Proteoglycans and collagenase in hypertrophic scar formation.

The collagen fibers of the nodules and whorl-like figures in hypertrophic scars are "coated" with proteoglycans, mainly chondroitin-4-sulfate. The latter was shown to prevent collagenase from breaking down collagen. This suggests that the presence of great amounts of chondroitin-4-sulfate in hypertrophic scars may contribute to the overabundance of collagen deposition which is characteristic of this abnormal healing process.     

Effect of Mederma on hypertrophic scarring in the rabbit ear model

Currently accepted conservative treatments of hypertrophic scars are limited to steroid injections, radiation therapy, and silicone occlusive therapy. However, the use of Mederma for these problematic lesions has become quite prevalent in the clinical setting. Little scientific evidence exists to support the efficacy of this product in reducing hypertrophic scars. The aim of this study was to study the effects of Mederma on hypertrophic scars in the rabbit hypertrophic scar model, allowing the histologic quantification of scar elevation, dermal collagen organization, vascularity, and inflammation and the gross examination of scar erythema. Full-thickness wounds down to cartilage, four per ear, were created in four New Zealand White rabbits, for a total of 32 scars. Twenty-eight days after the initial wounding, the hypertrophic scars were photographed, and treatment of half of the scars on each ear was begun with Mederma three times per day for a total of 4 weeks. The untreated scars served as control scars and were left exposed to air. After 4 weeks of treatment, the scars were once again photographed. The rabbits were then killed, and the scars were analyzed histologically. The pretreatment and posttreatment photographs were compared by using computer quantification of magenta, yellow, and cyan expression within the scars.Histologic analysis demonstrated no significant reduction in scar hypertrophy or scar elevation index. However, a significant improvement in dermal collagen organization was noted on comparing Mederma-treated scars with untreated control scars (p < 0.05). No significant difference in dermal vascularity or inflammation was noted. Computer analysis of the scar photographs demonstrated no significant reduction in scar erythema with Mederma treatment. The active product in Mederma, allium cepa, has as its derivative quercetin, a bioflavonoid noted for its antiproliferative effects on both normal and malignant cells, and its antihistamine release effects. These properties could theoretically prove beneficial in reversing the inflammatory and proliferative responses noted in hypertrophic scars. Despite the authors' inability to demonstrate a reduction in scar hypertrophy, the improvement in collagen organization noted in the Mederma-treated scars suggests it may have an effect on the pathophysiology of hypertrophic scar formation.     

Characterization of the collagen of human hypertrophic and normal scars.

The collagen produced in response to an injury of human skin is initially stabilized by a cross-link derived from hydroxyallysine, and characteristic of embryonic skin. In normal healing there is a change over with time to the cross-link derived from allysine, which is typical of young skin collagen. In contrast, hypertrophic scars fail to follow the time-related changes of normal skin, but retain the characteristics of embryonic collagen, indicating a continued rapid turnover of the collagen. This is further supported by the high proportion of the embryonic Type III collagen present in hypertrophic scars.     

Therapeutic effects of liposome-enveloped Ligusticum chuanxiong essential oil on hypertrophic scars in the rabbit ear model

Hypertrophic scarring, a common proliferative disorder of dermal fibroblasts, results from an overproduction of fibroblasts and excessive deposition of collagen. Although treatment with surgical excision or steroid hormones can modify the symptoms, numerous treatment-related complications have been described. In view of this, we investigated the therapeutic effects of essential oil (EO) from rhizomes of Ligusticum chuanxiong Hort. (Umbelliferae) on formed hypertrophic scars in a rabbit ear model. EO was prepared as a liposomal formulation (liposome-enveloped essential oil, LEO) and a rabbit ear model with hypertrophic scars was established. LEO (2.5, 5, and 10%) was applied once daily to the scars for 28 days. On postoperative day 56, the scar tissue was excised for masson's trichrome staining, detection of fibroblast apoptosis, assays of the levels of collagens I and III, and analysis of the mRNA expression of matrix metalloproteinase-1 (MMP-1), caspase-3 and -9, and transforming growth factor beta 1 (TGF-β(1)). In addition, the scar elevation index (SEI) was also determined. As a result, LEO treatment significantly alleviated formed hypertrophic scars on rabbit ears. The levels of TGF-β(1), MMP-1, collagen I, and collagen III were evidently decreased, and caspase -3 and -9 levels and apoptosis cells were markedly increased in the scar tissue. SEI was also significantly reduced. Histological findings exhibited significant amelioration of the collagen tissue. These results suggest that LEO possesses the favorable therapeutic effects on formed hypertrophic scars in the rabbit ear model and may be an effective cure for human hypertrophic scars.     

Intralesional cryotherapy for enhancing the involution of hypertrophic scars and keloids

Although therapeutic management of hypertrophic scars and keloids using contact or spray cryosurgery has yielded significant improvement or complete regression of hypertrophic scars and keloids, it requires one to 20 treatment sessions. This study was designed to assess the clinical safety and efficacy of an intralesional needle cryoprobe method in the treatment of hypertrophic scars and keloids.Ten patients, ranging in age from 3 to 54 years, with a total of 12 hypertrophic scars and keloids of more than 6 months duration and of diverse causes, were included in this study. The 18-month trial evaluated volume reduction of the hypertrophic scars and keloids after a single session of intralesional cryotherapy. Objective (hardness and color) and subjective (pain/tenderness and itchiness/discomfort) parameters were examined on a scale of 0 to 3 (low score was better). Pretreatment and posttreatment histomorphometric studies of the collagen fibers included spectral picrosirius red polarization and fast Fourier transformation orientation index. A specially designed cryo-needle was inserted into the long axis of the hypertrophic scars and keloids so as to maximize the volume of the hypertrophic scars and keloids to be frozen. The cryo-needle was connected by an adaptor to a cryogun filled with liquid nitrogen, which was introduced into the cryoprobe, thereby freezing the hypertrophic scars and keloids. After the hypertrophic scars and keloids were completely frozen, the cryoprobe defrosted and was withdrawn.An average of 51.4 percent of scar volume reduction was achieved after one session of intralesional cryosurgery treatment (average preoperative hypertrophic scars and keloids volume, 1.82 +/- 0.33; average posttreatment volume, 0.95 +/- 0.21; p < 0.0022). Significant alleviation of objective and subjective clinical symptoms was documented. Mild pain or discomfort during and after the procedure was easily managed. Only mild local edema and epidermolysis, followed by a short reepithelialization period, were evident. During the 18-month follow-up period, there was no evidence of bleeding, infection, adverse effects, recurrence, or permanent depigmentation. The histomorphometric analysis demonstrated rejuvenation of the treated scars (i.e., parallelization) and a more organized architecture of the collagen fibers compared with the pretreated scars.This study demonstrated the increased efficacy of this method as a result of increased freezing area of deep scar material compared with that obtained with contact/spray probes. As a result, fewer treatment cycles are needed. Because the reepithelialization period is short, treatment intervals, if any, can be shortened to 2 to 3 weeks. This intralesional cryoneedle method is simple to operate and safe to use, it necessitates less postoperative care of the wound, and it can easily be added to any preexisting cryosurgical unit.     

Effect of Hematoporphyrin Monomethyl Ether-Sonodynamic Therapy (HMME-SDT) on Hypertrophic Scarring

Objective

The aim of the present study was to explore the potential for hematoporphyrin monomethyl ether-Sonodynamic Therapy (HMME-SDT) treatment of hypertrophic scars within rabbit ears.

Methods

60 white rabbits were randomly divided into five groups: 1) untreated controls, 2) lesioned, 3) lesioned + HMME, 4) lesioned + US (Ultrasound), and 5) lesioned +HMME-SDT. After induction of a lesion upon the ears of the rabbits, hypertrophic scars were assessed at 14, 28, 42 and 56 days post-lesion +/− treatment. Assessments consisted of visual inspection in the change of the skin, scar formation pathological morphology by hematoxylin and eosin (HE) staining technique with optical microscopy, calculation of a hypertrophic index, fibroblastic density measures, and observation of collagen changes in the scar tissue by Van Gieson''s (VG)Stain along with calculation of collagen area density.

Results

With continued HMME-SDT treatment there was a gradual improvement in all parameters over the duration of the experiment. The lesion-induced scars of rabbits receiving HMME-SDT treatment were soft, the size was reduced, hyperplasia was flat and the color pale. The fibroblasts and collagens were reduced and the collagens were light red, sparse and orderly. The hypertrophic index was reduced, since the fibroblastic density was lowered and collagen area density was decreased.

Conclusion

HMME is an effective sonosensitizer and the combination of HMME-SDT treatment can exert significant benefits in reducing the formation of hypertrophic scars.     

Wnt5a在增生性瘢痕中的表达及其临床意义

目的：探讨wnt5a在增生性瘢痕中的表达及其临床意义。方法：选择12例增生性瘢痕患者,术中取成熟期增生性瘢痕6份,增殖期增生性瘢痕6份,正常皮肤组织6份。光镜下观察其形态学的差异,通过免疫组化技术检测和比较其Wnt5a阳性表达的细胞面积率。结果：与正常皮肤相比,增殖期增生性瘢痕中有大量的成纤维细胞,胶原纤维含量丰富,且排列紊乱,其间有大量的炎性细胞,成熟期增生性瘢痕也含有丰富的成纤维细胞和胶原,但炎性细胞很少。增殖期增生性瘢痕和成熟期增生性瘢痕组织中真皮浅层和真皮深层Wnt5a阳性表达的细胞面积率均显著高于正常皮肤组织（P〈0．05）,且增殖期增生性瘢痕组织中wnt5a阳性表达的细胞面积率显著高于成熟期增生性瘢痕（P〈0．05）。但正常皮肤组织、成熟期增生性瘢痕、增殖期增生性瘢痕各组间真皮浅层与真皮深层Wnt5a阳性表达的细胞面积率比较均无显著性差异（P〉0．05）。结论：Wnt5a的表达上调可能在增生性瘢痕的形成中起重要作用,并可能与增生性瘢痕的增殖活性有关。     

Endothelial cells enhance adipose mesenchymal stromal cell‐mediated matrix contraction via ALK receptors and reduced follistatin: Potential role of endothelial cells in skin fibrosis

Abnormal cutaneous wound healing can lead to formation of fibrotic hypertrophic scars. Although several clinical risk factors have been described, the cross‐talk between different cell types resulting in hypertrophic scar formation is still poorly understood. The aim of this in vitro study was to investigate whether endothelial cells (EC) may play a role in skin fibrosis, for example, hypertrophic scar formation after full‐thickness skin trauma. Using a collagen/elastin matrix, we developed an in vitro fibrosis model to study the interaction between EC and dermal fibroblasts or adipose tissue‐derived mesenchymal stromal cells (ASC). Tissue equivalents containing dermal fibroblasts and EC displayed a normal phenotype. In contrast, tissue equivalents containing ASC and EC displayed a fibrotic phenotype indicated by contraction of the matrix, higher gene expression of ACTA2, COL1A, COL3A, and less secretion of follistatin. The contraction was in part mediated via the TGF‐β pathway, as both inhibition of the ALK4/5/7 receptors and the addition of recombinant follistatin resulted in decreased matrix contraction (75 ± 11% and 24 ± 8%, respectively). In conclusion, our study shows that EC may play a critical role in fibrotic events, as seen in hypertrophic scars, by stimulating ASC‐mediated matrix contraction via regulation of fibrosis‐related proteins.     

Interaction between proteoglycan subunit and type II collagen from bovine nasal cartilage, and the preferential binding of proteoglycan subunit to type I collagen.

We studied the interaction of proteoglycan subunit with both types I and II collagen. All three molecular species were isolated from the ox. Type II collagen, prepared from papain-digested bovine nasal cartilage, was characterized by gel electrophoresis, amino acid analysis and CM-cellulose chromatography. By comparison of type I collagen, prepared from papain-digested calf skin, with native calf skin acid-soluble tropocollagen, we concluded that the papain treatment left the collagen molecules intact. Interactions were carried out at 4 degrees C in 0.06 M-sodium acetate, pH 4.8, and the results were studied by two slightly different methods involving CM-cellulose chromatography and polyacrylamide-gel electrophoresis. It was demonstrated that proteoglycan subunit, from bovine nasal cartilage, bound to cartilage collagen. Competitive-interaction experiments showed that, in the presence of equal amounts of calf skin acid-soluble tropocollagen (type I) and bovine nasal cartilage collagen (type II), proteoglycan subunit bound preferentially to the type I collagen. We suggest from these results that, although not measured under physiological conditions, it is unlikely that the binding in vivo between type II collagen and proteoglycan is appreciably stronger than that between type I collagen and proteoglycan.     

Abrogation of transforming growth factor-beta signaling by SMAD7 inhibits collagen gel contraction of human dermal fibroblasts

Human fibroproliferative disorders like hypertrophic scarring of the skin are characterized by increased contractility and excess extracellular matrix synthesis. A beneficial role of transforming growth factor (TGF)-beta in wound healing was proposed; however, chronic stimulation by this cytokine leads to fibrosis. In the present report, the intracellular TGF-beta signaling in fibroblasts derived from hypertrophic scars and normal skin was examined. In an attempt to intervene in profibrogenic TGF-beta functions, ectopic expression of Smad7 or dominant negative Smads3/4 completely inhibited contractility of scar-derived and normal fibroblasts after suspension in collagen gels. Both cell types displayed constitutive Smad2/3 phosphorylation and (CAGA)9-MLP-Luc activity with expression and phosphorylation of Smad3 being predominant in hypertrophic scar-derived fibroblasts. Down-regulation of intrinsic signaling with various TGF-beta antagonists, e.g. soluble TGF-beta receptor, latency-associated peptide, and anti-TGF-beta1 antibodies, confirms autocrine TGF-beta stimulation of both cell populations. Further, Smad7 expression inhibited alpha1 (I) collagen and alpha-smooth muscle actin expression. In summary, our data indicate that autocrine TGF-beta/Smad signaling is involved in contractility and matrix gene expression of fibroblasts from normal and hypertrophic scars. Smad7 inhibits these processes and may exert beneficial effects on excessive scar formation.     

The Anti-Scar Effects of Basic Fibroblast Growth Factor on the Wound Repair In Vitro and In Vivo

Hypertrophic scars (HTS) and keloids are challenging problems. Their pathogenesis results from an overproduction of fibroblasts and excessive deposition of collagen. Studies suggest a possible anti-scarring effect of basic fibroblast growth factor (bFGF) during wound healing, but the precise mechanisms of bFGF are still unclear. In view of this, we investigated the therapeutic effects of bFGF on HTS animal model as well as human scar fibroblasts (HSF) model. We show that bFGF promoted wound healing and reduced the area of flattened non-pathological scars in rat skin wounds and HTS in the rabbit ear. We provide evidence of a new therapeutic strategy: bFGF administration for the treatment of HTS. The scar elevation index (SEI) and epidermal thickness index (ETI) was also significantly reduced. Histological reveal that bFGF exhibited significant amelioration of the collagen tissue. bFGF regulated extracellular matrix (ECM) synthesis and degradation via interference in the collagen distribution, the α-smooth muscle actin (α-SMA) and transforming growth factor-1 (TGF-β1) expression. In addition, bFGF reduced scarring and promoted wound healing by inhibiting TGFβ1/SMAD-dependent pathway. The levels of fibronectin (FN), tissue inhibitor of metalloproteinase-1 (TIMP-1) collagen I, and collagen III were evidently decreased, and matrix metalloproteinase-1 (MMP-1) and apoptosis cells were markedly increased. These results suggest that bFGF possesses favorable therapeutic effects on hypertrophic scars in vitro and in vivo, which may be an effective cure for human hypertrophic scars.     

Comparative study of Langerhans cells in normal and pathological human scars. II. Hypertrophic scars.

Langerhans cells (LCs) seem to play a crucial role in the immune system of the skin. Changes in their density, distribution, phenotype and/or morphology have been described in a number of skin diseases, mostly immunologically mediated. For this reason, we investigated LCs in human hypertrophic scars, since these scars are presently believed to have an immunological basis. A preliminary analysis of the histological features was carried out on vertical serial sections, stained with hematoxylin and eosin. Both epidermal and dermal components of hypertrophic scar biopsies were examined. The total epidermal thickness and the thickness of the single epidermal layers were also measured; the values obtained were similar to those of control skin and normotrophic scars. Subsequently, CDla-positive LCs, revealed by indirect immunofluorescence and immunoperoxidase techniques, were studied to determine their position among the epidermal layers and within the dermis, their dimensions, their density and their morphology. According to these observations, two main types of hypertrophic scars were identified. In the first type (7 scars), LCs were widely clustered within both the whole epidermis and the dermis. Their density was increased (about 750 cells/mm2 of epidermal area), if compared to control skin and normotrophic scars (both about 400 cells/mm2 of epidermal area; p less than 0.001). The epidermal cell profiles, nearly three times larger than those of control skin, exhibited a dense network of interconnected dendrites. Further analysis for the presence of HLA-DR molecules revealed an anomalous expression of these antigens on keratinocytes. In the second type (3 scars), LCs density within the stratum Malpighii was unchanged, relative to control skin and normal scars, while CDla-positive cell bodies remained numerous in basal position and within the subpapillary corion. Epidermal LCs, only slightly larger than those evidentiated in control skin, displayed short and retracted dendritic projections. The aberrant expression of HLA-DR antigens on keratinocytes was very weak and sparse. The present results strongly suggest an immunologically activated state of the tissues examined; they provide morphological data that support the involvement of the immune system in hypertrophic scarring.     

Matrix metalloproteinase‐2 and ‐9 activities in human keloids,hypertrophic and atrophic scars: a pilot study

Proteolytic degradation of extracellular matrix is one of the principal features of cutaneous wound healing but little is known about the activities of gelatinases; matrix metalloproteinase‐2 (MMP‐2) and matrix metalloproteinase‐9 (MMP‐9) on abnormal scar formation. The aim of this study is to determine collagen levels and the gelatinase activities in tissue from hypertrophic scars, atrophic scars, keloids and donor skin in 36 patients and 14 donors. Gelatinase levels (proenzyme + active enzyme) were determined by ELISA and their activities by gelatin zymography. MMP‐9 activity was undetectable in gelatin zymography analysis. Pro‐MMP‐2 levels (median) were highest in normal skin group 53.58 (36.40–75.11) OD µg^?1 protein, while active MMP‐2 levels were highest in keloid group 52.53 (42.47–61.51) OD µg^?1 protein. The active/pro ratio was the highest in keloid group 0.97 followed by hypertrophic scar, normal skin and atrophic scar groups 0.69 > 0.54 > 0.48, respectively. According to results of our study, the two‐phase theory of the duration of hypertrophic scar and keloid formation can be supported by the data of tissue collagen and gelatinase analysis. This study is the first to relate scar formation relationship in regard to gelatinase activation ratio in a keloid, hypertrophic and atrophic scar patient group which is chosen appropriate in age and sex. Copyright © 2009 John Wiley & Sons, Ltd.     

Morphological analysis of the connective tissue reaction in linear hypertrophic scars treated with intralesional steroid or silicone-gel sheeting. A light and electron microscopic study

The linear hypertrophic scar has become the most common type of pathologic scarring. Silicone-gel sheeting is the first line therapy while intralesional steroid is the second. A light and electron microscopic analysis was carried out to reveal differences in tissue reaction following the two different treatments. Two groups of 12 patients each were treated for 4 months. For the first group, diluted Triamcinolone acetonide was injected until an inactive state was achieved. The other group of patients was treated with silicone-gel sheeting. The scars were examined every two weeks and their appearance documented. After reaching the expected therapeutic response, inactive scars were removed. The excised scars were evaluated through light microscopic histopathology and electron microscopy. The light and electron microscopic observations revealed marked differences following treatments. The activity of fibroblasts and the numbers of collagen fibers forming bundles decreased and the orientation of the collagen fibers was more variable in the treated scars. The amount of elastic fibers increased after both steroid and silicone-gel sheeting treatment. Vascularization was also slightly changed, with more capillaries and fewer pre-capillary arteries detected in the treated scars. Both treatments resulted in the same decrease in score but steroid treatment was more rapid in onset. We suggest that the two different treatments work through different mechanisms, although the final functional outcome is similar.     

Tissue alpha-globulins in keloid formation.

The deposition of alpha-1-antitrypsin and alpha-2-macroglobulin, both known to be inhibitors of human skin collagenase, is significantly increased in keloids and in hypertrophic scars (as compared to normal skin). However, following intralesional triamcinolone treatment, a marked resorption of these abnormal scars occurs along with a significant reduction of the alpha-1-antitrypsin deposits. These findings suggest that alpha-globulins are involved in abnormal scar formation, and that triamcinolone may remove collagenase and/or protease inhibitors--thereby allowing activation of the collagenase with subsequent breakdown and resorption of the excessive collagen.     

Human specific anti-type IV collagen monoclonal antibodies, characterization and immunohistochemical application

This paper describes two new monoclonal antibodies reactive with human specific type IV collagen epitopes in frozen as well as routinely fixed and processed tissue sections. The antibodies (1042 and 1043) were raised against human placental type IV collagen and were shown by immunoblotting and ELISA tests to react exclusively with type IV collagen determinants. Extensive immunohistochemical survey studies on panels of tissues from various species, using unfixed cryostat sections, demonstrated that antibody 1042 reacted only with human type IV collagen whereas antibody 1043 in addition reacted with rabbit type IV collagen. All tissues showed homogeneous staining of the basement membrane, indicating that the detected epitopes did not show organ-specific distribution. Tissue processing protocols for using these monoclonal antibodies on routinely processed paraffin embedded tissues were developed. It was found that whereas polyclonal anti-type IV collage antisera required pepsin digestion, our monoclonal antibodies required pronase or papain digestion to restore type IV collagen immunoreactivity in paraffin sections. It is concluded that these monoclonal anti-type IV collagen antibodies detect species specific epitopes which can be detected in routinely processed paraffin embedded tissues after appropriate enzyme pretreatment.     

Effect of P144? (Anti-TGF-β) in an “In Vivo” Human Hypertrophic Scar Model in Nude Mice

Background

Hypertrophic scars are one of the most important complications in surgery due to their cosmetic and functional impairments. Previous studies in tissue fibrotic disorders have shown promising results by inhibiting the biological activity effect of Transforming Growth Factor-beta 1 (TGF-β1). The aim of the current study was to determine the clinical effect of the inhibition of TGF-β1 signaling in human hypertrophic scars implanted in nude mice by topical application of an inhibitor of TGF-β1 (P144®).

Material and Methods

A total of 30 human hypertrophic scars were implanted in 60 nude mice. The animals were divided in two groups, group A (placebo) and group B (treatment). Group C (basal) was considered as the preimplanted scar samples and they were not implanted in the nude mice. After the shedding period, topical application of a lipogel containing placebo (group A) or P144 (group B) was daily administered during two weeks. The animals were sacrificed upon completion of the study. Total area, thickness and collagen fibers area were measure and compared across all groups. Immunohistochemistry was also performed in order to quantify collagen type I and type III and elastic fiber expressions present in the dermis.

Results

Successful shedding was achieved in 83,3% of the xenografts. The mean time for shedding was 35±5.4 days. Statistically significant differences were found in the total area, collagen fibers area and thickness between the groups. Increased elastic fibers and decreased collagen I were found in the P144-treated group compared to the basal group.

Conclusion

Topical application of an inhibitor of TGF-β1 may promote scar maturation and clinical improvement of hypertrophic scar morphology features in an “in vivo” model in nude mice after two weeks of treatment.     

Keloids have continuous high metabolic activity.

The adenosine triphosphate (ATP) content of keloids and scars resected from patients was demonstrated by high-performance liquid chromatography. The surface color of hypertrophic scars was red or pink and that of atrophic scars was white. The ATP content of red scars was (in mmol/g protein) 1.06 +/- 0.14, of pink scars 0.12 +/- 0.02, of white atrophic scars 0.19 +/- 0.06, and of keloids 1.06 +/- 0.19. The longer the elapsed time after the trauma, the lower the level of ATP in scar tissues (correlation coefficient = -0.506; p = 0.005 by Spearman's rank correlation). However, ATP levels in keloids were still high 10 years after the injury. Fibroblasts and fibrocytes in keloids and scars were counted in histologic preparations stained with hematoxylin and eosin. The average number of fibroblasts in a definite area (56 x 10(-4) mm at a magnification of x400) was 4.8 in keloids, 5.1 in red scars, 2.4 in pink scars, and 1.3 in white atrophic scars. The number of fibrocytes in the same area was 0.4 in keloids, 0.4 in red scars, 2.3 in pink scars, and 1.3 in white atrophic scars. These results indicate that keloids and red hypertrophic scars have higher ATP levels and contain more fibroblasts than pink or white scars, and they also suggest that the levels of ATP and the number of fibroblasts decrease when red hypertrophic scars change into atrophic scars. In keloids, ATP and fibroblasts seem to remain at high levels for a long time.     

Human specific anti-type IV collagen monoclonal antibodies,characterization and immunohistochemical application

Summary This paper describes two new monoclonal antibodies reactive with human specific type IV collagen epitopes in frozen as well as routinely fixed and processed tissue sections. The antibodies (1042 and 1043) were raised against human placental type IV collagen and were shown by immunoblotting and ELISA tests to react exclusively with type IV collagen determinants. Extensive immunohistochemical survey studies on panels of tissues from various species, using unfixed cryosat sections, demonstrated that antibody 1042 reacted only with human type IV collagen whereas antibody 1043 in addition reacted with rabbit type IV collagen. All tissues showed homogeneous staining of the basement membrane, indicating that the detected epitopes did not show organ-specific distribution.Tissue processing protocols for using these monoclonal antibodies on routinely processed paraffin embedded tissues were developed. It was found that whereas polyclonal antitype IV collagen antisera required pepsin digestion, our monoclonal antibodies required pronase or papain digestion to restore type IV collagen immunoreactivity in paraffin sections.It is concluded that these monoclonal anti-type IV collagen antibodies detect species specific epitopes which can be detected in routinely processed paraffin embedded tissues after appropriate enzyme pretreatment.