, it allows measures of microvascular physiology 1) in space by magnifying local perfused vasculature and microcirculation at any instant and 2) in time by providing the same volume of tissue for weekly viewing of an evolving process such as bone healing. This hollow screw's windows have revealed: 1) a consistent order for vascular and bone progression during healing, 2) vascular changes in response to implanted polymers and 3) preliminary data about effects of hyperbaric oxygenation and pulsed electromagnetic fields on vascular aspects of healing. The parameters measured are osteogenesis, angiogenesis, blood supply and permeability.

Evidence for reperfusion injury in cortical bone as a function of crush injury ischemia duration: a rabbit bone chamber study.

A model for critical limb ischemia was produced by occluding femoral vessels in 24 rabbits with a pneumatic cuff for 0, 2, 4, or 6 hours. Immediate sequelae and subsequent creeping substitution of cortical bone were observed in vivo using an implanted tibial window, the optical bone chamber implant (with intravital microscopy), and then by light and fluorescence microscopy of fluorochrome-labeled and surface-stained ground sections of retrieved implants. Six rabbits were used as controls (0 h) for each ischemia treatment, and the animals were monitored for 5 weeks postocclusion. A subpopulation of 13 implants was retrieved after euthanization and then histologically assessed for bone necrosis and remodeling. The hypothesis tested was that reperfusion injury during the 24 h after occluder release (reperfusion phase), and vessel perfusion/caliber, angiogenesis, and net bone resorption during the 5 subsequent weeks (creeping substitution phase), would exhibit ischemia duration-dependent effects. All animals could bear weight on the affected limb to ambulate by 1 week posttreatment. Two-way analysis of variance (ANOVA) comparison of the resulting data confirmed a significant difference between control and ischemia-treated rabbits for: (1) vessel perfusion/reperfusion; (2) vessel caliber; and (3) net bone resorption. Vascular responses to 4 vs. 6 h of ischemia were not significantly different, but net bone resorption was strictly ischemia duration-dependent. The conclusion that reperfusion injury was the mechanism spreading ischemia to more vessels was supported by a decrease in reperfusion and caliber of vessels, and an increase in vascular permeability and leukocyte adherence during the reperfusion phase. It is postulated that reperfusion injury produces a secondary ischemia that amplifies the occlusion-created primary ischemia and, in the present work, may have been succeeded by progressive episodes of ischemia, similar to the infarction pattern of ischemic hearts.

Acidity near eroding polylactide-polyglycolide in vitro and in vivo in rabbit tibial bone chambers.

Eroding poly(DL-lactide-co-glycolide)(PDLLG) washers were observed chronically in vitro and in vivo following loading in a bone chamber tibial implant (BCI). Images were recorded using intravital microscopy of the implanted rabbit and direct pH measurements were obtained of the tissue in the bone chamber using a combination probe-reference microelectrode. While statistically significant pH differences were found between the control (unloaded) and experimental (PDLLG-bearing) BCIs, they were only of the order of 0.2 pH units. This value proved to be physiologically insignificant as no statistically significant difference in bone defect healing, as indicated by angiogenesis, was detected. It was shown that the measured small pH changes during PDLLG washers erosion would result whether the buffer was phosphate-buffered saline replaced weekly or interstitial fluid subject to vascular exchanges.

A bone fluid flow hypothesis for muscle pump-driven capillary filtration: II. Proposed role for exercise in erodible scaffold implant incorporation.

A model is presented for enhancement of fluid flow through bone matrix and any porous tissue engineering scaffold implanted within it. The mechanism of enhancement is the skeletal muscle pump in compartments adjacent to the bone. Pressure waves from muscle pump contractions aided by increased blood pressure during exercise coupled with temporary occlusion of arteries leading to and veins from the bone, increase hydraulic pressure in cortical bone capillaries so as to amplify capillary filtration. It is proposed that capillary filtration increase is sufficiently convective to contribute to bone fluid flow and associated percolation through tissue engineered scaffold matrix implants. Importance of this contribution is its relative role in maintaining seeded cells in bioreactor scaffolds. Validation of the hypothesis starts at a minimum level of demonstrating that capillary filtration is convective. At a maximum level confirmation of the hypothesis requires demonstration that capillary filtration-based interstitial flow is sufficient to stimulate not only host bone cells (as proposed in part I of the hypothesis) but bioreactor-seeded cells as well. Preliminary data is presented supporting the prediction that skeletal muscle contraction generates convective capillary filtration.

Wound healing in the bone chamber 1. Neoosteogenesis during transition from the repair to the regenerative phase in the rabbit tibial cortex.

The optical bone chamber technique that includes intravital microscopy is described and is then applied to measuring primary wound healing neoostogenesis in rabbit tibia cortical bone during the period when fibrovascular tissue is being replaced by regenerating osteovascular tissue. The small population of rabbits sampled allowed only tentative conclusions. The quantitative measurements, a direct from of histomorphometry, are applied to determine the consistency of observations, with the hypothesis that healing into the bone chamber slit-gap follows the pattern of primary bone regeneration established as "characteristic" by other studies. The results supported the hypothesis and showed that bony ingrowth started during the third postoperative week (W3) with a maximum linear growth rate of 85.5 micron/day for a bone front. Evidence for remodeling by W6 was also obtained. While the bone chamber environment for tissue ingrowth is artificial, it can generate quantitative data that may provide a statistically valid basis for modeling pathophysiologic processes associated with bone wound healing.

Incorporation of polylactide-polyglycolide in a cortical defect: neoangiogenesis and blood supply in a bone chamber.

Erodible polymers are an alternative to metals for fracture fixation (for example, in the malleolus) and for maxillofacial reconstruction. In this study, the vascular response to eroding polylactide-polyglycolide copolymer threads was observed chronically in a bone chamber implant, with use of intravital microscopy. A bone chamber implant loaded with 100 microns thick polylactide-polyglycolide threads was implanted into the right tibia in 15 mature female New Zealand White rabbits. Periodic intravital microscopic observations were performed from the third to the tenth or twelfth week after implantation. Vascularization, blood flow, and trabecular growth into the chambers from the medial cortex were recorded on videotape and analyzed using digital image processing. A statistically significant delay of neo-osteogenesis in the presence of this copolymer was described in an earlier report. The present report describes the measures of neoangiogenesis and blood supply; there was a significant delay in neoangiogenesis. It is suggested that both delayed angiogenesis and osteogenesis were secondary consequences of the macrophage response to slowly eroding poly-L-lactide crystal nanoparticles and the influence of reduced nutrient exchange. The lesser effect on blood supply and vascular volume fraction was seen to be linked to the slowing down of angiogenesis, as the latter allowed vessels to mature, with a widening of their calibers. This homeostatic adjustment was interpreted as being only partially successful in restoring control levels of oxygen delivery, because resulting increases in vessel surface area did not reach control levels. Thus, in the presence of eroding polylactide-polyglycolide, the oxygen supply and extravasation of other nutrients may be below normal during healing phases when the need is critical.

The role of the periciliary fluid in mucociliary flows: flow velocity profiles in frog palate mucus.

Observations of flow velocity profiles over frog mucociliated palate are used to estimate viscosity, shear rate and shear stress in the periciliary flow field. The ability of cilia to generate significant shear stress at long distances and their utility as rhoeometers are examined. It is proposed that the depth of significant ciliary shear penetration into the periciliary fluid is sufficient to move mucus masses well beyond the ciliary tips, obviating the need for tip penetration where anchoring phenomena are sufficiently reduced.

A horizontal intravital microscope-plus-bone chamber system for observing bone microcirculation.

A horizontal intravital microscope has been built which utilizes the telescope optics format with infinity-corrected objectives. The intravital stage accomodates a rabbit into which a tibial bone chamber has been implanted. The chamber is itself an optical component and is considered an integral part of the system. Each animal is observed in supine position with its implanted leg held in an adjustable spring-loaded yoke which aligns the bone chamber in the optical axis. Long working-distance objectives and condensers allow for viewing of the slit-gap tissue in the bone chamber with both transmitted and epi-illumination; the latter being applied for fluorescence studies. A unique feature of the system is a transfer lens holder which can be relocated within the microscope tube. This adjustable lens holder allows for changing of transfer lenses, thereby freeing the investigator to utilize objective lenses with preferred numerical apertures for a given image size.

Ciliary propulsion of objects in tubes: wall drag on swimming Tetrahymena (Ciliata) in the presence of mucin and other long-chain polymers.

The lubrication effect of three long-chain polymers - mucin, methylcellulose and Ficoll - on ciliary propulsion in tubes is measured by plotting the relative velocities of swimming cilitates as a function of the tube bore diameter. Mucin shows the most unequivocal lubrication, which is found at concentrations between 0% and 9.1% (w/v). This observation, coupled with viscometric measurements which show that ciliary tip shear rates are sufficient to solate mucin, serve as the groundwork for a model of mucin lubrication which explains the optimized lubrication behaviour of thixotropic gelating polymers as an expression of the response to shear by the various stages of polymer clustering during the gelatin process. In addition to the lubricative effect, another wall drag reduction effect by mucin was measured in the clearance region beyond the lubrication layer. This apparent viscosity reduction is optimized in the concentration range between I.7% and 4.I% mucin and may also be explained in terms of the properties of gel clustering.

Comparative bone healing near eroding polylactide-polyglycolide implants of differing crystallinity in rabbit tibial bone chambers.

Eroding poly(DL-lactide-co-glycolide) (PDLLG) washers and poly(L-lactide-co-glycolide) (PLLG) threads were observed chronically in vivo following loading in a bone chamber tibial implant (BCI). Images were recorded using intravital microscopy of the implanted rabbit. Erosion and bone healing, as represented by angiogenesis and osteogenesis, was determined from changes in projected area of observed polymer, vessels and bone, respectively. Erosion rates of the two polymers were significantly different. Healing adjacent to both polymers differed significantly from controls. Healing response to each polymer was also different, with the faster eroding PDLLG causing more deviation from normal osteogenesis and angiogenesis than did PLLG. It was speculated that the faster eroding polymer released macrophage-stimulating fragments earlier in the healing process, thus altering the normal macrophage-endothelial cell interaction which in turn affected angiogenesis-linked components of osteogenesis.

Fibroblast growth factor-2 alters the effect of eroding polylactide-polyglycolide on angiogenesis in the bone chamber.

The effects of recombinant human fibroblast growth factor-2 in the presence of eroding 50:50 poly(DL-lactide-co-glycolide) on angiogenesis during acute bone defect healing were studied in the optical bone chamber. Bone chambers were loaded with disks of poly(DL-lactide-co-glycolide) surrounded by one of four doses of recombinant human fibroblast growth factor-2. Fifty-two female rabbit right tibias were implanted. Commencing with the third postimplantation week, healing into the bone chamber compartment was observed weekly, with use of intravital microscopy, until week 8. The treatment groups were as follows: unloaded, loaded with polymer only, and loaded with polymer plus-0.5, -1.0, and -10 microg recombinant human fibroblast growth factor-2. Videotaped and photographed bone images were measured and analyzed with a frame-grabber digitizing system. Comparison with controls or polymer alone revealed that angiogenesis rates were significantly above normal in rabbits loaded with polymer plus either of the two highest recombinant human fibroblast growth factor-2 doses. The effects of these doses were not, however, significantly different from each other, indicating a plateauing rather than monotonically increasing response. It was concluded that recombinant human fibroblast growth factor-2 in the dose- range studied can effectively overcome the retarding effects of eroding polymer on angiogenesis in bone, which has been reported previously by this laboratory. In contrast to these results, no statistically significant differences in angiogenesis rate as a function of time could be detected. It is suggested that this apparent lack of variation is linked to observed alterations in vessel patterns from week-to-week. The clinical relevance of these results is that inhibitory carrier effects can be compensated for by increasing the dose of loaded agent where the effect being inhibited is the one being treated, or by adding an agent specific for the inhibited effect. In either case, geometry of the carrier must be considered when programming for delivery rate.

Spirillum swimming: theory and observations of propulsion by the flagellar bundle.

The hydrodynamics and energetics of helical swimming by the bacterium Spirillum sp. is analysed using observations from medium speed cine photomicrography and theory. The photographic records show that the swimming organism's flagellar bundles beat in a helical fashion just as other bacterial flagella do. The data are analysed according to the rotational resistive theory of Chwang & Wu (1971) in a simple-to-use parametric form with the viscous coefficients Cs and Cn calculated according to the method of Lighthill (1975). Results of the analysis show that Spirillum dissipated biochemical energy in performing work against fluid resistance to motion at an average rate of about 6 X 10(-8) dyne cm s-1 with some 62-72% of the power dissipation due to the non-contractile body. These relationships yield a relatively low hydromechanical efficiency which is reflected in swimming speeds much smaller than a representative eukaryote. In addition the Cn/Cs ratio for the body is shown to lie in the range 0-86-1-51 and that for the flagellar bundle in the range 1-46-1-63. The implications of the power calculations for the Berg & Anderson (1973) rotating shaft model are discussed and it is shown that a rotational resistive theory analysis predicts a 5-cross bridge M ring for each flagellum of Spirillum.

Fibroblast growth factor-2 alters the effect of eroding polylactide-polyglycolide on osteogenesis in the bone chamber.

The effects of recombinant human fibroblast growth factor-2 (rhFGF-2) in the presence of eroding 50:50 poly(DL-lactide-co-glycolide) (PDLLG) on acute bone healing were studied in the optical bone chamber (BCI). BCIs were loaded with disks of PDLLG surrounded by one of four rhFGF-2 doses. Fifty-two female rabbit right tibias were implanted. Commencing the third week post implantation (W3) healing in the BCI compartment was observed weekly, using intravital microscopy, until W8. The doses were: unloaded, loaded with polymer only, and polymer plus 0.5, 1.0, and 10 microg rhFGF-2. Videotaped and photographed bone images were measured and analyzed using a frame-grabber digitizing system. Comparison with controls revealed that ossification rates were significantly above normal in rabbits loaded with polymer plus any of the rhFGF-2 doses. Comparison with polymer-only BCIs showed that PDLLG plus any of the three rhFGF-2 doses was linked with ossification rates significantly higher than baseline. The results indicated that FGF-2 in the dose range studied effectively can overcome the retarding effects of eroding polymer on ossification that has been reported by this laboratory. Interpretation of the retarding effects of the polymer disks, although consistent with previously studied washer-shaped devices of the same material, was complicated by a difference in erosion rate. This result supports the notion that erodible device geometry is a major factor in determining biocompatibility and must be considered in the design of carriers. Accordingly, programming of dose specificity for delivering a given polypeptide cytokine to a given host site must allow for the inhibitory effects of an eroding carrier and the influence of device geometry on these effects and erosion.

Incorporation of polylactide-polyglycolide in a cortical defect: neoosteogenesis in a bone chamber.

One hundred-micron-thick threads of 40-kDa copolymer polylactide:polyglycolide (PLGA) in a 50:50 molar ratio in bone chambers were implanted in rabbit tibias. Weekly intravital microscopic observations were used to assess trabecular ingrowth and PLGA absorption, processes referred to as incorporation. Nineteen rabbits were observed from the 3rd (W3) to the 10th or 12th (W10/W12) week postimplantation. Analysis of digitally processed video images obtained weekly showed that PLGA absorption was completed in about 8-10 weeks and trabecular ingrowth delayed to about 1 week slower than its control value. Four of the bone chambers showed PLGA unraveled by week 3, no trabecular ingrowth, and significantly reduced neovascularization. No such apparent inhibitory effects were observed in bone chambers that displayed PLGA absorption. No causal link could be established between the rate of neoosteogenesis and the rate of PLGA absorption, but it was clear that incorporation slowed and rapid degradation inhibited bone regeneration. It was concluded that copolymer synthesis and postsynthesis processing must result in a synthetic with a predictable micro- and macro-molecular structure before one can predict its incorporability in a given reconstruction or fixation site.

Effect of polymethylmethacrylate particles on mature bone in the optical bone chamber.

Reaction of mature bone and its vasculature to 3.33 +/- 0.19 microm polymethylmethacrylate (PMMA) particles at a concentration of 2.5 x 10(8)/cc was measured using optical bone chamber implant intravital microscopy. Twelve adult female New Zealand White rabbits were divided into six receiving Healon alone (controls) and six receiving Healon plus PMMA. The particles were introduced to the bone chamber compartment after removing its overlying optical element, which was immediately reinstalled. Reaction was monitored weekly over a 6-week period using video and photographic imaging. Bone was labeled before treatment with oxytetracycline and after treatment with alizarin complexone. Perfusing blood was labeled with fluorescein isothiocyanate dextran-70 kDa (FITC-D70). Parameters measured were net bone resorption, from black and white images, bone turnover, from color images, vascularity, and average vessel caliber. Neither bone turnover nor vessel caliber were significantly affected at the p < or = 0.050 level over time. In contrast, bone resorption was significantly greater and vascularity significantly less in the presence of PMMA. It was inferred that any differences in bone turnover were masked by resorption of new bone. It was concluded that the lack of a PMMA effect on average vessel caliber meant that the vascularity effects were not due to angiogenesis, but to vessel recruitment (or its opposite), an effect more consistent with inflammation than repair. The lack of vascularity increase in PMMA-treated compartments also suggested that increased resorption was a local phenomenon, because blood supply had not increased to provide the extra osteoclasts required for observed net bone loss.