Morphological and Histological Changes in the Human Anterior Cruciate Ligament after Rupture.

BACKGROUND: Injuries to the anterior cruciate ligament (ACL) are common and complete tears often fail to heal. ACL reconstruction is considered the surgical gold standard of care for ACL injuries in young active patients. OBJECTIVES: To determine the corresponding morphological and histological features of the torn ACL in different time periods after injury. METHODS: The study included 28 remnant specimens of torn ACLs from patients who had ACL reconstruction surgery of the knee. The remnant pathology was evaluated by its morphology during arthroscopy and by histopathologic measurements. RESULTS: At surgery there were three progressive and distinct morphological tear patterns. The first pattern was noticed within the first 3 months from injury and showed no scar tissue. The second pattern appeared later and was characterized by the appearance of scar tissue with adhesion to the femoral wall. The third pattern was characterized by adhesion of the ACL remnant to the posterior cruciate ligament. The histological changes of the first morphological pattern showed abundance of blood vessels and lymphocytes at the torn femoral end with few irregular collagen fibers. The second and third tear patterns showed decrement in the number of blood vessels and lymphocytes with longitudinally oriented collagen fibers. CONCLUSIONS: The morphological features of the ACL remnant in the first 3 months after injury showed no scar tissue and its histological features had the characteristics of a reparative phase. This phase was followed by a prolonged remodeling phase that ended with attachment of the remnant to the posterior cruciate ligament.

Efficacy of gelatin gel sheets sustaining epidermal growth factor for murine skin defects.

BACKGROUND: Epidermal growth factor (EGF) plays an important role in wound healing. However, EGF must be applied daily due to rapid inactivation in vivo. We investigated the sustained release of EGF from gelatin gel sheets (GGSs) and the efficacy of GGSs impregnated with EGF for promoting wound healing. MATERIALS AND METHODS: GGSs impregnated with EGF were prepared by cross-linking via glutaraldehyde to gelatin solution containing EGF. The sustained release of EGF and the bioactivity of released EGF were evaluated. Then, three kinds of GGSs containing NSS (normal saline solution; NSS group), 2.5 µg of EGF (EGF-L group), or 25 µg of EGF (EGF-H group) were applied to full-thickness skin defects created on the backs of mice. The wounds covered with polyurethane film without GGS were used as a control (PUF group). The wound area, neoepithelium length, regenerated granulation tissue, and newly formed capillaries were evaluated. RESULTS: EGF was sustained and released from GGS as it degraded. The bioactivity of released EGF was confirmed. EGF-L group promoted the neoepithelium length, regenerated granulation tissue, and newly formed capillaries compared with those in the PUF and NSS groups. The area of regenerated granulation tissue in the NSS group (week 1: 2.6 + 0.2 mm(2), week 2: 2.8 + 0.3 mm(2)) was larger than that in the PUF group (week 1: 0.6 + 0.1 mm(2), week 2: 1.0 + 0.1 mm(2)). The area of newly formed capillaries in the EGF-L group (9967 + 1903 µm(2)) was larger than that of the EGF-H group (3485 + 1050 µm(2)). CONCLUSIONS: GGSs impregnated with EGF-L showed promising results regarding wound healing.

[OBJECTIVE CRITERIA OF PATIENT'S READINESS TO FREE AUTOPLASTY IN CASE OF GRANULATING WOUNDS (CRITERIA OF READINESS OF GRANULATING WOUNDS TO OPERATION)].

The article based on the analysis of 84 follow-up of the patients. The authors suggested using the indices of microbiological and immunological investigations and data of laser Doppler ultrasonography to determine the readiness of granulating wound to free autoplasty. The data obtained allowed developing an algorithm of treatment, patient's preparation to surgery and determination of operation terms.

Regional disturbances in blood flow and metabolism in equine limb wound healing with formation of exuberant granulation tissue.

As in other fibroproliferative disorders, hypoxia has been suggested to play a key role in the pathogenesis of exuberant granulation tissue (EGT). The purpose of this study was to investigate metabolism and blood flow locally in full-thickness wounds healing with (limb wounds) and without (body wounds) formation of EGT. Microdialysis was used to recover endogenous metabolites from the wounds, and laser Doppler flowmetry was used to measure blood flow. Measurements were performed before wounding and 1-28 days after wounding. Blood flow was consistently lower in limb wounds than in body wounds throughout the study period with no change over time. After wounding and throughout the study period, the glucose concentration was significantly lower in limb wounds than in body wounds, whereas the lactate level showed a significantly higher concentration in limb wounds. The lactate/glucose ratio displayed a significant difference between body and limb wounds. In conclusion, the metabolic disturbances may suggest an inadequate oxygen supply during the wound healing process in equine limb wounds healing with EGT. This may be related to the inherently decreased perfusion in the wound bed of limb wounds.

Comparison of bacteria and fungus-binding mesh, foam and gauze as fillers in negative pressure wound therapy--pressure transduction, wound edge contraction, microvascular blood flow and fluid retention.

Bacteria- and fungus-binding mesh binds with and inactivates bacteria and fungus, which makes it an interesting alternative, wound filler for negative pressure wound therapy (NPWT). This study was conducted to compare the performance of pathogen-binding mesh, foam and gauze as wound fillers in NPWT with regard to pressure transduction, fluid retention, wound contraction and microvascular blood flow. Wounds on the backs of 16 pigs were filled with pathogen-binding mesh, foam or gauze and treated with NPWT. The immediate effects of 0, -40, -60, -80 and -120 mmHg, on pressure transduction and blood flow were examined in eight pigs using laser Doppler velocimetry. Wound contraction and fluid retention were studied during 72 hours of NPWT at -80 and -120 mmHg in the other eight pigs. Pathogen-binding mesh, gauze and foam provide similar pressure transduction to the wound bed during NPWT. Blood flow was found to decrease 0.5 cm laterally from the wound edge and increase 2.5 cm from the wound edge, but was unaltered 5.0 cm from the wound edge. The increase in blood flow was similar with all wound fillers. The decrease in blood flow was more pronounced with foam than with gauze and pathogen-binding mesh. Similarly, wound contraction was more pronounced with foam, than with gauze and pathogen-binding mesh. Wound fluid retention was the same in foam and pathogen-binding mesh, while more fluid was retained in the wound when using gauze. The blood flow 0.5-5 cm from the wound edge and the contraction of the wound during NPWT were similar when using pathogen-binding mesh and gauze. Wound fluid was efficiently removed when using pathogen-binding mesh, which may explain previous findings that granulation tissue formation is more rapid under pathogen-binding mesh than under gauze. This, in combination with its pathogen-binding properties, makes this mesh an interesting wound filler for use in NPWT.

[Influence of microbial wound dissemination and microcirculation on the results of skin engraftment].

The indices of Doppler laser flowmetery are proposed to be used for determination of the readiness of a granulating wound for free autoplasty. An analysis of capillary blood flow in the groups under test showed the information value of indicators of microcirculation obtained by Doppler laser flowmetery for determination of the granulating wound condition before autotransplantation and prediction of the results of skin engraftment. It is stated, that the disorder of microcirculation has been developed against the background of progression of wound invasive infection. The obtained data can allow the development of an algorithm of treatment and the preparation of the patients to surgery, determination of the terms of operation, the development the strategy of postoperative management of the patients, which can reduce unfavorable results of operations.

Contrast-enhanced ultrasonography for the detection of joint vascularity in arthritis--subjective grading versus computer-aided objective quantification.

PURPOSE: To compare joint inflammation assessment using subjective grading of power Doppler ultrasonography (PDUS) and contrast-enhanced ultrasonography (CEUS) versus computer-aided objective CEUS quantification. MATERIALS AND METHODS: 37 joints of 28 patients with arthritis of different etiologies underwent B-mode ultrasonography, PDUS, and CEUS using a second-generation contrast agent. Synovial thickness, extent of vascularized pannus and intensity of vascularization were included in a 4-point PDUS and CEUS grading system. Subjective CEUS and PDUS scores were compared to computer-aided objective CEUS quantification using Qontrast® software for the calculation of the signal intensity (SI) and the ratio of SI for contrast enhancement. RESULTS: The interobserver agreement for subjective scoring was good to excellent (κ = 0.8 - 1.0; P < 0.0001). Computer-aided objective CEUS quantification correlated statistically significantly with subjective CEUS (P < 0.001) and PDUS grading (P < 0.05). The Qontrast® SI ratio correlated with subjective CEUS (P < 0.02) and PDUS grading (P < 0.03). Clinical activity did not correlate with vascularity or synovial thickening (P = N. S.) and no correlation between synovial thickening and vascularity extent could be found, neither using PDUS nor CEUS (P = N. S.). CONCLUSION: Both subjective CEUS grading and objective CEUS quantification are valuable for assessing joint vascularity in arthritis and computer-aided CEUS quantification may be a suitable objective tool for therapy follow-up in arthritis.

Effect of transplantation of bone marrow monomuclears on angiogenesis in rats.

We studied the effect of non-selective intracoronary transplantation of bone marrow mononuclears on day 30 after acute coronary infarction on angiogenesis in rats. On days 14 and 30 after transplantation of mononuclear cells, stable formation of new vessels was observed. The number of venules considerably increased after transplantation of mononuclear cells, which was seen from increased volume density of blood vessels and their caliber. Stable vascularization after transplantation of mononuclear cells improves blood supply, which is essential for reparation of the myocardium.

Time- and Dose-Dependent Effects of Pulsed Ultrasound on Dermal Repair in Diabetic Mice.

Chronic wounds, including diabetic, leg and pressure ulcers, impose a significant health care burden worldwide. Some evidence indicates that ultrasound can enhance soft tissue repair. However, therapeutic responses vary among individuals, thereby limiting clinical translation. Here, effects of pulsed ultrasound on dermal wound healing were assessed using a murine model of chronic, diabetic wounds. An ultrasound exposure system was developed to provide daily ultrasound exposures to full-thickness, excisional wounds in genetically diabetic mice. Wounds were exposed to 1 MHz ultrasound (2 ms pulse, 100 Hz pulse repetition frequency, 0-0.4 MPa) for 2 or 3 wk. Granulation tissue thickness and wound re-epithelialization increased as a function of increasing ultrasound pressure amplitude. At 2 wk after injury, significant increases in granulation tissue thickness and epithelial ingrowth were observed in response to 1 MHz pulsed ultrasound at 0.4 MPa. Wounds exposed to 0.4 MPa ultrasound for 3 wk were characterized by collagen-dense, revascularized granulation tissue with a fully restored, mature epithelium. Of note, only half of wounds exposed to 0.4 MPa ultrasound showed significant granulation tissue deposition after 2 wk of treatment. Thus, the db+/db+ mouse model may help to identify biological variables that influence individual responses to pulsed ultrasound and accelerate clinical translation.

Study on the Effect and Mechanism of Antibacterial Adhesive Hydrogel on Wound Healing.

Bleeding and infection can cause significant increases in mortalities. Hydrogel sealants have attracted extensive attention for their ability to control bleeding. In this study, the adjuvant treatment with antibacterial adhesive hydrogel dressings was applied to patients with deep second-degree burns/scalds. The traditional medical dressing was regarded as control adjuvant treatment. The results indicated that the total positive rate of bacteria in wound secretions and the pain during dressing change in patients who used antibacterial adhesive hydrogel dressings were significantly reduced. The number of fibroblasts and new capillaries in the granulation tissue of the wound increased, and the patient's wound healing is accelerated. The overall clinical effectiveness has been significantly improved. It is proven that the antibacterial adhesive hydrogel dressing has a significant effect on wound healing.

Vascularity during wound maturation correlates with fragmentation of serum albumin but not ceruloplasmin, transferrin, or haptoglobin.

Reduced vascularity during wound maturation is mediated by endothelial apoptosis. Albumin has an anti-apoptotic activity for endothelium, which increases up to 100-fold on albumin fragmentation (AF). We now report that levels of AF correlate with changing vascularity during wound maturation. Both scarring and adipogenic wound-healing models were established in mice. Western blots of granulation tissue revealed AF concurrent with periods of high vascularity as determined by thin-section microscopy, with reduced AF on wound maturation (p<0.02). In profiling AF, the levels of 27.5 and 39 kDa fragments were reduced on maturation of both scarring and adipogenic wounds (p<0.005), as were the levels of an additional 17.5 kDa fragment prominent only in adipogenic wounds (p<0.001). A 49 kDa albumin fragment was found to be reduced during maturation of scarring (p<0.001) but not adipogenic wounds. For comparison, we probed for transferrin, ceruloplasmin, and haptoglobin fragmentation on the basis that like albumin, these are considered acute-phase transport proteins. Minimal fragmentation of transferrin and ceruloplasmin was seen, along with partial dissociation of haptoglobin subunits, but these did not correlate with AF or vascularity. Our findings are consistent with a role for AF in regulating granulation tissue vascularity during healing.

Angiogenesis after transplantation of auto- and allogenic cells.

Neoangiogenesis after transplantation of auto- and allogenic mononuclears and multipotent stromal cells from the bone marrow was studied on the model of inflammatory angiogenesis. Transplanted auto- and allogenic cells stimulate the formation of new blood vessels in the granulation tissue, this manifesting in an increase in the quantity and volume density of blood vessels. The most pronounced angiogenesis was observed after transplantation of allogenic mononuclears and multipotent stromal cells. It was associated with intense inflammatory infiltration, with less numerous and mature collagen fibers in the granulation tissue. Injection of allogenic cells led to stimulation and chronization of inflammation, infiltration with inflammatory and poorly differentiated cells, and more pronounced and lasting angiogenesis. However, neither auto-, nor allogenic transplanted labeled cells were detected in the walls of new blood vessels. Hence, it seems that bone marrow mononuclears and multipotent stromal cells stimulated angiogenesis mainly at the expense of production of angiogenic factors, and after transplantation of allogenic cells also by stimulating the inflammation.

Requirement of vascular integrin alpha v beta 3 for angiogenesis.

Angiogenesis depends on the adhesive interactions of vascular cells. The adhesion receptor integrin alpha v beta 3 was identified as a marker of angiogenic vascular tissue. Integrin alpha v beta 3 was expressed on blood vessels in human wound granulation tissue but not in normal skin, and it showed a fourfold increase in expression during angiogenesis on the chick chorioallantoic membrane. In the latter assay, a monoclonal antibody to alpha v beta 3 blocked angiogenesis induced by basic fibroblast growth factor, tumor necrosis factor-alpha, and human melanoma fragments but had no effect on preexisting vessels. These findings suggest that alpha v beta 3 may be a useful therapeutic target for diseases characterized by neovascularization.

[Effects of reinforcing qi and nourishing blood recipes on vascular endothelial growth factor and microvessel count in granulation tissue in rats with chronic skin ulcers].

OBJECTIVE: To explore the effects of reinforcing qi and nourishing blood recipes on vascular endothelial growth factor (VEGF) and the microvessel count (MVC)in granulation tissue in rats with chronic skin ulcers. METHODS: A total of 90 male SD rats with back full-thickness skin lesion were used in this study. Except for the normal control group, the rats were injected with hydrocortisone for inducing chronic skin ulcers, and were randomly divided into untreated group, Sijunzi Decoction group, Siwu Decoction group and Bazhen Decoction group. The time of wound healing was observed; immunohistochemical method and image analytical method were used to test the expressions of VEGF and MVC in granulation tissue of dermal chronic ulcers in the rats. RESULTS: In the untreated group, the time of wound healing was significantly longer than that in the normal group (P<0.01). The time of wound healing was obviously shortened in the three treated groups as compared with the untreated group (P<0.01, P<0.05). In the Sijunzi Decoction group, the time of wound healing was obviously shorter than those in Siwu Decoction group and Bazhen Decoction group (P<0.05, P<0.01). In the untreated group, the expression of VEGF and MVC were significantly lower than those in the normal control group (P<0.01). The expression of VEGF and MVC were obviously increased in the three treated groups as compared with those in the untreated group (P<0.01, P<0.05). In the Sijunzi Decoction group, the expression of VEGF and MVC were obviously lower than those in Siwu Decoction group and Bazhen Decoction group (P<0.05, P<0.01). CONCLUSION: Reinforcing qi and nourishing blood recipes can promote wound healing in rats through up-regulating the expression of VEGF in granulation tissue of dermal chronic ulcers, and inducing angiogenesis.

Giant arachnoid granulation with a thrombosed dural arteriovenous fistula.

Arachnoid granulations are common incidentally detected small dural lesions which are usually asymptomatic and follow cerebrospinal fluid density/signal intensity on CT/MRI. Such lesions reaching a size of more than 1 cm are termed as giant arachnoid granulations (GAGs) which have been previously reported to cause venous hypertension and headaches. We report a case of GAG involving the proximal half of the superior sagittal sinus in a 45-year-old male patient which was associated with left temporal thrombosed dural arteriovenous fistula (AVF) whose thrombosed draining veins were seen converging towards the site of GAG. The patient presented with three episodes of generalised tonic-clonic seizures and improved with conservative treatment. No reports of such association of GAG with AVF is available in the literature, and we believe it could have occurred due to venous hypertension induced by GAG.

Negative-Pressure Wound Therapy Promotes Wound Healing by Enhancing Angiogenesis Through Suppression of NLRX1 via miR-195 Upregulation.

Negative-pressure wound therapy (NPWT) is one of the most advanced therapeutic methods in the treatment of various hard-to-heal acute and refractory chronic wounds. Recent emerging evidence points to a role of the microRNA-mediated regulation of angiogenesis in ischemic tissues, and a series of microRNAs associated with angiogenesis have been successively identified. In this study, we found that miR-195 expression was significantly upregulated and the microvessel density (MVD) was increased in granulation tissue collected 7 days after NPWT compared with those in the pre-NPWT tissue. Moreover, the expression of NLRX1, the potential target gene of miR-195, was down-regulated in post-NPWT compared with that in pre-NPWT tissue. Significant negative correlations were detected between miR-195 and NLRX1 expression levels ( r = -.856, P < .001) and between NLRX1 expression and MVD ( r = -.618, P < .05), whereas miR-195 expression was positively correlated with MVD in the granulation tissue ( r = .630, P < .05). In summary, NPWT may suppress NLRX1 expression through the upregulation of miR-195 expression, thus efficaciously promoting angiogenesis in the granulation tissue to enhance wound healing.

Wound healing process of a full-thickness skin wound model in rats.

Using a rat full-thickness skin wound model, confocal laser scanning microscopy was carried out for examination of angiogenesis in granulation tissue formation. By semi-quantitation analysis with reverse transcription-polymerase chain reaction (RT-PCR), assessment was made of mRNA expression in the vascular endothelial growth factor (VEGF) and connective tissue growth factor (CTGF). Immunohistological analysis was also made to determine the location of these factors in granulation tissue at the protein level. Comparison was made of the cytokines in granulation and angiogenesis. Chronic granulation tissue with advanced fibrosis could be clearly demonstrated by the inhibition of wound contraction and epithelialization. From the results of the present study, VEGF would appear quite closely involved in angiogenesis in granulation tissue formation and pericytes may possibly give rise to VEGF and have significant roles in angiogenesis even with inhibition of epithelialization. CTGF expression in parenchyma is considerable during granulation and thus possibly may be related to the progress of fibrosis.

Endothelial cell hypertrophy is associated with microvascular occlusion in horse wounds.

Wound repair in horse limbs is often complicated by excessive fibroplasia and scarring. Occlusion of the microvessels populating the granulation tissue appears to be involved in the excessive accumulation of extracellular matrix during the repair of limb wounds. This study aimed to determine whether endothelial cell hypertrophy or hyperplasia, or both, contribute to microvascular occlusion and whether the pericyte is involved in this anomaly. We created 5 wounds, each 2.5 x 2.5 cm, on both forelimbs and on the body of 6 horses. One limb was bandaged to stimulate excessive wound fibroplasia. Weekly biopsy specimens were evaluated by transmission electron microscopy to measure microvessel luminal diameters and the surface area of endothelial cells and to count endothelial cells and pericytes. Microvessels were occluded significantly more often in limb wounds than in body wounds. The surface area of endothelial cells lining occluded microvessels (mean +/- standard error, 28.4013 +/- 1.5154 microm2) was significantly greater (P = 0.05) than that of cells lining patent microvessels (26.2220 +/- 1.5268 microm2). Conversely, neither the number of endothelial cells nor the number of pericytes differed between patent and occluded microvessels or between limb and body wounds. Furthermore, the wound location and the status of the microvessels (patent or occluded) did not alter the ratio of endothelial cells to pericytes. These data suggest that endothelial cell hypertrophy might play a role in the microvascular occlusion present in granulation tissue of limb wounds in horses, but the contribution of the pericyte remains obscure.

[Evaluation of intensity of angiogenesis in granulation tissue in chronic otitis media--preliminary report]. / Ocena intensywnosci angiogenezy w ziarninie w przewleklym zapaleniu ucha srodkowego--doniesienie wstepne.

In chronic otitis media molecular pathogenetic mechanisms are still unknown, however, angiogenesis may play a role. The aim of the study was to determine the intensity of angiogenesis in granulation tissue in chronic otitis media of different clinical course. Twenty six granulation tissue specimens (twenty two--from chronic otitis media, four--from prolongated otitis externa) taken during surgery were analyzed. The angiogenesis (microvessel density) was measured in paraffin-embedded tissue by an immunohistochemical method, by staining for endothelial cells with a monoclonal antibody against CD 34. The presence of CD 34 was found in all specimens. The expression was more intense in tissue samples from the group with good clinical course (good healing and without recurrence) than those in the group with poor healing and recurrence (mean number of dots for mm2 589,2 vs 533,3, respectively) and from the group without bacterial infection by Pseudomonas aeruginosa than those with this infection (mean number of dots for mm2 645,5 vs 440,8, respectively), but differences were not significant. In conclusion it is suggested that angiogenesis may contribute to different clinical course of chronic otitis media.

Albumin permeation of new vessels is increased in diabetic rats.

125I-bovine serum albumin (BSA) permeation of the vasculature of 3-wk-old granulation tissue (induced by subcutaneous implantation of polyester fabric) formed in the diabetic milieu was assessed in female BB/W, spontaneously diabetic rats and in male, Sprague-Dawley rats with streptozocin-induced diabetes as well as in corresponding nondiabetic controls. Albumin permeation of new granulation tissue vessels was markedly increased in both groups of diabetic animals relative to that of nondiabetic controls, while albumin permeation of vessels in most other tissues did not differ for controls and diabetics. These observations indicate that the functional integrity of new vessels formed in the diabetic milieu is impaired: (1) to a greater extent than that of older vessels formed before induction of diabetes and (2) relative to new vessels in nondiabetics. The implication of these observations is that molecular constituents of vessels synthesized in the diabetic milieu are quantitatively and/or qualitatively abnormal and/or their incorporation into vessels is defective.