Short-term Changes After Corticosteroid Injections Into the Normal Tendons of Rabbits: A Controlled Randomized Study.

BACKGROUND: Corticosteroid injections in or around tendons for the treatment of athletic injuries are a common practice among orthopaedic surgeons and are apparently efficacious in the short term, although controversies persist related to local complications. PURPOSE: This study evaluated short-term (48 hours) biomechanical, biochemical, and histological alterations after a single injection of betamethasone into the normal tendons of rabbits. STUDY DESIGN: Controlled laboratory study. METHODS: A total of 72 New Zealand White rabbits were randomly divided into 2 groups: the test group-in which 36 animals underwent 1 intratendinous injection of betamethasone (1.4 mg / 0.2 mL) in the right calcaneal tendon; the control group-in which the right calcaneal tendon of 36 animals was injected with saline (placebo control group) and the left calcaneal tendon was left untreated for normal standards (normal control). Forty-eight hours later, animals were euthanized and tendons were harvested. Metalloproteinase (MMP1 and MMP2) and interleukin (IL1 and IL6) expression levels, biomechanical resistance (load × elongation parameters), and histomorphometry (hematoxylin and eosin and picrosirius red stains for collagen fibers, tenocytes, and inflammatory cells) were analyzed in the tendons. RESULTS: The test group had a significant reduction in MMP2 expression as compared with the control groups ( P = .027). Regarding the other parameters, there were no additional significant differences between the groups. CONCLUSION: A single injection of corticosteroid into normal calcaneal tendons did not trigger acute local morphological, structural, or biomechanical injuries at 48 hours, but it did promote a significant decrease in MMP2 levels. Additional studies are needed with increased duration of follow-up, various doses, and multiple injections and in tendinopathic models. CLINICAL RELEVANCE: Some previous studies demonstrated early structural changes in tendons after a single corticosteroid injection, which was not corroborated by the present study. Metalloproteinase decrease is usually associated with a reduction in collagen degradation, which would be protective for the healing process. More studies are necessary to confirm the possible beneficial effect of these results in the long term and for tendinopathies.

Knockout of hyaluronan synthase 1, but not 3, impairs formation of the retrocalcaneal bursa.

Hyaluronan (HA), a high molecular weight non-sulfated glycosaminoglycan, is an integral component of the extracellular matrix of developing and mature connective tissues including tendon. There are few published reports quantifying HA content during tendon growth and maturation, or detailing its effects on the mechanical properties of the tendon extracellular matrix. Therefore, the goal of the current study was to examine the role of HA synthesis during post-natal skeletal growth and maturation, and its influence on tendon structure and biomechanical function. For this purpose, the morphological, biochemical, and mechanical properties of Achilles tendons from wild type (WT) and hyaluronan synthase 1 and 3 deficient mouse strains (Has1-/- (Has1KO), Has3-/- (Has3KO), and Has1-/- 3-/- (Has1/3KO)) were determined at 4, 8, and 12 weeks of age. Overall, HAS-deficient mice did not show any marked differences from WT mice in Achilles tendon morphology or in the HA and chondroitin/dermatan sulfate (CS/DS) contents. However, HAS1-deficiency (in the single or Has1/3 double KO) impeded post-natal formation of the retrocalcaneal bursa, implicating HAS1 in regulating HA metabolism by cells lining the bursal cavity. Together, these data suggest that HA metabolism via HAS1 and HAS3 does not markedly influence the extracellular matrix structure or function of the tendon body, but plays a role in the formation/maintenance of peritendinous bursa. Additional studies are warranted to elucidate the relationship of HA and CS/DS metabolism to tendon healing and repair in vivo. © 2018 Orthopaedic Research Society. Published by Wiley Periodicals, Inc. J Orthop Res 36:2622-2632, 2018.

Influence of repetitive mechanical loading on MMP2 activity in tendon fibroblasts.

Matrix metalloproteinase2 has been implicated in tendon pathology caused by repetitive movements. However, its activity in the early stages of the tendon's response to overuse, and its presence in the circulation as a possible indicator of tendon degradation, remain unknown. Human tendon cells were repetitively stretched for 5 days, and the rabbit Achilles tendon complex underwent repetitive motion 3× per week for 2 weeks. Quantitative polymer chain reaction analysis was performed to detect matrix metalloproteinase2/14 and tissue inhibitor of matrix metalloproteinase2 messenger ribonucleic acid of cells and rabbit tissue, and matrix metalloproteinase2 protein levels were determined with an enzyme linked immunoassay. Matrix metalloproteinase2 activity was examined using zymography of the conditioned media, tendon and serum. Immunohistochemistry was used to localize matrix metalloproteinase2 in tendon tissue, and the density of fibrillar collagen in tendons was examined using second harmonic generation microscopy. Tendon cells stretched with high strain or high frequency demonstrated increased matrix metalloproteinase2 messenger ribonucleic acid and protein levels. Matrix metalloproteinase2 activity was increased in the rabbit Achilles tendon tissue at weeks 1 and 2; however, serum activity was only increased at week 1. After 2 weeks of exercise, the collagen density was lower in specific regions of the exercised rabbit Achilles tendon complex. Matrix metalloproteinase2 expression in exercised rabbit Achilles tendons was detected surrounding tendon fibroblasts. Repetitive mechanical stimulation of tendon cells results in a small increase in matrix metalloproteinase2 levels, but it appears unlikely that serum matrix metalloproteinase2 will be a useful indicator of tendon overuse injury. © 2016 Orthopaedic Research Society. Published by Wiley Periodicals, Inc. J Orthop Res 34:1991-2000, 2016.

Treatment of heterotopic ossification through remote ATP hydrolysis.

Heterotopic ossification (HO) is the pathologic development of ectopic bone in soft tissues because of a local or systemic inflammatory insult, such as burn injury or trauma. In HO, mesenchymal stem cells (MSCs) are inappropriately activated to undergo osteogenic differentiation. Through the correlation of in vitro assays and in vivo studies (dorsal scald burn with Achilles tenotomy), we have shown that burn injury enhances the osteogenic potential of MSCs and causes ectopic endochondral heterotopic bone formation and functional contractures through bone morphogenetic protein-mediated canonical SMAD signaling. We further demonstrated a prevention strategy for HO through adenosine triphosphate (ATP) hydrolysis at the burn site using apyrase. Burn site apyrase treatment decreased ATP, increased adenosine 3',5'-monophosphate, and decreased phosphorylation of SMAD1/5/8 in MSCs in vitro. This ATP hydrolysis also decreased HO formation and mitigated functional impairment in vivo. Similarly, selective inhibition of SMAD1/5/8 phosphorylation with LDN-193189 decreased HO formation and increased range of motion at the injury site in our burn model in vivo. Our results suggest that burn injury-exacerbated HO formation can be treated through therapeutics that target burn site ATP hydrolysis and modulation of SMAD1/5/8 phosphorylation.

High glucose concentration up-regulates the expression of matrix metalloproteinase-9 and -13 in tendon cells.

BACKGROUND: Diabetes mellitus is associated with tendinopathy or tendon injuries. However, the mechanism underlying diabetic tendinopathy is unclear. The purpose of this study was to examine the effects of high glucose concentrations on the activity and expression of matrix metalloproteinases, type I collagen, and type III collagen in tendon cells. METHODS: Tendon cells from rat Achilles tendons were treated with 6 mM, 12 mM, and 25 mM glucose, and then cell proliferation was evaluated by the 3-[4,5-Dimethylthiazol-2-yl]-2,5-diphenyltetrazolium bromide (MTT) assay. Messenger RNA (mRNA) expression of MMP-2, MMP-8, MMP-9, and MMP-13 and type I and type III collagen was assessed by quantitative real-time polymerase chain reaction (PCR). The enzymatic activity of MMP-2 and MMP-9 was measured by gelatin zymography. RESULTS: The MTT assay results showed that the glucose concentration did not affect tendon cell proliferation. The results of the real-time PCR assay revealed that the mRNA expression of MMP-9 and MMP-13 was up-regulated by treatment with 25 mM glucose, whereas the mRNA expression of type I and III collagen was not affected. Gelatin zymography showed that 25 mM glucose increased the enzymatic activity of MMP-9. CONCLUSIONS: High glucose concentration up-regulates the expression of MMP-9 and MMP-13 in tendon cells, which may account for the molecular mechanisms underlying diabetic tendinopathy.

Local NO synthase inhibition produces histological and functional recovery in Achilles tendon of rats after tenotomy: tendon repair and local NOS inhibition.

Repair of injured tendon is a very slow process and involves the release of many molecules, including nitric oxide. We investigate the influence of local nitrergic inhibition in histological and functional recovery of injured Achilles tendon. A standard murine model of tendon injury by rupture was used. The animals were divided into three experimental groups: control, injury + vehicle (normal saline) and injury + Nω-nitro-L-arginine methyl ester (L-NAME). The products were injected into the paratendinous region every 2 days and body weight gain and Achilles functional index (AFI) were evaluated on days 0, 7, 14 and 21 after tendon injury. On day 21 post-injury, the animals were killed to evaluate nitric oxide production and tissue organization. We observed that tendon surgical division led to increased tissue nitrite levels, which were reduced in L-NAME-treated rats. The AFI revealed functional recovery of L-NAME-treated animals on day 21 post-injury, which was not observed in the saline-treated group. Microscopic analysis of hematoxylin-eosin staining and collagen autofluorescence showed that L-NAME-treated rats had more aligned areas of collagen fibers and that the diameter of newly organized collagen in this group was also greater than that in the vehicle-treated one. We demonstrate that local treatment with L-NAME significantly improves the functional parameters and accelerates histomorphological recovery.

Aging is associated with increased activities of matrix metalloproteinase-2 and -9 in tenocytes.

BACKGROUND: Most tendon pathology is associated with degeneration, which is thought to involve cyclic loading and cumulative age-related changes in tissue architecture. However, the association between aging and degeneration of the extracellular matrix (ECM) in tendons has not been investigated extensively. METHODS: We examined tenocytes from Achilles tendons taken from rats of three different ages (2, 12, and 24 months). Tenocyte viability was assessed using the 3-[4,5-dimethylthiazol-2-yl]-2,5-diphenyltetrazolium bromide (MTT) assay. Quantitative real-time polymerase chain reaction (PCR) was used to determine the levels of mRNAs that encode type-I collagen, matrix metalloproteinase (MMP)-2 and -9, tissue inhibitor of metalloproteinase (TIMP)-1 and -2 and transforming growth factor (TGF)-ß1. Gelatin zymography was used to evaluate the enzymatic activities of MMP-2 and -9. Furthermore, the concentration of TGF-ß1 in conditioned medium was evaluated using enzyme-linked immunosorbent assay (ELISA). RESULTS: The results of the MTT assay showed that the number of viable tenocytes decreased with age. No differences were observed in the levels of mRNAs that encode type-I collagen and TGF-ß1 among the three age groups, and the TGF-ß1 concentration did not change with age. However, mRNAs that encode MMP-2 and -9 were significantly more abundant in tenocytes from the aging group, and gelatin zymography revealed that the enzymatic activities of MMP-2 and -9 also increased significantly with age. Furthermore, as compared with young group, mRNAs that encode TIMP-1 and -2 were significantly decreased in tenocytes from the aging group. CONCLUSIONS: Activities of MMP-2 and MMP-9 in tenocytes increase with age. This might provide a mechanistic explanation of how aging contributes to tendinopathy or tendon rupture with age.

Effects of ovariectomy and resistance training on MMP-2 activity in rat calcaneal tendon.

Tendon remodeling relies on extracellular matrix (ECM) restructuring by the matrix metallopeptidases (MMPs). The aim of this study was to investigate MMP-2 activity in different regions of the calcaneal tendon (CT) after resistance training (RT) in ovariectomized rats. Wistar adult female rats were grouped into sedentary (Sed-Intact), ovariectomized sedentary (Sed-Ovx), acute exercise (AcuteEx-Intact), ovariectomized acute exercise (AcuteEx-Ovx), resistance trained (ChronicEx-Intact), and ovariectomized resistance trained (ChronicEx-Ovx) (n = 10 each group). The RT protocol required the animals to climb a 1.1-m vertical ladder with weights attached to their tail. The sessions were performed once every 3 days with 4-9 climbs and 8-12 dynamic movements per scaling. The acute groups performed one session and the chronic groups underwent 12 weeks of RT. There was an increase in total MMP-2 activity in Sed-Ovx, AcuteEx-Intact, and ChronicEx-Intact compared with that in Sed-Intact in the proximal region of CT. AcuteEx-Ovx exhibited higher total MMP-2 than Sed-Ovx and AcuteEx-Intact in the distal region of CT. Chronic-Ovx presented lower total MMP-2 activity than Sed-Ovx and Chronic-Intact in the distal region of tendon. The active MMP-2 was higher for the AcuteEx-Ovx than Sed-Ovx and AcuteEx-Intact in proximal region of tendon. There was higher active MMP-2 in the distal region of tendon in the Acute-Ovx than in the Sed-Ovx and AcuteEx-Intact. Ovariectomy and resistance exercise modulate MMP-2 activity according to specific tendon region, indicating a differentiated tissue remodeling.

Calcaneal tendon regions exhibit different MMP-2 activation after vertical jumping and treadmill running.

Increased activity of matrix metalloproteinases (MMPs) -2 and -9 was found in calcaneal tendon after physical training. However, little attention has been given to the distinct biomechanical and tissue structure of the calcaneal tendon's proximal and distal regions. Herein, we evaluated the effect of two types of physical activities on tendon morphology and matrix metalloproteinase activities in the proximal and distal regions of rat calcaneal tendon, separately. Adult male Wistar rats from control, water-adapted, vertical-jumping, and treadmill-running groups were sacrificed after 1 or 4 days of physical exercise, 6 hr after the end of that day's exercise session. Tendons were processed for histology, morphometry, and gelatin zymography. Tendons from adapted and trained animals showed active secretory cells and increased thickness, cellularity, and blood vessel volume fraction of peritendinous sheath, but without inflammatory process. In the proximal region, both pro- and active MMP-2 were increased after vertical jumping, but only pro-MMP-2 was increased after treadmill running. In contrast, in the distal region, both exercise types increased the activity of pro- and active MMP-2, especially treadmill running, which increased the active MMP-2 by about 11- and eightfold, respectively, after 1 and 4 days of training. No activity of MMP-9 was observed in either tendon region in this study. In conclusion, distal and proximal regions of calcaneal tendon exhibit differential intensities of tissue remodeling after treadmill running or vertical jumping and MMP-2, in the absence of inflammation, plays a major role in this adaptive response.

Androgenic-anabolic steroids associated with mechanical loading inhibit matrix metallopeptidase activity and affect the remodeling of the achilles tendon in rats.

BACKGROUND: The indiscriminate use of anabolic-androgenic steroids has been shown to induce pathologic changes in the Achilles tendon in several situations. PURPOSE: To study tendon remodeling in rats treated with anabolic-androgenic steroids combined with an exercise program. STUDY DESIGN: Controlled laboratory study. METHODS: Wistar rats were grouped as follows: sedentary (group I), injected with anabolic-androgenic steroids only (group II), trained only (group III), and trained and injected with anabolic-androgenic steroids (group IV). The trained groups performed jumps in water: 4 series of 10 jumps each, with an overload of 50% to 70% of the animal's body weight and a 30-second rest interval between series, for 6 weeks. Anabolic-androgenic steroids (5 mg/kg) were injected subcutaneously. Activity of matrix metallopeptidases, a marker for tendon remodeling, was analyzed in tissue extracts by zymography on gelatin-sodium dodecyl sulfate-polyacrylamide gel electrophoresis. RESULTS: Morphological analyses of tendons showed that in group II, the most external layer that covers the tendon was thicker with aggregation of the collagen fibers, suggesting an increase in collagen synthesis. In group IV, an inflammatory infiltrate and fibrosis in tendons as well as a pronounced increase of the serum corticosterone level were observed. This training protocol upregulated matrix metallopeptidase activity, whereas anabolic-androgenic steroid treatment strongly inhibited this activity. The appearance of lytic bands with molecular masses of approximately 62 and 58 kDa suggests the activation of matrix metallopeptidase-2. CONCLUSION: Anabolic-androgenic steroid treatment can impair tissue remodeling in the tendons of animals undergoing physical exercise by down-regulating matrix metallopeptidase activity, thus increasing the potential for tendon injury. CLINICAL RELEVANCE: Since the AAS abuse is so widespread, a better comprehension of the pathological effects induced by these drugs may be helpful for the development of new forms of therapy of AAS-induced lesions.

Expression profiling of metalloproteinases and tissue inhibitors of metalloproteinases in normal and degenerate human achilles tendon.

OBJECTIVE: To profile the messenger RNA (mRNA) expression for the 23 known genes of matrix metalloproteinases (MMPs), 19 genes of ADAMTS, 4 genes of tissue inhibitors of metalloproteinases (TIMPs), and ADAM genes 8, 10, 12, and 17 in normal, painful, and ruptured Achilles tendons. METHODS: Tendon samples were obtained from cadavers or from patients undergoing surgical procedures to treat chronic painful tendinopathy or ruptured tendon. Total RNA was extracted and mRNA expression was analyzed by quantitative real-time reverse transcription-polymerase chain reaction, normalized to 18S ribosomal RNA. RESULTS: In comparing expression of all genes, the normal, painful, and ruptured Achilles tendon groups each had a distinct mRNA expression signature. Three mRNA were not detected and 14 showed no significant difference in expression levels between the groups. Statistically significant (P < 0.05) differences in mRNA expression, when adjusted for age, included lower levels of MMPs 3 and 10 and TIMP-3 and higher levels of ADAM-12 and MMP-23 in painful compared with normal tendons, and lower levels of MMPs 3 and 7 and TIMPs 2, 3, and 4 and higher levels of ADAMs 8 and 12, MMPs 1, 9, 19, and 25, and TIMP-1 in ruptured compared with normal tendons. CONCLUSION: The distinct mRNA profile of each tendon group suggests differences in extracellular proteolytic activity, which would affect the production and remodeling of the tendon extracellular matrix. Some proteolytic activities are implicated in the maintenance of normal tendon, while chronically painful tendons and ruptured tendons are shown to be distinct groups. These data will provide a foundation for further study of the role and activity of many of these enzymes that underlie the pathologic processes in the tendon.

Contrasting effects of fluoroquinolone antibiotics on the expression of the collagenases, matrix metalloproteinases (MMP)-1 and -13, in human tendon-derived cells.

OBJECTIVES: Fluoroquinolone antibiotics may cause tendon pain and rupture. We reported previously that the fluoroquinolone ciprofloxacin potentiated interleukin (IL)-1beta-stimulated expression of matrix metalloproteinases (MMP)-3 and MMP-1 in human tendon-derived cells. We have now tested additional fluoroquinolones and investigated whether they have a similar effect on expression of MMP-13. METHODS: Tendon cells were incubated for two periods of 48 h with or without fluoroquinolones and IL-1beta. Total ribonucleic acid (RNA) was assayed for MMP messenger RNA by relative quantitative reverse transcriptase polymerase chain reaction, with normalization for glyceraldehyde-3-phosphate dehydrogenase mRNA. Samples of supernatant medium were assayed for MMP output by activity assays. RESULTS: MMP-13 was expressed by tendon cells at lower levels than MMP-1, and was stimulated typically 10- to 100-fold by IL-1beta. Ciprofloxacin, norfloxacin and ofloxacin each reduced both basal and stimulated expression of MMP-13 mRNA. In contrast, ciprofloxacin and norfloxacin increased basal and IL-1beta-stimulated MMP-1 mRNA expression. Both the inhibition of MMP-13 and the potentiation of MMP-1 expression by fluoroquinolones were accompanied by corresponding changes in IL-1beta-stimulated MMP output. The non-fluorinated quinolone nalidixic acid had lesser or no effects. CONCLUSIONS: Fluoroquinolones show contrasting effects on the expression of the two collagenases MMP-1 and MMP-13, indicating specific effects on MMP gene regulation.

Physical exercise can influence local levels of matrix metalloproteinases and their inhibitors in tendon-related connective tissue.

Microdialysis studies indicate that mechanical loading of human tendon tissue during exercise or training can affect local synthesis and degradation of type I collagen. Degradation of collagen and other extracellular matrix proteins is controlled by an interplay between matrix metalloproteinases (MMPs) and their tissue inhibitors (TIMPs). However, it is unknown whether local levels of MMPs and TIMPs are affected by tendon loading in humans in vivo. In the present experiment, six healthy young men performed 1 h of uphill (3%) treadmill running. Dialysate was collected from microdialysis probes (placed in the peritendinous tissue immediately anterior to the Achilles tendon) before, immediately after, 1 day after, and 3 days after an exercise bout. MMP-2 and MMP-9 were measured in dialysate by gelatin zymography, and amounts were quantified by densitometry in relation to total protein in the dialysate. TIMP-1 and TIMP-2 were analyzed by reverse gelatin zymography and semiquantitated visually. Pro-MMP-9 increased markedly after exercise and remained high for 3 days after exercise. Pro-MMP-2 dropped from the basal level immediately after exercise and remained low 1 day after exercise but was slightly elevated 3 days after exercise. The MMP-2 inhibitory activity of TIMP-1 was clearly elevated 1 and 3 days after exercise, and the MMP-2 inhibitory activity of TIMP-2 rose 1 day after loading. The present findings demonstrate enhanced interstitial amounts of MMPs and TIMPs after exercise in the human peritendinous tissue in vivo, and the magnitude and time pattern of these changes may well indicate that MMPs and TIMPs are playing a role in extracellular matrix adaptation to exercise in tendon tissue.

The cell specific temporal expression of nitric oxide synthase isoforms during achilles tendon healing.

OBJECTIVE AND DESIGN: We have previously shown that nitric oxide synthase (NOS) activity is upregulated following tendon injury, and that this activity is important to Achilles tendon healing. The aim of this study was to identify the cellular distribution of nitric oxide synthase isoforms during tendon healing. MATERIAL OR SUBJECTS: Surgical division of the right Achilles tendon was performed in eighty-five male Sprague-Dawley rats. Healing Achilles tendons were harvested at 4, 7, 14 and 21 days following the surgery. The un-injured left Achilles tendons were used as controls. Using RNase protection assays, in situ hybridization and immunohistochemistry, mRNA and protein of NOS isoforms were evaluated. RESULTS: Minimal NOS expression was found in un-injured tendon. A cell specific temporal pattern for the mRNA and protein for all three NOS isoforms was found following injury to the Achilles tendon. iNOS was maximal on day 4 in macrophages and fibroblasts. eNOS was maximal on day 4 in endothelial cells and fibroblasts. bNOS expression gradually increased up to day 21 and was found only in fibroblasts. CONCLUSIONS: These results suggest that all three nitric oxide synthase isoforms are expressed by fibroblasts in a coordinated temporal sequence during tendon healing. The sequential pattern of NOS expression in healing fibroblasts suggests that each NOS isoform may play a different role in the healing process and provides opportunities to modify tendon healing in the clinical setting.

Temporal expression of nitric oxide synthase isoforms in healing Achilles tendon.

We investigated the temporal expressions of the three nitric oxide synthase (NOS) isoforms by semi-quantitative polymerase chain reaction (PCR) assays and by immunoblot analysis, following Achilles tendon transection in rats. Four days after injury, there were increases in the steady-state levels of mRNA for all three NOS isoforms, with peaks for the inducible isoform (iNOS) (23-fold increase) at day 4, the endothelial isoform (eNOS) (24-fold increase) at day 7 and the neuronal isoform (bNOS) (seven-fold increase) at day 21. The temporal expression of NOS isoforms at a protein level was consistent with the results at the mRNA level. We have previously shown a five-fold increase in the NOS activity, as detected by 3H-arginine to 3H-citrulline conversion, at day 7 postinjury. These findings indicate that all three NOS isoforms are expressed during tendon healing with differential expression patterns during the various phases of tendon healing. These findings may prove clinically relevant with respect to strategies for regulating tendon healing.

Search for an endogenous collagenase in chicken endochondral bone matrix vesicles.

While isolating matrix vesicles from avian endochondral bone, collagenase activity was discovered unexpectedly. The question was raised whether this collagenase activity was endogenous or if it was due to the bacterial collagenase used to release the matrix vesicles from bone. In related experiments done, collagen partially degraded by collagenase mineralized better than undegraded collagen. This study would then attempt to show if an endogenous collagenase is found, whether it facilitates collagen mineralization by allowing better access for matrix vesicles by the "nicking" of collagen. From this two-month study evidence was found that an endogenous collagenase as well as a gelatinase was present on matrix vesicles. SDS gel electrophoresis and zymography were used to determine the presence of collagenases in purified matrix vesicle fractions. Western blots with antibodies to bacterial collagenase, matrix metallo-proteinases 13 (MMP-13), an endogenous collagenase, and MMP-2, an endogenous gelatinase, were also done to determine the presence of an endogenous collagenase. Another facet was added to this study to determine if contamination by exogenous collagenase during matrix vesicle isolation could be removed. From the final Western blots done, it was verified that even after exhaustive washings of the column and centrifugation, purified matrix vesicles contained contaminating bacterial collagenase. Due to the research completed, the matrix vesicle isolation protocol was changed from using bacterial collagenase to a trypsin isolation method to prevent any exogenous collagenase contamination.

Interleukin 1 alpha mediates collagenase synthesis stimulated by phorbol 12-myristate 13-acetate.

Stimulation of collagenase expression in cultures of normal diploid fibroblasts by the tumor promotor phorbol 12-myristate 13-acetate (PMA) occurs secondarily to synthesis of unknown intermediary proteins. We have investigated the hypothesis that a form of the cytokine interleukin 1 (IL-1) is one intermediate controlling PMA-stimulated collagenase expression. Treatment with an IL-1 receptor antagonist inhibits the constitutive synthesis of collagenase in early passage fibroblast cultures from rabbit. Radioimmunoassay demonstrates that, of the two known IL-1 forms, IL-1 alpha and IL-1 beta, only IL-1 alpha is synthesized and released into the medium of corneal fibroblast cultures. PMA treatment of cells increases the level of IL-1 alpha mRNA; this occurs prior to the increase in collagenase mRNA and corresponds with increased synthesis and release of IL-1 alpha protein. Neutralizing antiserum to IL-1 alpha inhibits constitutive collagenase synthesis. Reagents that inhibit the activity of IL-1 alpha (IL-1 receptor antagonist or neutralizing antibody) also inhibit the PMA-mediated stimulation of collagenase synthesis. These results indicate that constitutive and PMA-stimulated expression of collagenase is regulated through an IL-1 alpha intermediate. In vivo, regulation of the lytic phase of tissue remodeling through the IL-1 alpha intermediate may ensure the recruitment of cells adjacent to the one that received the initial stimulus.

Chronic Achilles paratenonitis in athletes: a histological and histochemical study.

Pathological alterations of chronic Achilles paratenonitis were studied histologically and histochemically in tissue samples obtained operatively from 16 athletes with this complaint and from 3 control patients. The activities of 11 different enzymes--lactate, succinate, malate, glucose-6-phosphate and glutamate dehydrogenases, lipoamide dehydrogenase and glutathione reductase (NADH2- and NADPH2-diaphorases), acid and alkaline phosphatases, phosphorylase and leucylaminopeptidase--were studied. Pathological findings were located diffusely around the tendon. A slight inflammatory cell reaction was found in all cases. The fatty areolar tissue was clearly thickened and edematous, and showed fibrinous exudations, widespread fat necrosis, considerable connective tissue proliferation and adhesion formation. The blood vessels showed profound degenerative and necrotizing changes. The thin membranes of the paratenon were clearly hypertrophied. Increased enzyme activities were mainly found in the fibroblasts, inflammatory cells and vascular walls. A moderate activity of lysosomal enzymes, an increased activity of enzymes of electron transport, anaerobic glycolysis, pentose phosphate shunt and decreased activity of those of aerobic energy metabolism were found. Simultaneously an increased amount of both neutral and acid mucopolysaccharides and a locally increased amount of elastic fibres were found in the inflamed paratenon. These results indicate that marked metabolic changes occur in paratenonitis, i.e. an increased catabolism and decreased oxygenation of the inflamed areas. The morphological alterations suggest that the gliding function of the paratenon may be impaired.

The distribution of lactate dehydrogenase isoenzymes in adult human Achilles tendons.

The authors have studied the LDH isoenzymes distribution in the human Achilles tendons. Connection between the age, sex, and enzyme activity were not found. In the Achilles tendon were found strong activity of isoenzymes IV and V (M-type), and controversy them moderate activity of isoenzymes I and II (H-type).