Browsing by Author "Klitzman, Bruce"

Many implanted devices fail due to the formation of an avascular capsule. Fat is known to promote healing and vascularization. It is possible that isolating and attaching ASCs (adipose stem cells) to an implanted device improves the healing in the adjacent tissue.

Various attachment methods were studied, and the fibronectin treatment was found comparable to or better than other treatments. Next, bare and ASC coated polyurethane were implanted into rats. The fibrous capsule surrounding the bare polyurethane was thicker and contained more collagen at 8 weeks. Additionally, the microvessel density in the tissue surrounding the ASC coated polyurethane was significantly higher at 4 and 8 weeks. Quantification of glucose sensor response following ASC attachment for 1 week found no measurable significant differences in function.

The bioluminescence technique, which quantifies the tissue glucose concentration around the implant at the moment of freezing, was used to determine if ASC attachment to biomaterials impacts the tissue glucose concentration profile. ASC attachment to polyurethane and to glucose sensors did not significantly change the glucose profiles in the tissue. However, a quantifiable glucose concentration profile was observed around all glucose sensors.

The final experiments were performed to identify a possible mechanism that adipose tissue uses to alter the foreign body response. In vitro experiments showed that VEGF (VEGF-A specifically) secretion following ASC attachment to polyurethane was 10-20 times higher than with fibroblast attachment after three days and 40-70 times higher after six days. This high secretion of VEGF would likely have in vivo physiological affects on microvasculature.

In conclusion, the attachment of ASCs to polyurethane reduced the thickness and collagen content of the fibrous capsule surrounding ASC coated implants and increased the microvessel density in adjacent tissue. In addition, ASC attachment did not enhance glucose sensor function, nor did it decrease the glucose concentration in the adjacent tissue. Finally, ASCs were found to secrete high amounts of pro-vascular cytokines, which likely plays a key role in the observed improvement of the foreign body response.

Burn injuries in the United States account for over one million hospital admissions per year, with treatment estimated at four billion dollars. Of severe burn patients, 30-90% will develop hypertrophic scars (HSc). Current burn therapies rely upon the use of bioengineered skin equivalents (BSEs), which assist in wound healing but do not prevent HSc. HSc contraction occurs of 6-18 months and results in the formation of a fixed, inelastic skin deformity, with 60% of cases occurring across a joint. HSc contraction is characterized by abnormally high presence of contractile myofibroblasts which normally apoptose at the completion of the proliferative phase of wound healing. Additionally, clinical observation suggests that the likelihood of HSc is increased in injuries with a prolonged immune response. Given the pathogenesis of HSc, we hypothesize that BSEs should be designed with two key anti-scarring characterizes: (1) 3D architecture and surface chemistry to mitigate the inflammatory microenvironment and decrease myofibroblast transition; and (2) using materials which persist in the wound bed throughout the remodeling phase of repair. We employed electrospinning and 3D printing to generate scaffolds with well-controlled degradation rate, surface coatings, and 3D architecture to explore our hypothesis through four aims.

In the first aim, we evaluate the impact of elastomeric, randomly-oriented biostable polyurethane (PU) scaffold on HSc-related outcomes. In unwounded skin, native collagen is arranged randomly, elastin fibers are abundant, and myofibroblasts are absent. Conversely, in scar contractures, collagen is arranged in linear arrays and elastin fibers are few, while myofibroblast density is high. Randomly oriented collagen fibers native to the uninjured dermis encourage random cell alignment through contact guidance and do not transmit as much force as aligned collagen fibers. However, the linear ECM serves as a system for mechanotransduction between cells in a feed-forward mechanism, which perpetuates ECM remodeling and myofibroblast contraction. The electrospinning process allowed us to create scaffolds with randomly-oriented fibers that promote random collagen deposition and decrease myofibroblast formation. Compared to an in vitro HSc contraction model, fibroblast-seeded PU scaffolds significantly decreased matrix and myofibroblast formation. In a murine HSc model, collagen coated PU (ccPU) scaffolds significantly reduced HSc contraction as compared to untreated control wounds and wounds treated with the clinical standard of care. The data from this study suggest that electrospun ccPU scaffolds meet the requirements to mitigate HSc contraction including: reduction of in vitro HSc related outcomes, diminished scar stiffness, and reduced scar contraction. While clinical dogma suggests treating severe burn patients with rapidly biodegrading skin equivalents, these data suggest that a more long-term scaffold may possess merit in reducing HSc.

In the second aim, we further investigate the impact of scaffold longevity on HSc contraction by studying a degradable, elastomeric, randomly oriented, electrospun micro-fibrous scaffold fabricated from the copolymer poly(l-lactide-co-ε-caprolactone) (PLCL). PLCL scaffolds displayed appropriate elastomeric and tensile characteristics for implantation beneath a human skin graft. In vitro analysis using normal human dermal fibroblasts (NHDF) demonstrated that PLCL scaffolds decreased myofibroblast formation as compared to an in vitro HSc contraction model. Using our murine HSc contraction model, we found that HSc contraction was significantly greater in animals treated with standard of care, Integra, as compared to those treated with collagen coated-PLCL (ccPLCL) scaffolds at d 56 following implantation. Finally, wounds treated with ccPLCL were significantly less stiff than control wounds at d 56 in vivo. Together, these data further solidify our hypothesis that scaffolds which persist throughout the remodeling phase of repair represent a clinically translatable method to prevent HSc contraction.

In the third aim, we attempt to optimize cell-scaffold interactions by employing an anti-inflammatory coating on electrospun PLCL scaffolds. The anti-inflammatory sub-epidermal glycosaminoglycan, hyaluronic acid (HA) was used as a coating material for PLCL scaffolds to encourage a regenerative healing phenotype. To minimize local inflammation, an anti-TNFα monoclonal antibody (mAB) was conjugated to the HA backbone prior to PLCL coating. ELISA analysis confirmed mAB activity following conjugation to HA (HA+mAB), and following adsorption of HA+mAB to the PLCL backbone [(HA+mAB)PLCL]. Alican blue staining demonstrated thorough HA coating of PLCL scaffolds using pressure-driven adsorption. In vitro studies demonstrated that treatment with (HA+mAB)PLCL prevented downstream inflammatory events in mouse macrophages treated with soluble TNFα. In vivo studies using our murine HSc contraction model suggested positive impact of HA coating, which was partiall impeded by the inclusion of the TNFα mAB. Further characterization of the inflammatory microenvironment of our murine model is required prior to conclusions regarding the potential for anti-TNFα therapeutics for HSc. Together, our data demonstrate the development of a complex anti-inflammatory coating for PLCL scaffolds, and the potential impact of altering the ECM coating material on HSc contraction.

In the fourth aim, we investigate how scaffold design, specifically pore dimensions, can influence myofibroblast interactions and subsequent formation of OB-cadherin positive adherens junctions in vitro. We collaborated with Wake Forest University to produce 3D printed (3DP) scaffolds with well-controlled pore sizes we hypothesized that decreasing pore size would mitigate intra-cellular communication via OB-cadherin-positive adherens junctions. PU was 3D printed via pressure extrusion in basket-weave design with feature diameter of ~70 µm and pore sizes of 50, 100, or 150 µm. Tensile elastic moduli of 3DP scaffolds were similar to Integra; however, flexural moduli of 3DP were significantly greater than Integra. 3DP scaffolds demonstrated ~50% porosity. 24 h and 5 d western blot data demonstrated significant increases in OB-cadherin expression in 100 µm pores relative to 50 µm pores, suggesting that pore size may play a role in regulating cell-cell communication. To analyze the impact of pore size in these scaffolds on scarring in vivo, scaffolds were implanted beneath skin graft in a murine HSc model. While flexural stiffness resulted in graft necrosis by d 14, cellular and blood vessel integration into scaffolds was evident, suggesting potential for this design if employed in a less stiff material. In this study, we demonstrate for the first time that pore size alone impacts OB-cadherin protein expression in vitro, suggesting that pore size may play a role on adherens junction formation affiliated with the fibroblast-to-myofibroblast transition. Overall, this work introduces a new bioengineered scaffold design to both study the mechanism behind HSc and prevent the clinical burden of this contractile disease.

Together, these studies inform the field of critical design parameters in scaffold design for the prevention of HSc contraction. We propose that scaffold 3D architectural design, surface chemistry, and longevity can be employed as key design parameters during the development of next generation, low-cost scaffolds to mitigate post-burn hypertrophic scar contraction. The lessening of post-burn scarring and scar contraction would improve clinical practice by reducing medical expenditures, increasing patient survival, and dramatically improving quality of life for millions of patients worldwide.

Bacterial colonization of a medical device has been seen to precede clinical infection as well as adversely affect function of the indwelling device. This is a major cause of implant failure with the most common bacterial infections being due to Staphylococcus aureus. This strain has been known to be anti-biotic resistant, therefore it is very important to test and find biomaterials with low bacterial adhesion properties to avoid device-associated infections when implanted into the body. In the present study, three specific aims were preformed to characterize the performance of Polyethersulfone (PES) and polyvinylidene difluoride (PVDF) membranes. These polymeric membranes are commonly used in nanofiltration applications in the water and waste water market; therefore they are of high interest for use in the medical device market. PES and PVDF hydrophilic filter disks of 25mm diameter were purchased from Millipore with pore sizes of 0.22 µm. Bacterial migration (N=4), bacterial adhesion (N=4) and outflow resistance (N=4) studies were tested for each filter. Bacteria cultured to a concentration of 1 McFarland (3x108 cells/mL) were used for migration and adhesion studies. Migration was tested by pumping bacterial broth through the membranes and collecting the perfusate to quantify the bacterial migration. Adhesion studies were quantified by incubating filters in bacterial broth for 24hrs and plating attached bacteria after detachment by sonication. SEM images were taken for visual analysis of bacteria and filters. Lastly, outflow resistance was measured by pumping deionized water while recording pressure readings throughout 5 minutes. Results of the studies demonstrated that bacteria did not migrate through both PES and PVDF filters, thus properly filtering S. aureus cells. PES membranes were found to have more bacterial adherence to the surface and a lower outflow resistance than PVDF. Both filters could be considered to be used as a part of biomedical devices depending on the specific applications and resistance requirement. However, further studies are needed to inhibit bacterial adherence such as antibacterial coatings or incorporating antimicrobial compounds within polymeric biomaterials. The use of selenium as an antibacterial agent in biomedical devices has sparked a great interest in recent years this, incorporating this in PES and PVDF membranes are the next goal for these studies.

Efforts to create a reliable, long–term implantable glucose sensor have been stymied by the effects of the foreign body response and wound healing that introduce delayed response times as well as unpredictable sensor performance. Loss of vascularization from fibrotic encapsulation around implanted sensors is purported as a key contributor to sensor failure, as glucose and oxygen transport to the sensor becomes impeded. Improving sensor performance by increasing angiogenesis and/or reducing capsule thickness using tissue-modifying textured coatings is attractive because texturing is not dependent upon a depletable drug reservoir. A significant range of materials and pore sizes are capable of promoting angiogenesis and reducing capsule thickness, provided pores have open-architecture with dimensions sufficiently large enough to allow inflammatory cell infiltration.

Poly–L–lactic acid was gas foamed/salt leached with ammonium bicarbonate to produce porous coatings for Medtronic MiniMed SOF–sensor glucose sensors. Coating properties included 30μm pore diameters, 90% porosity, and 50μm wall thickness. Cytotoxicity, degradation, and sensor response time studies were performed to ensure the porous coatings were non–toxic and negligibly retarded glucose diffusion prior to in vivo testing. Histology was used to evaluate angiogenesis and collagen deposition adjacent to porous coated and bare (i.e. smooth, uncoated) non–functional sensor strips after three weeks in the rat dorsal subcutis. Functional Medtronic glucose sensors, with and without porous coatings, were percutaneously implanted in the rat dorsum to assess if the angiogenic–inducing properties observed around the non–functional porous coated sensor strips translated into stable, non–attenuated sensor signals over two and three weeks. MiniLinkTM transmitters were attached to the rats, permitting continuous glucose monitoring. Vessel counts and collagen deposition adjacent to sensors were determined from histological analysis. A one–sided dorsal window model was developed to further evaluate the interplay between vascularization and sensor performance Sensors were inserted beneath the windows, allowing visualization of microvascular changes adjacent to sensor surfaces, with simultaneous evaluation of how vascular changes impacted interstitial glucose monitoring.

Porous coating did have angiogenic–inducing effects on the surrounding tissue. When fully implanted in the rat dorsum, sensor strips with porous coatings induced three–fold more vessels within 100μm2 of the sensor strip surface after three weeks and two-fold more cumulative vessel lengths within 1mm2 after two weeks, compared to bare surfaces. In contrast, when percutaneously implanted in the rat dorsum, porous coated and bare sensors were equally highly vascularized, with two–fold more vessels than fully implanted bare sensors.

Despite increased angiogenesis adjacent to percutaneous sensors, sensor signal attenuation occurred over 14 days, suggesting that angiogenesis plays a secondary role in maintaining sensor function. Percutaneously implanted porous coated sensors had greater reductions in baseline current (20 to 50+%) over two weeks than bare sensors (10 to 30%). Mechanical stresses imposed by percutaneous tethering may override the beneficial effects of porous coatings. Furthermore, integration of the porous coating with the surrounding tissue may have increased tissue tearing at the porous coating–tissue, increasing inflammation and collagen deposition resulting in greater signal attenuation compared with bare sensors. Future investigations of the role mechanical irritation has on wound healing around percutaneous glucose sensors are warranted.

Performance of biomedical implants has been hindered by fibrosis, infection, and deficient tissue integration due in part to the body's foreign body response. In addition, diabetes mellitus is affecting a greater number of people worldwide and in the United States. As the percentage of the population affected by diabetes increases, a larger fraction of these implanted devices will be placed in diabetic patients. Unfortunately, diabetes is often complicated by poor wound healing and a greater risk of infection, issues that could adversely affect proper acceptance of an implant. Diabetic animal models are useful in studying the response to infection as well as the tissue response to an implanted device. Therapies such as nitric oxide release have been applied to indwelling devices to mediate the foreign body response and improve the wound healing response around implants. Particularly relevant to diabetic patients are implantable glucose sensors, and so determining the diabetic tissue response to these devices is crucial to improving their lifetime and performance.

A novel outcome-based streptozotocin dosing regimen was developed to induce Type 1 diabetes in a rodent model. Male CD (Sprague-Dawley type) rats weighing 150-200 g were given three consecutive daily doses of 40 mg/kg streptozotocin (STZ) on Days 1, 2, and 3. On Day 5, tail vein blood glucose was checked. If blood glucose was not within the target diabetic range of 350-600 mg/dl, rats received an additional dose of STZ. This procedure was repeated every 48 hours until all rats achieved target hyperglycemia. Control rats were given similar doses of vehicle (saline/citrate), which had no effect on blood glucose. After the last injection of streptozotocin, two weeks were allowed to ensure the full effects of the diabetic state would be present at device implantation. Blood glucose was measured every 2 days for diabetics and 4 days for controls for the duration of the experiment. The developed diabetic model resulted in a stable hyperglycemia for the duration of the experiment (in some cases, up to 2 months). Animals also exhibited typical symptoms of diabetes, such as minimal changes in weight, excessive thirst, and polyuria.

Infection response in the presence of implanted devices was investigated in healthy and diabetic animal models. In the healthy animal model, control stainless steel compression plates and plates coated with a nitric-oxide releasing xerogel (20% AHAP) were attached to the femurs of 12 adult rabbits. Both femurs were inoculated with 3x106 CFU MSSA (methicillin-sensitive Staph aureus) for a period of 20 minutes before the surgical sites were sutured. After 7 days, the wound, device, and a portion of bone were cultured. A muscle biopsy was removed and homogenized to quantify bacterial infection. Since the microbiologic data were not normally distributed, they were compared using an unpaired Wilcoxon Rank Sum Test. No significant differences in bacterial burden were observed between the control and NO-eluting devices, however the study did find a high correlation of temperature of the adjacent muscle at implantation with the ensuing bacterial content. In the diabetic animal model, a novel dosing regimen of streptozotocin based on a target blood glucose of 350-600 mg/dl was used to induce type-1 diabetes. Stable hyperglycemia was maintained for 21 days. Two weeks after achieving the target hyperglycemia, stainless steel fracture plates were secured to each femur with stainless steel screws approximately 3 mm in length. The implant site on the right side of the animal was inoculated with 3 x 107 CFU of methicillin-sensitive S. aureus while the left side served as a control. After seven days, quantitative bacterial count was performed at explantation and no cross-over of bacteria was detected from the inoculated side to the non-inoculated side. Infection after S aureus inoculation in the presence of an implanted device was significantly higher in diabetic animals when compared to that of control animals (p = 0.0003, Wilcoxin Rank-Sum Test) supporting the hypothesis that diabetes adversely affects the ability to fight infection in the presence of an indwelling device in an animal model. There was not a significant difference detected in the infectious burden for the non-inoculated limb (left) between the diabetic and non-diabetic groups when compared using a Wilcoxon Rank-Sum Test (p = 0.0682), however this near-significance suggests that even in the absence of an introduced inoculum, diabetes increases infection susceptibility in the presence of an implant.

Nitric oxide (NO) release can be used to mediate the foreign body response around implanted devices. NO also has antibacterial properties that may enhance the body's immune response to implant-associated infection. Additionally, diabetic wounds are characterized by nitric oxide deficiency, and thus NO supplementation may promote better wound healing and implant acceptance in diabetics. The use of nitric oxide to modulate the tissue response to indwelling implants was explored in two studies. In vivo glucose recovery of subcutaneously implanted NO-releasing microdialysis probes was evaluated in a healthy rat model using saturated NO solutions that provided a steady release of NO. A constant NO flux of 162 pmol cm-2 s-1 was perfused through the probe membrane for 8 hours daily. The in vivo effects of increased localized NO were evaluated by monitoring glucose recovery over a 14-day period. Beginning at 7 days, significant differences in glucose recovery between the control and NO-releasing probes were observed. At the 14-day time point, histological analysis revealed decreased inflammatory cell density at the probe surface and a thinner collagen capsule. In the second study, polyurethane-coated wires with varying NO release properties were implanted subcutaneously in 17 Yorkshire piglets with time points of 3, 7, 21, and 42 days. To create the NO-releasing coating, the NO-releasing vehicle (i.e., PROLI/NO, AEAP3 or MPTMS nanoparticles) was dispersed into EtOH (2.5 mL) at concentrations of 36 or 72 mg/mL. This solution was then mixed with an equal volume of 50:50 wt% HPU/TPU (160 mg/mL total PU). Effects of NO release were analyzed using histological data. These data were analyzed using a non-parametric Wilcoxon rank-sum test. Coatings with short NO release durations (i.e., 24 h) failed to reduce collagen capsule thickness at 3 and 6 weeks. Longer release durations (3 and 14d) however significantly reduced collagen capsule thickness at longer timepoints. The acute inflammatory response was significantly affected by coatings with the longest duration and greatest dose of NO release. However these benefits were not realized at later timepoints, suggesting that NO must be actively released in order to influence inflammatory response.

The tissue response to percutaneously implanted glucose sensors was investigated in healthy and diabetic rats. A multi-dose regimen of streptozotocin was used to induce diabetes in experimental rats. Three types of functional, implantable glucose sensors, supplied by Medtronic® were used: SofTM sensor, EnliteTM sensor, and Enlite 2TM sensor. The sensors were percutaneously implanted in the rat dorsum subcutaneous space. MiniLinkTM transmitters were attached to the rats, permitting continuous glucose monitoring. At 3 days, 1 week, and 4 weeks, tissue directly adjacent to the sensors was evaluated for collagen encapsulation, density of any collagen encapsulation, inflammatory response as measured via inflammatory cell density, and microvessel density. These endpoints were evaluated histologically via Masson's trichrome, Hoechst, H&E, and CD31 staining. Additionally, continuous functional sensor data was evaluated for sensor accuracy, attenuation, and lag time. Histological analyses revealed few significant differences in collagen thickness among different sensors, in different tissue types, or over time. In general, Masson's trichrome-stained images seem to suggest a balance between collagen capsule formation and inflammatory cell density. As inflammation increased adjacent to sensors over time, collagen capsule thickness decreased somewhat and stabilized. Collagen capsule formation was most evident adjacent to the plastic tubing portion of the sensor whereas inflammation was greatest adjacent to the sensing electrode. Likewise, few significant differences in collagen density index (CDI) were observed among sensor types, tissue types, or over time. CDI remained relatively constant over time for all sensors. Analysis of inflammatory cell density in general revealed a greater inflammatory response adjacent to percutaneous Enlite sensors, though these results were not significant. Additionally, inflammatory cell density was generally greater adjacent to non-diabetic sensors, however this result was also not significant. Inflammatory cell density increased or remained stable over time for all sensor types, suggesting that the presence of percutaneously-implanted sensors produces a chronic inflammatory response that does not resolve. Vascularity adjacent to implanted sensors remained generally stable over time, sometimes decreasing but not significantly. At the 1-month timepoint, no significant differences in vasculature were seen among sensor types. A balance also appears to exist for microvessel and inflammatory cell densities. The non-diabetic percutaneous Enlite sensor had the greatest microvessel density at earlier timepoints, while also having the greatest inflammatory cell density. However, at later timepoints, microvessel density decreased somewhat as inflammation increased somewhat. Finally, analysis of sensor performance showed significant sensor failure at longer timepoints. Sensitivity decreased somewhat for all sensors except for the non-diabetic Enlite sensor, which in general had greater overall sensitivity in comparison to the non-diabetic Sof sensor. Lag time was relatively similar among all sensor types, tissue types, and over time. MARD values were considerably lower for diabetic sensors for the Day 1 bolus, but were generally similar for all sensors at the 1-week bolus. These results suggest that the diabetic foreign body response, while somewhat decreased, is not significantly different than that in non-diabetic tissue. In addition, the design of the EnliteTM and Enlite 2TM sensors promoted a more aggressive inflammatory response despite being smaller and more flexible in design. Most evident from the results was the presence of a chronic inflammatory response adjacent to the percutaneously-implanted sensors, which likely contributed to the high rate of sensor failure over time.