Joint capsule matrix turnover in a rabbit model of chronic joint contractures: Correlation with human contractures.

To evaluate changes in matrix molecules of the joint capsule, the right knees of 24 skeletally mature female NZW rabbits were immobilized while the contralateral limb served as an unoperated control. The immobilization was discontinued at 8 weeks and the rabbits were divided among four groups (n = 6) based on the number of weeks the right knees were remobilized: 0, 8, 16, or 32. Three rabbits (six knees) that did not have operations provided normal control joint capsules. The mRNA levels for collagen types I, II, and III, and MMP-1 and -13 were significantly increased in the joint capsules of the contracture knees in all groups when compared to normal and contralateral limb joint capsules. In contrast, the mRNA levels for TIMP-1, -2, and -3 were decreased in the joint capsules of the contracture knees in all groups when compared to normal and contralateral limb joint capsules. The mRNA levels for lumican and decorin were increased in the joint capsules of the contracture knees in all groups when compared to normal capsules. Many of the changes observed in this animal model are similar to those observed in human joint capsules from posttraumatic elbow contractures, supporting the value of this rabbit model.     

A case of bilateral inguinal hernia recurrence in infancy: Investigations on collagen metabolism

Background. Recurrent inguinal hernias in early infancy are rare. We report on a case of a 3-month-old male infant suffering bilateral inguinal hernia recurrence (RINGH). Due to previous observations of an altered collagen metabolism in hernia patients, a severe connective-tissue pathology in the infant was hypothesised. Methods. Hernial sac tissue of the infant was analysed and compared to specimens from five children operated upon one-sided primary inguinal hernias (controls). In paraffin-embedded sections, we determined the distribution of collagen types I and III by crosspolarisation microscopy and the expression of matrix metalloproteinase 2 (MMP-2) by immunohistochemistry. In fibroblast cultures, expression of collagen types I and III and of MMP-2 was investigated by RT-PCR (real-time polymerase chain reaction) and zymography. Electron microscopical investigations were performed exemplarily in two fibroblast cultures to compare cell morphology. Results. No differences in collagen I/III ratios between RINGH and controls were found either on protein or on mRNA level. Immunohistochemical and RT-PCR analysis of MMP-2 showed a lowered expression in the RINGH patient, as compared to controls, whereas the gelatinolytic activity of MMP-2 did not differ between the groups. Electron microscopical investigations showed similar cell arrangement and morphology. Conclusions. To conclude, a marked biochemical correlate to a severe connective-tissue pathology in the infant suffering inguinal hernia recurrence could not be found. With regard to the slight differences in the expression of MMP-2, a possible role in the genesis of inguinal hernia recurrence cannot be ruled out.     

Gelatinase A activity in Dupuytren's disease

PURPOSE: Dupuytren's contracture is a fibroproliferative disorder of the hand characterized by an abnormal myofibroblast and fibroblast proliferation and extracellular matrix deposition leading to retraction and deformation of the palm. Recent studies have shown that molecules of extracellular matrix may coordinate morphogenesis, cell differentiation, and most importantly, fibrogenesis in tissue. Gelatinase A (MMP-2) is a member of the matrix metalloproteinase family of proteolytic enzymes that contribute to remodeling the extracellular matrix by degrading its components. The aim of this study was to determine the level of MMP-2 activation in the palmar fascia of patients with Dupuytren's contracture with reference to the clinical stages of disease progression and recurrence of the contracture after surgery. METHODS: The level of relative MMP-2 activation, expressed by the active to latent MMP-2 ratio, was investigated with use of zymography and computerized densitometry in 16 normal and 71 pathologic tissues characterizing different clinical stages of the disease progression. RESULTS: We found that the level of MMP-2 activation was significantly elevated in the palmar fascias with Dupuytren's contracture compared with normal tissues. We did not find statistically significant differences between groups with different stages of the disease progression. We also did not find a relation between a high level of MMP-2 activation and the recurrence in the area of surgically treated Dupuytren's contracture. CONCLUSIONS: The differences in MMP-2 activation between contractured and normal fascia suggest a participation of this enzyme in the promotion of Dupuytren's disease. We did not find a relationship, however, between the level of MMP-2 activation and the secondary contracture.     

The extracellular matrix of the lung and its role in edema formation

The extracellular matrix is composed of a three-dimensional fiber mesh filled with different macromolecules such as: collagen (mainly type I and III), elastin, glycosaminoglycans, and proteoglycans. In the lung, the extracellular matrix has several functions which provide: 1) mechanical tensile and compressive strength and elasticity, 2) low mechanical tissue compliance contributing to the maintenance of normal interstitial fluid dynamics, 3) low resistive pathway for an effective gas exchange, d) control of cell behavior by the binding of growth factors, chemokines, cytokines and the interaction with cell-surface receptors, and e) tissue repair and remodeling. Fragmentation and disorganization of extracellular matrix components comprises the protective role of the extracellular matrix, leading to interstitial and eventually severe lung edema. Thus, once conditions of increased microvascular filtration are established, matrix remodeling proceeds fairly rapidly due to the activation of proteases. Conversely, a massive matrix deposition of collagen fiber decreases interstitial compliance and therefore makes the tissue safety factor stronger. As a result, changes in lung extracellular matrix significantly affect edema formation and distribution in the lung.     

Joint capsule mast cells and neuropeptides are increased within four weeks of injury and remain elevated in chronic stages of posttraumatic contractures

The purpose of this article was to determine mast cell and neuropeptide nerve fiber numbers in joint capsules in posttraumatic contractures, as elevated numbers have been implicated in other fibrotic and contracture conditions. Twelve skeletally mature rabbits had intraarticular cortical windows removed from the medial and lateral femoral condyles and the knee joint immobilized. The contralateral unoperated limb served as a control. Equal numbers of rabbits were sacrificed 4 weeks after surgery or 40 weeks after the first surgery that included 32 weeks of remobilization. Six patients with chronic posttraumatic elbow joint contractures and six age‐matched organ donor controls free of elbow contractures were also studied. Joint capsule myofibroblast, mast cell, and neuropeptide containing nerve fiber numbers were assessed with immunohistochemistry. The numbers of myofibroblasts, mast cells, and neuropeptide containing nerve fibers expressed as a percentage of total cells were significantly greater in the contracture capsules when compared to the control capsules at all time points (p < 0.0001). The range of percentages for the three components in the contracture capsules versus the controls were 41–48% versus 9–10%, 44–50% versus 11–13%, and 45–50% versus 10–12% for the acute and chronic stages of the rabbit model and the chronic stages in the human elbows, respectively. These data support the hypothesis that a myofibroblast–mast cell–neuropeptide fibrosis axis may underlie some of the pathologic changes in the joint capsule in posttraumatic contractures. Approaches designed to manipulate this axis, such as preventing degranulation of mast cells, warrant further investigation. © 2008 Orthopaedic Research Society. Published by Wiley Periodicals, Inc. J Orthop Res 26:1313–1319, 2008     

Plasmin inhibition increases MMP-9 activity and decreases vein wall stiffness during venous thrombosis resolution

INTRODUCTION: Deep venous thrombosis (DVT) resolution involves the plasmin and the matrix metalloproteinase (MMP) system. This study tested the hypothesis that pharmacological inhibition of the plasmin system would impair DVT resolution and worsen vein wall damage. METHODS: A rat model of stasis DVT by inferior vena cava (IVC) ligation was performed with intravenous control saline or aprotinin (AP; 2.8 mg/kg at operation), and harvest of thrombosed IVC at 7 days. After laser Doppler imaging, DVT were separated and weighed, and vein wall stiffness was assessed by tensiometry. Thrombus and vein wall tissue analysis included total collagen by colorimetric assay, cytokines, chemokines, and d-dimer by ELISA, urokinase-plasminogen activator (uPA), and plasminogen activator inhibitor-1 (PAI-1) by immuno-blotting, MMP-2 and -9 by zymography, and neutrophil (PMN) and monocyte (ED-1) leukocytes by immunohistochemistry. RESULTS: DVT weights were 2-fold greater in the AP-treated rats (P < 0.05), but no significant differences in thrombus perfusion, collagen, or d-dimer levels were found. Vein wall stiffness was reduced 50% (P < 0.05), suggesting less biomechanical injury. The total vein wall MMP-9 was increased (P < 0.05) 5-fold in the AP group compared with controls, while MMP-2 was elevated but did not reach significance. No difference was found in vein wall tumor necrosis factor-alpha, tissue growth factor-beta, vein wall or thrombus monocytes, PMN, or uPA/PAI-1 ratio between groups. DISCUSSION: AP inhibition of the plasmin system was associated with larger thrombi but less vein wall injury, but no difference in other measures of resolution, possibly because of increased vein wall MMP-9 activity. These data suggest an important redundant mechanism for DVT resolution.     

Matrix metalloproteinase expression in normal skin associated with basal cell carcinoma and in distal skin from the same patients

OBJECTIVE: To obviate the difficulty of ruling out confounding variables (eg, age, individual variability) as the source of differences seen when comparing tumor tissue and control tissue from unrelated individuals, we examined the expression of matrix metalloproteinase (MMP)-1 (interstitial collagenase) and MMP-9 (92-kDa gelatinase B) in histologically normal skin immediately adjacent to basal cell carcinomas (peritumoral tissue) after Mohs micrographic surgery and postauricular skin from the same patients. DESIGN: Peritumoral and postauricular skin samples were obtained from 17 patients undergoing Mohs surgery. Expression of MMP-1 and MMP-9 was examined in these specimens using a combination of approaches including zymography, collagen-degradation assays, and immunohistology. RESULTS: The expression levels of MMP-1 and MMP-9 were consistently elevated in the peritumoral tissue compared with skin from the distal site. CONCLUSION: This finding indicates that even when potentially important variables such as age and individual variability are controlled for, tumor-specific effects on the expression of MMP-9 and MMP-1 remain.     

Ilizarov牵张技术对挛缩跟腱组织学改变的实验研究

 目的 探讨 Ilizarov牵张技术的牵拉应力对挛缩跟腱组织学的影响。方法 18只狗右后肢应用 Ilizarov外固定支架中立位固定 1周后开始牵拉, 1 mm /d, 分 2次调整, 连续 3周, X线摄片确定 足下垂 15°, 再维持固定 3周, 随机选取 9只动物处死取材(挛缩组);余动物再以同样的速度和方法牵 拉矫正 3周后处死取材(牵拉组);所取健侧标本合并为对照组。(1) HE染色观察腱细胞排列分布, 胶 原纤维形态, 并分析腱网膜厚度变化;(2)以苦味酸-天狼猩红染色法, 偏振光显微镜观察玉、芋胶原, 并 分析二者含量变化;(3)采用间苯三酚分光光度法, 测定蛋白多糖(主要为葡糖氨基葡聚糖)含量。结果 (1)挛缩组腱细胞与胶原纤维间隙不明显, 腱细胞呈梭形, 囊状, 胶原纤维束扭曲, 排列散乱, 难见细胞 间质。腱网膜厚度与健侧(对照组)有明显差异;牵拉组, 腱细胞呈圆形或卵圆形, 在胶原束间成串排列, 细胞与胶原纤维间有基质间隔, 见炎症细胞, 形态与正常腱组织相似。腱网膜厚度与健侧无明显差异。 (2)偏振光显微镜下, 挛缩组玉、芋胶原构成比与对照组有明显差异, 牵拉组玉、芋胶原构成比与健侧无 明显差异。(3)挛缩组跟腱蛋白多糖含量明显减少, 牵拉组基本恢复到正常水平。结论 在 Ilizarov牵张 技术牵拉下, 挛缩退化的跟腱组织在组织学上可基本恢复正常。     

Breast capsule contracture: Is fibroblast activity associated with severity?

Factors responsible for breast capsule contracture remain elusive. Using an in vitro model of wound contraction, the fibroblast-populated collagen lattice (FPCL), breast capsule fibroblasts and control dermal fibroblasts from ten patients were analyzed. Comparison was made to determine (1) if the activity of dermal fibroblasts on a collagen lattice correlated with the activity of breast capsule fibroblasts or if capsular fibroblasts are unique, and (2) if the degree of fibroblast-driven collagen contraction correlated with clinical severity of breast capsule contracture. If so, a preoperative predictor of breast capsule contracture would be available. Dermal fibroblasts and breast capsule fibroblasts were cultured and mixed with media and collagen to form a matrix, and then the degree of lattice contraction was measured. A correlation between breast capsule fibroblasts and control dermal fibroblasts with respect to collagen matrix contraction was confirmed. Collagen lattice contraction coordinated by fibroblasts derived from breast capsules did not correlate with clinical severity of capsular contracture. These results indicate that the degree of breast capsule contracture can not be predicted by fibroblast activity alone. An interaction between inflammatory cells, extracellular matrix, and fibroblasts is hypothesized. Further work is needed to delineate the mechanisms responsible for breast capsule contracture.     

Collagens,Proteoglycans, MMP-2, MMP-9 and TIMPs in Human Achilles Tendon Rupture

Tendon integrity depends on the extracellular matrix (ECM) metabolism which is regulated by proteolytic enzymes. However, it is unclear which enzymes play a role in tendon rupture. We studied the ECM of 19 ruptured human Achilles tendons, comparing the composition of specimens harvested close to the rupture with specimens harvested from an apparently healthy area in the same tendon. We compared gene expression of collagen Type I, decorin, and versican including enzymes involved in their metabolism as matrix metalloproteases (MMP-2 and -9) and tissue inhibitory of metalloproteinase (TIMP-1 and -2) using real-time PCR, zymography and FACE analysis. We found greater gene expression of proteoglycan core protein decorin and versican, collagen Type I, MMPs and TIMPs in the tendon rupture. Zymography analysis, reflecting expression of enzymatic activity, confirmed the gene expression data at protein level. Carbohydrate content was greater in the macroscopically healthy area than in the ruptured area. In the ruptured area, we found increased core protein synthesis but without the normal glycosaminoglycan production. The tissue in the area of rupture undergoes marked rearrangement at molecular levels and supports the role of MMPs in the pathology. Each author certifies that he or she has no commercial associations (eg, consultancies, stock ownership, equity interest, patent/licensing arrangements, etc) that might pose a conflict of interest in connection with the submitted article. Each author certifies that his or her institution has approved the reporting of these cases, that all investigations were conducted in conformity with ethical principles of research, and that informed consent for participation in the study was obtained.     

The role of matrix metalloproteinases in rheumatoid tendon disease

In rheumatoid arthritis (RA) invasive tenosynovitis is associated with an increase in tendon rupture, although little is known about the mechanisms involved. We obtained specimens of noninvasive encapsulating tenosynovium, invasive tenosynovium, and wrist joint synovium from 28 rheumatoid patients. In vitro production of the matrix metalloproteinase (MMP) enzymes, MMP-8 and -9, and total collagenase activity were measured. Invasive tenosynovium produced highest levels of the collagenase MMP-8 and displayed significantly greater ability to degrade collagen type I than encapsulating tenosynovium. Levels of the gelatinase enzyme MMP-9 were similar in all groups. These results show that invasive tenosynovium is more destructive than encapsulating tenosynovium at a molecular level, providing an explanation for the increased tendon rupture associated with invasive tenosynovitis in RA.     

Effect of oxidised regenerated cellulose/collagen matrix on proteases in wound exudate of patients with chronic venous ulceration

Oxidised regenerated cellulose/collagen matrix (ORC/collagen matrix) modifies wound microenvironments by binding and inactivating excess levels of proteases such as elastase, plasmin and gelatinases in wound exudates. To compare levels of the gelatinases matrix metalloproteinase 2 (MMP-2), elastase and plasmin in wound exudates collected from chronic venous insufficiency patients with venous leg ulcers treated with either an ORC/collagen matrix or a standard control therapy. During a 12-week treatment period, wound exudate samples were obtained from a control group of 10 patients treated with a hydrocolloid dressing and a treatment group of 17 patients treated with a combination of ORC/collagen matrix and hydrocolloid dressing. On admission and days 5, 14 and every subsequent 14th day, ulcers were photographed to determine healing rate and changes in ulcer appearance, and MMP-2 concentration and the gelatinase, elastase and plasmin activities were analysed from wound exudates. The patients treated with ORC/collagen matrix showed a significant decrease in elastase, plasmin and gelastinase activity as compared with the control group, with no significant difference in the MMP-2 concentrations between the two groups. The results show a significant and immediate reduction in protease activity in wound exudates from venous leg ulcers treated with ORC/collagen.     

Action of matrix metalloproteinases at restricted sites in colon anastomosis repair: an immunohistochemical and biochemical study

BACKGROUND: Dehiscence of colon anastomosis is a common, serious and potentially life-threatening complication after colorectal operation. In experimental models, impaired biomechanic strength of colon anastomoses is preventable by general inhibitors of matrix metalloproteinases (MMPs) and associated with collagen loss, which indicates a possible link between MMP-mediated collagen degradation and dehiscence. The precise localization of collagen degradation within the anastomotic area and the specific MMPs responsible are unknown. METHODS: We have analyzed distinct zones within anastomoses using a novel microdissection technique for collagen levels, collagenolytic activity exerted directly by endogenous proteinases, and MMP-8 and MMP-9 immunoreactivity and their collagenolytic activity. RESULTS: The most pronounced collagen loss was observed in the suture-holding zone, showing a 29% drop compared with adjacent micro-areas of 3-day-old anastomoses. Only this specific tissue compartment underwent a dramatic and significant increase in collagenolysis, amounting to a loss of 10% of existing collagen molecules in 24 hours, and was abolished by metalloproteinase inhibitors. The tissue surrounding suture channels was heavily infiltrated with CD68-positive histiocytes that expressed MMP-8 and to a lesser extent MMP-9. The collagenolytic effect of the interstitial collagenase MMP-8 was synergistically potentiated by the gelatinase MMP-9 when added to colon biopsies incubated in vitro. CONCLUSIONS: The unique finding of this study was that the specific tissue holding the sutures of a colon anastomosis lost the most collagen presumably through induction and activation of multiple MMPs that may explain the beneficial effects of treatment with non-selective MMP antagonists.     

Gas-related impact of pneumoperitoneum on systemic wound healing

Introduction The aim of the present study was to investigate the gas-dependent effects of pneumoperitoneum on wound healing distant from the abdomen. Materials and methods Dorsal skin incisions were performed in 54 male Sprague–Dawley rats. A CO₂ or a helium pneumoperitoneum of 3 mmHg was maintained before, with an overall duration of 30 min (each n = 18). Rats in the control group received laparotomy only (n = 18). Animals were killed after 5 and 10 days. The infiltration of macrophages (CD 68), expression of matrix metalloproteinase 13 (MMP-13) and collagen I/III ratios were analysed in the dorsal skin wounds. Results Both after 5 and 10 days, infiltration of macrophages and expression of MMP-13 were greatest after helium pneumoperitoneum. After 5 days, collagen I/III ratio was significantly increased in the helium group. After 10 days, collagen I/III ratio was lowest in the CO₂ group. Conclusion Our results suggest beneficial effects on systemic wound healing for helium pneumoperitoneum as compared to CO₂.     

Matrix metalloproteinase-2 in a murine model of infantile-type polycystic kidney disease

It was previously found that elevated levels of matrix metalloproteinase (MMP)-2 (gelatinase A) and -9 (gelatinase B) were synthesized and secreted into the medium by cultured kidney tubules derived from cystic C57BL/6J-cpk mice. To determine whether increased synthesis and secretion occur in vivo in this mouse model of polycystic kidney disease, kidney protein extracts, mRNA, and tissue sections were compared for expression and activity of MMP-2 and -9. Although both MMP were detected in tissue extracts, the differences in expression levels and activity in normal and cystic kidneys were far greater for MMP-2. High levels of MMP-2 seemed to result from increased expression by the cystic kidneys predominantly in the second and third postnatal weeks (a time when the kidneys are undergoing rapid cystic enlargement). Much of the increased MMP was present in the inactive zymogen form, although active enzyme was readily detected by sodium dodecyl sulfate-polyacrylamide gel zymography and in situ zymography. MMP-2 was abnormally localized to the interstitium and to foci between cysts, suggesting that MMP-2 may regulate collagen accumulation at those sites, thus allowing cyst enlargement and limiting the severity of interstitial fibrosis.     

基质金属蛋白酶及抑制因子与腹主动脉瘤临床病理特点的相关性研究

目的：研究基质金属酶（MMP）-2、MMP-9及抑制因子TIMP-1在腹主动脉瘤中的表达及与临床病理特征之间的关系。方法：应用免疫组化PV-9000通用型二步法对70例腹主动脉瘤和15例正常腹主动脉标本中的MMP-2、MMP-9及TIMP-1表达进行检测。结果：腹主动脉瘤组织中MMP-2和MMP-9蛋白表达阳性率明显高于正常腹主动脉组织,TIMP-1蛋白表达阳性率和正常腹主动脉没有统计学差异,（X^2=0.103,P=0.991）;MMP-2蛋白的表达与腹主动脉瘤的直径呈负相关（X^2=13.785,P=0.032）,MMP-9蛋白的表达与患者临床症状,腹主动脉瘤直径、破裂有相关性,（P〈0.05）,TIMP-1蛋白表达阳性率与临床病理特征无相关性（X^2=0.103,P=0.991）。结论：腹主动脉瘤组织中MMP高表达和TIMP的相对弱表达在腹主动脉瘤发生、发展过程中起重要作用,MMP-9可以预测腹主动脉瘤的自然病程从而作为腹主动脉瘤手术治疗的指征之一。