Richard C. Baynosa

Levels of Evidence in Research Published in The Journal of Bone and Joint Surgery (American Volume) Over the Last Thirty Years

BACKGROUND The recent emphasis on evidence-based medicine has led to increasing levels of evidence being published in surgical journals. The purpose of the present study was to review the levels of evidence in reports published in The Journal of Bone and Joint Surgery (American Volume) over the last thirty years. METHODS We reviewed all of the articles published in The Journal in the years 1975, 1985, 1995, and 2005. Cadaver studies, animal studies, basic-science studies, review articles, Instructional Course Lectures, and correspondence were excluded. Articles were scored according to The Journals levels of evidence for a primary research question. RESULTS A total of 1058 articles were reviewed. Of these, 134, 123, 120, and 174 articles met the inclusion criteria for the years 1975, 1985, 1995, and 2005, respectively, and were ranked according to level of evidence. The number of articles for each level of evidence rating was then expressed as a percentage of the total number of articles meeting the inclusion criteria for that year. There was a significant trend toward higher levels of evidence, with the combined percentage of Level-I, II, and III studies increasing from 17% to 52% (p < 0.01). The percentage of Level-I studies increased from 4% in 1975 to 21% in 2005. The average level of evidence rating improved from 3.72 to 2.90 during the study period. CONCLUSIONS The level of evidence in The Journal has improved significantly over the last thirty years.

Update on ischemia-reperfusion injury for the plastic surgeon: 2011.

Ischemia-reperfusion injury occurs when tissue is reperfused following a prolonged period of ischemia. It is a subject of interest to plastic surgeons involved in replantation, free tissue transfer, and composite tissue allotransplantation, as it can have a significant impact on the overall success of these procedures. The purpose of this article is to review the recent progress in the investigation of ischemia-reperfusion injury in skeletal muscle and skin and to highlight the potential clinical implications of therapeutic interventions aimed at reducing ischemia-reperfusion injury.

Microcirculatory effects of melatonin in rat skeletal muscle after prolonged ischemia.

Abstract:  The purpose of this study was to determine microcirculatory effects and response of nitric oxide synthase (NOS) to melatonin in skeletal muscle after prolonged ischemia. A vascular pedicle isolated rat cremaster muscle model was used. Each muscle underwent 4 hr of zero‐flow warm ischemia followed by 2 hr of reperfusion. Melatonin (10 mg/kg) or saline as a vehicle was given by intraperitoneal injection at 30 min prior to reperfusion and the same dose was given immediately after reperfusion. After reperfusion, microcirculation measurements including arteriole diameter, capillary perfusion and endothelial‐dependent and ‐independent vasodilatation were performed. The cremaster muscle was then harvested to measure endothelial NOS (eNOS) and inducible NOS (iNOS) gene expression and enzyme activity. Three groups of rats were used: sham‐ischemia/reperfusion (I/R), vehicle + I/R and melatonin + I/R. As compared with vehicle + I/R group, administration of melatonin significantly enhanced arteriole diameter, improved capillary perfusion, and attenuated endothelial dysfunction in the microcirculation of skeletal muscle after 4 hr warm ischemia. Prolonged warm ischemia followed by reperfusion significantly depressed eNOS gene expression and constitutive NOS activity and enhanced iNOS gene expression. Administration of melatonin did not significantly alter NOS gene expression or activity in skeletal muscle after prolonged ischemia and reperfusion. Melatonin provided a significant microvascular protection from reperfusion injury in skeletal muscle. This protection is probably attributable to the free radical scavenging effect of melatonin, but not to its anti‐inflammatory effect.

Analysis for apoptosis and necrosis on adipocytes, stromal vascular fraction, and adipose-derived stem cells in human lipoaspirates after liposuction.

Background: Adipose-derived stem cells have become the most studied adult stem cells. The authors examined the apoptosis and necrosis rates for adipocyte, stromal vascular fraction, and adipose-derived stem cells in fresh human lipoaspirates. Methods: Human lipoaspirate (n = 8) was harvested using a standard liposuction technique. Stromal vascular fraction cells were separated from adipocytes and cultured to obtain purified adipose-derived stem cells. A panel of stem cell markers was used to identify the surface phenotypes of cultured adipose-derived stem cells. Three distinct stem cell subpopulations (CD90+/CD45−, CD105+/CD45−, and CD34+/CD31−) were selected from the stromal vascular fraction. Apoptosis and necrosis were determined by annexin V/propidium iodide assay and analyzed by flow cytometry. Results: The cultured adipose-derived stem cells demonstrated long-term proliferation and differentiation evidenced by cell doubling time and positive staining with oil red O and alkaline phosphatase. Isolated from lipoaspirates, adipocytes exhibited 19.7 ± 3.7 percent apoptosis and 1.1 ± 0.3 percent necrosis; stromal vascular fraction cells revealed 22.0 ± 6.3 percent of apoptosis and 11.2 ± 1.9 percent of necrosis; stromal vascular fraction cells had a higher rate of necrosis than adipocytes (p < 0.05). Among the stromal vascular fraction cells, 51.1 ± 3.7 percent expressed CD90+/CD45−, 7.5 ± 1.0 percent expressed CD105+/CD45−, and 26.4 ± 3.8 percent expressed CD34+/CD31−. CD34+/CD31− adipose-derived stem cells had lower rates of apoptosis and necrosis compared with CD105+/CD45− adipose-derived stem cells (p < 0.05). Conclusions: Adipose-derived stem cells had a higher rate of apoptosis and necrosis than adipocytes. However, the extent of apoptosis and necrosis was significantly different among adipose-derived stem cell subpopulations.

Nitrite attenuates ischemia-reperfusion-induced microcirculatory alterations and mitochondrial dysfunction in the microvasculature of skeletal muscle.

Background: Recently, nitrite has been rediscovered as a physiologically relevant storage reservoir of nitric oxide in blood and it can readily be converted to nitric oxide under hypoxic and acidic conditions. In this study, the authors evaluated the therapeutic efficacy of nitrite on reperfusion-induced microcirculatory alterations and mitochondrial dysfunction in the microvasculature of skeletal muscle. Methods: The authors used a vascular pedicle isolated rat cremaster model that underwent 4 hours of warm ischemia followed by 2 hours or 17 hours of reperfusion. At 5 minutes before reperfusion, normal saline, sodium nitrite (0.20 &mgr;M/minute/kg), or nitrite mixed with 2-(4-carboxyphenyl)-4,5-dihydro-4,4,5,5-tetramethylimidazoline-3-oxide-1-oxyl (potassium salt) (0.2 mg/minute/kg) was infused into the microcirculation of ischemic cremaster by means of intraarterial infusion. Ischemia-reperfusion–induced microcirculatory alterations were measured after 2 hours of reperfusion. Microvasculature of the cremaster muscle including the vascular pedicle was harvested to determine the mitochondrial dysfunction. The blood concentration of methemoglobin was also measured to determine the toxicity of nitrite. Results: The authors found that nitrite significantly attenuated ischemia-reperfusion–induced vasoconstriction, arteriole stagnation, and capillary no-reflow in the early phase of reperfusion and the depolarization of mitochondrial membrane potential and cytochrome c release in the late phase of reperfusion. Nitrite-induced protection was significantly blocked by a nitric oxide scavenger (potassium salt). The methemoglobin results showed that the doses of nitrite we used in the present study were safe. Conclusion: The supplementation of a low dose of nitrite, directly into the microcirculation of ischemic muscle through local intraarterial infusion, significantly attenuated ischemia-reperfusion–induced microcirculatory alterations in vivo and mitochondrial dysfunction in vitro in the microvasculature of skeletal muscle.

Use of a novel penetrating, sutureless anastomotic device in arterial microvascular anastomoses.

The advent of microsurgery and microvascular anastomosis has helped to alter the treatment of many traumatic defects as well as revolutionize the reconstructive options after extirpative surgery. These procedures, however, continue to be technically demanding and intensive. In addition, increased ischemia and operative times are known to raise complications in microsurgery, particularly in older patients. The nitinol U-clip (Medtronic, Inc., Minneapolis, MN) is a new penetrating clip applied in an interrupted fashion that eliminates knot tying and decreases operative time. This technology holds the potential for rapid, reproducible sutureless microvascular anastomoses. In addition, the surgical technique is very similar to conventional suture microanastomosis, lending itself to comparatively decreased learning curves and suitability for training programs. We present three consecutive cases of successful microsurgical anastomoses using this technology.

Objective evaluation of skill acquisition in novice microsurgeons.

INTRODUCTION Medical training is increasingly focused on patient safety, limiting the ability to practice technical skills in the operative arena. Alternative methods of training residents must be designed and implemented. METHODS Three expert microsurgeons were solicited to develop two drills to help residents acquire the basic subset of skills in microsurgery. The first drill was performance of five consecutive simple interrupted sutures on a rubber glove. Expert proficiency was considered a drill time of two standard deviations from expert mean. The drill was performed up to 10 times until completion of the task at expert proficiency. The second drill was performance of an anastomosis on silastic tubing. Residents performed the drill sequentially until performing two consecutive drills at expert proficiency. RESULTS Eight residents with no microsurgical experience volunteered. Six of the eight residents were able to perform the rubber glove drill at expert proficiency within 10 attempts, with an average of 5.3. All of the residents were able to perform two consecutive silastic tubing drills at expert proficiency within nine attempts, with an average of 5.4. CONCLUSION Residents were able to acquire a basic subset of microsurgical skills within a reasonable time period using these drills.

The effect of hyperbaric oxygen on nitric oxide synthase activity and expression in ischemia-reperfusion injury

BACKGROUND Hyperbaric oxygen (HBO) mitigates ischemia-reperfusion (IR) injury via a nitric oxide mechanism that is nitric oxide synthase (NOS) dependent. The purpose of this study was to investigate this NOS-dependent mechanism by examining isoform-specific, tissue-specific, and time-specific upregulation of NOS mRNA, protein, and enzymatic activity. METHODS We raised a gracilis flap in Wistar rats that were separated into early and late phases. Treatment groups included nonischemic control, IR, HBO-treated ischemia-reperfusion (IR-HBO), and nonischemic HBO control. We harvested tissue-specific samples from gracilis, rectus femoris, aorta, and pulmonary tissues and processed them by reverse transcription polymerase chain reaction and Western blot to determine upregulation of isoform-specific NOS mRNA and protein. We also harvested tissue for NOS activity to investigate upregulation of enzymatic activity. Data are presented as mean ± standard error of the mean with statistics performed by analysis of variance. P ≤ 0.05 was considered significant. RESULTS There was no increase in NOS mRNA in the early phase. In the late phase, there was a significant increase in endothelial-derived NOS (eNOS) mRNA in IR-HBO compared with IR in gracilis muscle (79.4 ± 22.3 versus 36.1 ± 4.5; P < 0.05) and pulmonary tissues (91.0 ± 31.2 versus 30.2 ± 3.1; P < 0.01). There was a significant increase in the late-phase eNOS pulmonary protein IR-HBO group compared with IR (235.5 ± 46.8 versus 125.2 ± 14.7; P < 0.05). Early-phase NOS activity was significantly increased in IR-HBO compared with IR in pulmonary tissue only (0.049 ± 0.009 versus 0.023 ± 0.003; P < 0.05). CONCLUSIONS The NOS-dependent effects of HBO on IR injury may result from a systemic effect involving an early increase in eNOS enzymatic activity followed by a late-phase increase in eNOS protein expression within the pulmonary tissues.

The Effect of Lipoaspirates Cryopreservation on Adipose-Derived Stem Cells

BACKGROUND Autologous fat grafting has gained popularity, particularly with the discovery of adipose-derived stem cells (ADSC). The possibility of freezing lipoaspirates (LA) for later use has intriguing clinical potential. However, the effect of LA cryopreservation on ADSC is unclear. OBJECTIVES The authors explore the effect of LA cryopreservation on ADSC viability. METHODS Human LA (n = 8) were harvested using a standard technique. Lipoaspirate samples were either processed immediately as fresh LA (A) or stored at -20°C and then at -80°C for 30 days with (B) or without (C) freezing medium. Stromal vascular fraction (SVF) was separated from adipocytes and either cultured to obtain purified ADSC or processed for the isolation of 3 distinct ADSC subpopulations (CD90(+)/CD45(-), CD105(+)/CD45(-), and CD34(+)/CD31(-)). Apoptosis and necrosis were determined by an annexin V/propidium iodide assay and quantified by flow cytometry. The capability of ADSC for long-term proliferation and differentiation was also examined. RESULTS There were no significant differences in the apoptosis and necrosis of adipocytes, SVF, or ADSC between groups A and B. However, cell viability in SVF and ADSC was significantly compromised in group C as compared with group B (P < .01) due to higher ADSC apoptosis but not necrosis. The viable ADSC isolated from fresh or frozen LA were cultured for more than 20 passages and demonstrated similar patterns and speed of proliferation with strong capability to differentiate, evidenced by cell doubling time and positive staining with Oil Red O (Sigma-Aldrich, St Louis, Missouri) and alkaline phosphatase. CONCLUSIONS Lipoaspirates cryopreservation had a significant impact on ADSC apoptosis but not on ADSC necrosis, proliferation, or differentiations. Freezing medium provides significant protection against ADSC apoptosis.

Evaluation of artificial dermis neovascularization in an avascular wound.

Soft tissue coverage for avascular wounds is necessary in reconstructive surgery. Several authors have demonstrated successful treatment of problem wounds using artificial dermis. This study evaluates in an animal model the potential for neovascularization of artificial dermis in devascularized and avascular wound beds. Forty rats were assigned to four groups: (1) control, full-thickness skin graft was replaced on the vascular wound bed; (2) Integra, Integra placed over the full-thickness wound; (3) fascia, the spinotrapezius fascia exposed and Integra placed over the wound; and (4) fascia/Parafilm, a Parafilm layer placed under the raised fascia with Integra over the fascia. Laser Doppler readings were taken at baseline over the intact skin and then over the created wound beds. Biopsies of the full-thickness skin graft and the neodermis were obtained on postoperative day 14 and histologically evaluated for neovascularization. The laser Doppler readings confirmed the nature of the surgically created, poorly vascularized and avascular wound beds. Subsequent biopsies of the artificial dermis in these wound beds, however, demonstrated active neovascularization. This study demonstrates that Integra artificial dermis can serve as an effective dermal substitute in avascular wounds. Lateral ingrowth of capillaries into the dermal substitute may explain the successful integration of this artificial dermis.