Design matters: A comparison of natural versus synthetic skin substitutes across benchtop and porcine wound healing metrics: An experimental study.

Background and Aims: Skin substitutes, essential tools for helping close full thickness wounds with minimal scarring, are available in both collagen-based and synthetic polyurethane constructions. Here we explore fundamental differences between two frequently used skin substitutes and discuss how these differences may impact in vivo performance. Methods: Polyurethane- and collagen-based matrices were characterized in vitro for pore size via scanning electron microscopy, hydrophobicity via liquid contact angle, conformability via bending angle, and biocompatibility via fibroblast and keratinocyte adhesion and proliferation. These matrices were then evaluated in a full-thickness excisional pig wound study followed by histological analysis. Statistical analysis was performed using t-tests or one-way analysis of variances with Tukey's multiple post hoc comparisons, where appropriate. Results: Average pore diameter in the tested polyurethane matrix was over four times larger than that of the collagen matrix (589 ± 297 µm vs. 132 ± 91 µm). Through liquid contact angle measurement, the collagen matrix (not measurable) was found to be hydrophilic compared to the hydrophobic polyurethane matrix (>90°). The collagen matrix was significantly more conformable than the polyurethane matrix (9 ± 2° vs. 84 ± 5° bending angle, respectively). Fibroblast and keratinocyte adhesion and proliferation assays elucidated a significantly greater ability of both cell types to attach and proliferate on collagen versus polyurethane. While the porcine study showed minimal contraction of either matrix material, histological findings between the two treatments were markedly different. Collagen matrices were associated with early fibroblast infiltration and fibroplasia, whereas polyurethane matrices elicited a strong multinucleated giant cell response and produced a network of comparatively aligned collagen fibrils. Conclusions: The more favorable in vitro properties of the collagen matrix led to less inflammation and better overall tissue response in vivo. Overall, our findings demonstrate how the choice of biomaterial and its design directly translate to differing in vivo mechanisms of action and overall tissue quality.

Integra® Dermal Regeneration Template: From Design to Clinical Use.

Integra® Dermal Regeneration Template (IDRT, Integra LifeSciences, Princeton, NJ, USA) is a bilayer membrane developed, by Yannas and Burke in the 1980s, to fulfill the unmet need of surgeons having a readily available off-the-shelf dermal regeneration method. IDRT is composed of a sheet of porous cross-linked type I collagen and glycosaminoglycans, with a semi-permeable silicone sheet cover. IDRT is bio-engineered, from adult bovine Achilles tendons and chondroitin-6-sulfate derived from shark cartilage, in a multi-step process involving cross-linking using glutaraldehyde. By design, the composition, porosity, and biodegradation rate of IDRT guides the mechanism of wound repair towards a regenerative pathway. Its mechanism of action involves four distinct phases: imbibition, fibroblast migration, neovascularization, and remodeling/maturation. Originally developed for the post-excisional treatment of deep-partial to full-thickness burns where autograft is limited, over the years its use has expanded to reconstructive surgery.

Skin substitutes with noncultured autologous skin cell suspension heal porcine full-thickness wounds in a one-stage procedure.

Clinical application of skin substitute is typically a two-stage procedure with application of skin substitute matrix to the wound followed by engraftment of a split-thickness skin graft (STSG). This two-stage procedure requires multiple interventions, increasing the time until the wound is epithelialised. In this study, the feasibility of a one-stage procedure by combining bioengineered collagen-chondroitin-6-sulfate (DS1) or decellularised fetal bovine skin substitute (DS2) with autologous skin cell suspension (ASCS) in a porcine full-thickness wound healing model was evaluated. Twelve full-thickness excisional wounds on the backs of pigs received one of six different treatments: empty; ASCS; DS1 with or without ASCS; DS2 with or without ASCS. The ASCS was prepared using a point-of-care device and was seeded onto the bottom side of DS1, DS2, and empty wounds at 80 000 cells/cm2 . Wound measurements and photographs were taken on days 0, 9, 14, 21, 28, 35, and 42 post-wounding. Histological analysis was performed on samples obtained on days 9, 14, 28, and 42. Wounds in the empty group or with ASCS alone showed increased wound contraction, fibrosis, and myofibroblast density compared with other treatment groups. The addition of ASCS to DS1 or DS2 resulted in a marked increase in re-epithelialisation of wounds at 14 days, from 15 ± 11% to 71 ± 20% (DS1 vs DS1 + ASCS) or 28 ± 14% to 77 ± 26 (DS2 vs DS2 + ASCS) despite different mechanisms of tissue regeneration employed by the DS used. These results suggest that this approach may be a viable one-stage treatment in clinical practice.

Biophysical Characterization of a Novel Tri-Layer Placental Allograft Membrane.

Objective: Placental tissues, including membranes composed of amnion and chorion, are promising options for the treatment of chronic wounds. Amnion and chorion contain multiple extracellular matrix (ECM) proteins and a multitude of growth factors and cytokines that, when used clinically, assist in the progression of difficult to heal wounds through restoration of a normal healing process. The objective of this study was to characterize the in vitro physical and biological properties of a dehydrated tri-layer placental allograft membrane (TPAM) consisting of a chorion layer sandwiched between two layers of amnion. Approach: Mechanical properties were evaluated by mechanical strength and enzyme degradation assays. The ECM composition of TPAM membranes was evaluated by histological staining while growth factors and cytokine presence was evaluated by a multiplex enzyme-linked immunosorbent assay. Proliferation, migration, and ECM secretion assays were performed with fibroblasts. Immunomodulatory properties were assessed by a pro-inflammatory cytokine reduction assay while the macrophage phenotype was determined by quantifying the ratio of M1 versus M2 secreted factors. Results: The unique three-layer construction improves mechanical handling properties over single- and bi-layer membranes. Results demonstrate that TPAM is rich in ECM proteins, growth factors, cytokines, and tissue inhibitors of metalloproteinases, and favorably influences fibroblast migration, proliferation, and ECM secretion when compared to negative controls. Furthermore, after processing and preservation, these membranes maintain their intrinsic immunomodulatory properties with the ability to suppress pro-inflammatory processes and modulate the M1 and M2 macrophage phenotype toward a pro-regenerative profile when compared to a negative control. Innovation: This is the first study to characterize both the biophysical and biological properties of a tri-layer placental membrane. Conclusion: This work demonstrates that TPAM has improved handling characteristics over single- and bi-layer membranes, stimulates pro-healing cellular responses, and advantageously modulates inflammatory responses, altogether making this scaffold a promising option for treating wounds, especially those that are complex or difficult to heal.

Biological characterization of dehydrated amniotic membrane allograft: Mechanisms of action and implications for wound care.

There is a growing clinical demand in the wound care market to treat chronic wounds such as diabetic foot ulcers. Advanced cell and tissue-based products (CTPs) are often used to address challenging chronic wounds where healing has stalled. These products contain active biologics such as growth factors and cytokines as well as structural components that support and stimulate cell growth and assist in tissue regeneration. This study addresses the in vitro biologic effects of a clinically available dehydrated amniotic membrane allograft (DAMA). The broad mechanism of action results from DAMA's biologic composition that leads to stimulation of cell migration cell proliferation, and reduction of pro-inflammatory cytokines. Results show that DAMA possesses growth factors and cytokines such as EGF, FGF, PDGFs, VEGF, TGF-ß, IL-8, and TIMPs 1 and 2. Furthermore, in vitro experiments demonstrate that DAMA stimulates cell proliferation, cell migration, secretion of collagen type I, and the reduction of pro-inflammatory cytokines IL-1ß, IL-6, and TNF-α. This study findings are consistent with the clinical benefits previously published for DAMA and other CTPs in chronic wounds suggesting that the introduction of DAMA to non-healing, complex wounds helps to improve the wound milieu by providing essential structural components, cytokines, and growth factors to create an appropriate environment for wound healing.

Synthesis and evaluation of anti-inflammatory, analgesic, ulcerogenic and lipid peroxidation properties of new 2-(4-isobutylphenyl)propanoic acid derivatives.

Synthesis and pharmacological evaluation of various 2-(4-isobutylphenyl)propanoic acid derivatives containing 1,3,4-thiadiazole and thiadiazolo[3,2-a][1,3,5]triazine-5-thione nucleus is reported here. The structures of new compounds are supported by IR, (1)H & (13)C NMR data. These compounds were tested in vivo for their anti-inflammatory activity. The compounds which showed activity comparable to the standard drug ibuprofen were screened for their analgesic, ulcerogenic and lipid peroxidation activities. The compounds, which showed less ulcerogenic action, also showed reduced malondialdehyde production (MDA). Compound 4i and 5f showed 89.50 and 88.88% of inhibition in paw edema, 69.80 and 66.25% protection against acetic acid-induced writhings and 0.7 and 0.65 of severity index, respectively, compared to 90.12, 72.50 and 1.95 values of ibuprofen.

The effects of low-intensity pulsed ultrasound upon diabetic fracture healing.

In the United States, over 17 million people are diagnosed with type 1 diabetes mellitus (DM) with its inherent morbidity of delayed bone healing and nonunion. Recent studies demonstrate the utility of pulsed low-intensity ultrasound (LIPUS) to facilitate fracture healing. The current study evaluated the effects of daily application of LIPUS on mid-diaphyseal femoral fracture growth factor expression, cartilage formation, and neovascularization in DM and non-DM BB Wistar rats. Polymerase chain reaction (PCR) and ELISA assays were used to measure and quantify growth factor expression. Histomorphometry assessed cartilage formation while immunohistochemical staining for PECAM evaluated neovascularization at the fracture site. In accordance with previous studies, LIPUS was shown to increase growth factor expression and cartilage formation. Our study also demonstrated an increase in fracture callus neovascularization with the addition of LIPUS. The DM group showed impaired growth factor expression, cartilage formation, and neovascularization. However, the addition of LIPUS significantly increased all parameters so that the DM group resembled that of the non-DM group. These findings suggest a potential role of LIPUS as an adjunct for DM fracture treatment.

The relationship between functional sciatic nerve block duration and the rate of release of lidocaine from a controlled-release matrix.

BACKGROUND: Nerve blocks of long duration are often desirable in perioperative and postoperative situations. The relationship between the duration of such blocks and the rate at which a local anesthetic is released is important to know for developing a localized drug delivery system that will optimize block duration. METHODS: Lidocaine concentration was varied in 1 series of formulations (OSB-L) containing a constant amount of release rate modifier. In another series (OST-R), the release rate modifier was varied while the lidocaine content was held constant. Release kinetics were measured in vitro and correlated to the in vivo duration of antinociceptive and motor block effects when the formulation was implanted next to the rat sciatic nerve. In parallel studies, rats receiving different formulations of slow-release lidocaine were fixed by intracardiac perfusion with 4% paraformaldehyde and nerve-muscle tissue taken for histopathological analysis. RESULTS: In this study, we have demonstrated that the most important variable for effecting functional nerve block, i.e., the blockade of impulses in the relevant fibers of the sciatic nerve, is the rate of lidocaine release at that time. For the OSB-L formulations (lidocaine concentrations of 1.875%, 3.75%, 7.5%, and 15% at a constant release rate modifier of 5%), the average in vitro release rates at 50% recovery of motor block and nociceptive block were 0.91 +/- 0.28 and 1.75 +/- 0.61 mg/h, respectively. For the OST-R formulations (16% lidocaine with release rate modifier concentrations of 1.875%, 3.75%, 7.5%, and 15%), the average in vitro release rates at 50% recovery of motor block and nociceptive block were 2.33 +/- 1.39 and 4.34 +/- 1.09 mg/h, respectively. The OSB-L formulations showed a dose-dependent increase in block duration proportional to an increase in initial lidocaine concentration, whereas the OST-R formulations showed a nonmonotonic relationship between release rate modifier concentration and block duration. The histopathological studies at 24 hours, 3, 5, or 7 days, and 4 weeks after the implantation revealed inflammatory reactions with degrees correlated with lidocaine content, but limited to the connective tissue and muscle immediately surrounding the implanted material. Despite these observed inflammatory reactions, nociceptive and motor block function returned to normal, preimplantation values in all animals. CONCLUSIONS: Increasing initial lidocaine content proportionately increased the duration of functional sciatic nerve block. However, decreasing the release rate per se does not give a proportional increase in block duration. Instead, there seems to be an optimal, intermediate release rate for achieving the maximum duration of block.

An absorbable local anesthetic matrix provides several days of functional sciatic nerve blockade.

BACKGROUND: Functional blockade of peripheral nerves is the primary objective of local anesthesia, and it is often desirable to have a persistent blockade, sustained throughout and beyond a surgical procedure. Current local anesthetics give effective analgesia for <8-12 h after a single bolus injection. We report on an implantable, controlled-release drug delivery system intended for use in bone and consisting of a Food and Drug Administration-approved matrix containing lidocaine that is capable of local delivery for several days. METHODS: Xybrex, an absorbable, controlled-release delivery system containing 16% (w/w) lidocaine, was implanted next to the sciatic nerve of male rats (300-350 gm), at lidocaine doses of 5.3, 10.6, 16, and 32 mg lidocaine per rat. For comparison, a lidocaine HCl solution (0.2 mL, 2% = 4 mg) was injected in close proximity to the sciatic nerve. Rats were assessed behaviorally for analgesia by a forceps pinch of the lateral digits, and for motor block by quantifying the extensor postural thrust. Potential neurotoxicity of sciatic nerves was evaluated histologically at 24 h, 4 days, and 4 wk after implantation. The kinetics of lidocaine's release from the matrix was measured in vitro by ultraviolet detection of lidocaine in samples collected at 2.5, 6.5, 20, and 24.25 h. RESULTS: Xybrex at the highest doses (300 and 600 mg/kg, containing 16 and 32 mg of lidocaine free base, respectively) provided complete analgesia to an intense pinch for 7.0 +/- 2.0 h, 6.9 +/- 1.7 h and partial analgesia for 60.0 +/- 5.4 h, 58.8 +/- 4.2 h, respectively, compared to 0.61 +/- 0.03 h of complete analgesia and 0.96 +/- 0.03 h of partial analgesia by sciatic block from the 2% lidocaine solution (containing 4 mg lidocaine). These same high doses of Xybrex produced complete motor block for 17.0 +/- 3.3 h, 17.6 +/- 3.3 h with full recovery in 352.0 +/- 55.7 h (14.7 +/- 2.3 days), 579.0 +/- 36.1 h (24.1 +/- 1.5 days) respectively. Data are reported as mean +/- SE. P < 0.001 for all Xybrex groups compared to the 2% lidocaine group. Minor local tissue inflammation/pathology, primarily in the connective tissue and muscle 0.1 mm adjacent to the nerve, was observed equally in animals treated with Xybrex and 2% lidocaine solution. There were no behavioral signs of systemic toxicity. The in vitro release followed exponential kinetics and its comparison to the time-course of functional nociceptive deficit implied that the duration of nociception represented the local, immediate interaction of lidocaine between the nerve and the matrix and not a cumulative effect of previously released drug. CONCLUSIONS: Xybrex is an absorbable, controlled-release drug delivery system that provides several days of analgesia for rat peripheral nerves without apparent significant local neurotoxicity or systemic toxicity.

Complications of ankle fracture in patients with diabetes.

Ankle fractures in patients with diabetes mellitus have long been recognized as a challenge to practicing clinicians. Complications of impaired wound healing, infection, malunion, delayed union, nonunion, and Charcot arthropathy are prevalent in this patient population. Controversy exists as to whether diabetic ankle fractures are best treated noninvasively or by open reduction and internal fixation. Patients with diabetes are at significant risk for soft-tissue complications. In addition, diabetic ankle fractures heal, but significant delays in bone healing exist. Also, Charcot ankle arthropathy occurs more commonly in patients who were initially undiagnosed and had a delay in immobilization and in patients treated nonsurgically for displaced ankle fractures. Several techniques have been described to minimize complications associated with diabetic ankle fractures (eg, rigid external fixation, use of Kirschner wires or Steinmann pins to increase rigidity). Regardless of the specifics of treatment, adherence to the basic principles of preoperative planning, meticulous soft-tissue management, and attention to stable, rigid fixation with prolonged, protected immobilization are paramount in minimizing problems and yielding good functional outcomes.

Diabetic fracture healing.

Patients with diabetic ankle fractures consistently are at greater risk of sustaining a complication during treatment than nondiabetics.other medical comorbidities, especially Charcot neuroarthropathy and peripheral vascular disease, play distinct roles in increasing these complication rates. Many options for nonoperative and operative treatment exist, but respect for soft tissue management and attention to stable, rigid fixation with prolonged immobilization and prolonged restricted weight bearing are paramount in trying to minimize problems and yield functions.

The effects of local platelet rich plasma delivery on diabetic fracture healing.

Several studies have documented that diabetes impairs bone healing clinically and experimentally. The percutaneous delivery of platelet rich plasma (PRP) was used in the diabetic BB Wistar femur fracture model to investigate the use of PRP as a concentrated source of critical early growth factors on bone healing. PRP delivery at the fracture site normalized the early (cellular proliferation and chondrogenesis) parameters while improving the late (mechanical strength) parameters of diabetic fracture healing. These results suggest a role for PRP in mediating diabetic fracture healing and potentially other high risk fractures.

The role of platelet-rich plasma in foot and ankle surgery.

Platelet-rich plasma (PRP), derived from autologous blood, is defined as a volume of plasma that has a platelet concentration that typically is five times greater (approximately 1,000,000/microl) than physiologic levels. PRP serves as a reservoir of critical growth factors, including platelet-derived growth factor, transforming growth factor-beta, and insulin-like growth factor-I. Although there is an abundance of literature pertaining to dental applications, this article highlights the use of PRP in orthopedic applications, ranging from PRP preparation to in vitro and in vivo studies to clinical research.

The effects of local insulin delivery on diabetic fracture healing.

Several studies have documented that diabetes impairs bone healing clinically and experimentally. Systemic insulin treatment has been shown to ameliorate impaired diabetic bone healing. However, these studies failed to distinguish between a direct and a systemic effect of insulin upon bone healing. A novel intramedullary insulin delivery system was used in the diabetic BB Wistar femur fracture model to investigate the potential direct effects of insulin on bone healing. Insulin delivery at the fracture site normalized the early (cellular proliferation and chondrogenesis) and late (mineralized tissue, cartilage content and mechanical strength) parameters of diabetic fracture healing without affecting the systemic parameters of blood glucose. These results suggest a critical role for insulin in directly mediating fracture healing and that decreased systemic insulin levels in the diabetic state lead to reduced localized insulin levels at fracture site with concomitant increases in diabetic fracture healing time.

Effects of triiodothyronine pretreatment on beta-adrenergic responses in stunned cardiac myocytes.

OBJECTIVE: To investigate whether triiodothyronine pretreatment enhanced beta-adrenergic responses in stunned myocardium and whether this acute effect of triiodothyronine was mediated through the cyclic adenosine 3',5'-monophosphate (AMP) system. DESIGN: A prospective study. SETTING: University laboratory. PARTICIPANTS: Rabbits. INTERVENTIONS: Rabbit ventricular myocytes were isolated and placed in a medium equilibrated with either air (control) or with 95% N(2) and 5% CO(2) (stunned) for 15 minutes at 37 degrees C. The stunned myocytes were reoxygenated with air for 30 minutes. Triiodothyronine (10 nmol/L) and/or isoproterenol (0.1 nmol/L) was added to the myocytes. Myocyte shortening was measured by using a video-edge detector. MEASUREMENTS AND MAIN RESULTS: In electrically stimulated cells, the basal values of the percent shortening (22%-30%) and the maximum rate of shortening (22%-25%) were significantly reduced in the stunned myocytes. Isoproterenol (5 minutes) alone significantly increased the percent shortening in the control (from 3.70 +/- 0.36 to 4.14 +/- 0.37) but not in the stunned myocytes (from 2.60 +/- 0.30 to 3.15 +/- 0.27). Triiodothyronine (5 minutes) alone significantly increased the percent shortening in the control (from 3.75 +/- 0.36 to 4.34 +/- 0.45) and in the stunned myocytes (from 2.91 +/- 0.2 to 3.85 +/- 0.26). After triiodothyronine pretreatment for 5 minutes, isoproterenol caused greater increases in the percent shortening in both the control (37%) and the stunned myocytes (62%) than either agent alone. Isoproterenol or triiodothyronine caused small increases in the maximum rate of shortening in the control (14%-16%) and the stunned myocytes (34%-49%). After triiodothyronine pretreatment, isoproterenol caused greater increases in the maximum rate of shortening in both groups (control: 41%, stunned: 73%) than either agent alone. Isoproterenol caused an increase in the level of cyclic AMP (rho;moles/10(5) myocytes) in the control (from 2.92 +/- 0.47 to 3.77 +/- 0.43) but not in the stunned myocytes (from 2.42 +/- 0.25 to 2.42 +/- 0.20). Triiodothyronine pretreatment did not cause any change in cyclic AMP levels in the control (2.50 +/- 0.29) or in the stunned myocytes (2.60 +/- 0.40). After triiodothyronine pretreatment, isoproterenol caused a small increase in the cyclic AMP level in the control but not in the stunned myocytes. CONCLUSIONS: The data showed that the myocardial beta-adrenergic responses were more sensitive to ischemic insult than the triiodothyronine responses. Triiodothyronine pretreatment enhanced beta-adrenergic responses in both the control and the stunned myocytes. However, this acute positive inotropic effect of triiodothyronine might not be mediated through the cyclic AMP system.

Negative effect of nitric oxide on shortening-frequency relationship in cardiac myocytes is diminished after simulated ischemia-reperfusion.

Increasing stimulation rate increases function in cardiac myocytes and nitric oxide and cyclic GMP inhibit this effect. We tested the hypothesis that myocyte stunning would blunt both the effects of increases in rate and of nitric oxide and cyclic GMP. Ventricular myocytes from 11 rabbits were used to determine maximum rate of shortening (R(max), microm/s) and % shortening during control and after simulated ischemia [15 min 95% N(2)- 5% CO(2)] and reperfusion [reoxygenation]. Measurements were obtained at 1-4 Hz with vehicle, 1H[1,2,4]oxadiazolo[4,3,alpha] quinoxaline-1-one (ODQ) 10(-6) M, soluble guanylyl cyclase inhibitor, or N(G)-nitro-L-arginine methyl ester, nitric oxide synthase inhibitor (L-NAME) 10(-5) M. In control, increases in rate increased R(max) from 69 +/- 3 to 254+/-12 and % shortening from 5.3 +/- 0.3 to 8.7 +/- 0.5. Both ODQ and L-NAME shifted values higher. With stunning, the effects of pacing on Rmax and % shortening were blunted and ODQ and L-NAME failed to alter these values. Cyclic GMP was 322+/-37 pmol/10(5) myocytes at baseline and these values were lowered by ODQ (244 +/- 31) and LNAME (207 +/- 23), and similar changes were observed in stunned myocytes. Increasing frequency increased function, and reducing nitric oxide/cyclic GMP enhanced this relationship. The effect of nitric oxide was diminished by stunning, but this was not related to altered cyclic GMP levels. This suggested changes in effects of cyclic GMP downstream to its production during myocardial stunning.