Experimental Granulation Research Articles

A physiological role for connective tissue growth factor in early wound healing

Mesenchymal stem cells (MSCs) that overexpress secreted frizzled-related protein 2 (sFRP2) exhibit an enhanced reparative phenotype. The secretomes of sFRP2-overexpressing MSCs and vector control-MSCs were compared through liquid chromatography tandem mass spectrometry. Proteomic profiling revealed that connective tissue growth factor (CTGF; CCN2) was overrepresented in the conditioned media of sFRP2-overexpressing MSCs and MSC-derived CTGF could thus be an important paracrine effector. Subcutaneously implanted, MSC-loaded polyvinyl alcohol (PVA) sponges and stented excisional wounds were used as wound models to study the dynamics of CTGF expression. Granulation tissue generated within the sponges and full-thickness skin wounds showed transient upregulation of CTGF expression by MSCs and fibroblasts, implying a role for this molecule in early tissue repair. Although collagen and COL1A2 mRNA were not increased when recombinant CTGF was administered to sponges during the early phase (day 1–6) of tissue repair, prolonged administration (>15 days) of exogenous CTGF into PVA sponges resulted in fibroblast proliferation and increased deposition of collagen within the experimental granulation tissue. In support of its physiological role, CTGF immunoinhibition during early repair (days 0–7) reduced the quantity, organizational quality and vascularity of experimental granulation tissue in the sponge model. However, CTGF haploinsufficiency was not enough to reduce collagen deposition in excisional wounds. Similar to acute murine wound models, CTGF was transiently present in the early phase of human acute burn wound healing. Together, these results further support a physiological role for CTGF in wound repair and demonstrate that when CTGF expression is confined to early tissue repair, it serves a pro-reparative role. These data also further illustrate the potential of MSC-derived paracrine modulators to enhance tissue repair.

Hemoglobin expression in rat experimental granulation tissue

The general opinion that hemoglobin is only a carrier protein for oxygen and carbon dioxide has been challenged by several recent studies showing hemoglobin expression in other cells than those of the erythroid series, for example, in macrophages. We discovered β-globin expression in rat experimental granulation tissue induced by subcutaneously implanted cellulose sponges. Closer investigation revealed also α-globin expression. The first peak of the biphasic globin expression noticed during granulation tissue formation correlated with the invasion of monocytes/macrophages, whereas the second one seemed to be connected to the appearance of hematopoietic progenitors. Data presented in this study indicate globin expression both in macrophages and in immature erythroid cells as validated by erythroid-specific markers.

SFRP2 Suppression of Bone Morphogenic Protein (BMP) and Wnt Signaling Mediates Mesenchymal Stem Cell (MSC) Self-renewal Promoting Engraftment and Myocardial Repair

Transplantation of mesenchymal stem cells (MSCs) is a promising therapy for ischemic injury; however, inadequate survival of implanted cells in host tissue is a substantial impediment in the progress of cellular therapy. Secreted Frizzled-related protein 2 (sFRP2) has recently been highlighted as a key mediator of MSC-driven myocardial and wound repair. Notably, sFRP2 mediates significant enhancement of MSC engraftment in vivo. We hypothesized that sFRP2 improves MSC engraftment by modulating self-renewal through increasing stem cell survival and by inhibiting differentiation. In previous studies we demonstrated that sFRP2-expressing MSCs exhibited an increased proliferation rate. In the current study, we show that sFRP2 also decreased MSC apoptosis and inhibited both osteogenic and chondrogenic lineage commitment. sFRP2 activity occurred through the inhibition of both Wnt and bone morphogenic protein (BMP) signaling pathways. sFRP2-mediated inhibition of BMP signaling, as assessed by levels of pSMAD 1/5/8, was independent of its effects on the Wnt pathway. We further hypothesized that sFRP2 inhibition of MSC lineage commitment may reduce heterotopic osteogenic differentiation within the injured myocardium, a reported adverse side effect. Indeed, we found that sFRP2-MSC-treated hearts and wound tissue had less ectopic calcification. This work provides important new insight into the mechanisms by which sFRP2 increases MSC self-renewal leading to superior tissue engraftment and enhanced wound healing.

The Wnt modulator sFRP2 enhances mesenchymal stem cell engraftment, granulation tissue formation and myocardial repair

Cell-based therapies, using multipotent mesenchymal stem cells (MSCs) for organ regeneration, are being pursued for cardiac disease, orthopedic injuries and biomaterial fabrication. The molecular pathways that regulate MSC-mediated regeneration or enhance their therapeutic efficacy are, however, poorly understood. We compared MSCs isolated from MRL/MpJ mice, known to demonstrate enhanced regenerative capacity, to those from C57BL/6 (WT) mice. Compared with WT-MSCs, MRL-MSCs demonstrated increased proliferation, in vivo engraftment, experimental granulation tissue reconstitution, and tissue vascularity in a murine model of repair stimulation. The MRL-MSCs also reduced infarct size and improved function in a murine myocardial infarct model compared with WT-MSCs. Genomic and functional analysis indicated a downregulation of the canonical Wnt pathway in MRL-MSCs characterized by significant up-regulation of specific secreted frizzled-related proteins (sFRPs). Specific knockdown of sFRP2 by shRNA in MRL-MSCs decreased their proliferation and their engraftment in and the vascular density of MRL-MSC-generated experimental granulation tissue. These results led us to generate WT-MSCs overexpressing sFRP2 (sFRP2-MSCs) by retroviral transduction. sFRP2-MSCs maintained their ability for multilineage differentiation in vitro and, when implanted in vivo, recapitulated the MRL phenotype. Peri-infarct intramyocardial injection of sFRP2-MSCs resulted in enhanced engraftment, vascular density, reduced infarct size, and increased cardiac function after myocardial injury in mice. These findings implicate sFRP2 as a key molecule for the biogenesis of a superior regenerative phenotype in MSCs.

The Wnt Inhibitor sFRP2 Enhances Mesenchymal Stem Cell Engraftment, Granulation Tissue Formation and Myocardial Repair

Cell-based therapies using bone marrow-derived mesenchymal stem cells (MSCs) for organ regeneration are being pursued for cardiac disease, orthopedic injuries and biomaterial fabrication. The molecular pathways that regulate MSC-mediated regeneration or enhance their therapeutic efficacy are, however, poorly understood. In an attempt to elucidate a way to strengthen the regenerative potential of MSCs, preliminary studies in our lab were performed comparing MSCs isolated from wildtype and regenerative mouse strains. The MRL/MpJ mouse has been described as a “super healer” mouse that is able to repair soft tissue with minimal scaring. MSCs were isolated from the MRL/MpJ mouse (MRL-MSCs) and from C57/Bl6 mice (WT-MSCs) and their differing qualities assessed. Compared to WT-MSCs, MRL-MSCs demonstrated increased proliferation in vitro. We utilized a Poly-vinyl alcohol (PVA) sponge model of repair stimulation to assess their capacity to generate wound repair tissue. We observed that the MRL-MSCs demonstrated increased in vivo engraftment, experimental granulation tissue reconstitution, and tissue vascularity. The MRL-MSCs also reduced infarct size and improved cardiac function as compared to WT-MSCs in a murine acute myocardial infarct model. Genomic and functional analyses indicated a downregulation of the canonical Wnt pathway in MRL-MSCs characterized specifically by upregulation of secreted frizzled related proteins (sFRPs). In vitro proliferation studies confirmed that recombinant sFRP2 mediated enhanced proliferation of both mouse and human MSCs. Based on these observations, we hypothesized that sFRP2 served an important role in MSC-mediated repair and regeneration. We generated WT-MSCs overexpressing sFRP2 (sFRP2-MSCs) by retroviral transduction to test this hypothesis. sFRP2-MSCs maintained their ability for multilineage differentiation in vitro and proliferated faster than the vector only control MSCs (GFP-MSCs). When implanted in vivo in the PVA sponge model, the sFRP2-MSCs recapitulated the MRL phenotype by mediating greater, more vascularized granulation tissue. Moreover, periinfarct intramyocardial injection of sFRP2-MSCs resulted in reduced infarct size, favorable remodeling and better preserved left ventricular function following acute myocardial infarct in mice. These findings implicate sFRP2 as a key molecule for the biogenesis of a superior regenerative phenotype of MSCs.

CARP, a Cardiac Ankyrin Repeat Protein, Is Up-Regulated during Wound Healing and Induces Angiogenesis in Experimental Granulation Tissue

Cardiac ankyrin repeat protein (CARP) was identified by subtractive hybridization as one of a group of genes that are rapidly modulated by acute wounding of mouse skin. Quantitative RT-PCR showed that CARP was strongly induced during the first day after wounding (157.1-fold), and the high level persisted for up to 14 days. Immunohistochemistry and in situ hybridization revealed that CARP was expressed in skeletal muscle, vessel wall, hair follicle, inflammatory cells, and epidermis in the wound area. To examine the effects of CARP on wound healing, we developed an adenoviral CARP vector to treat subcutaneously implanted sponges in either rats or Flk-1(LacZ) knock-in mice. Four days after infection, CARP-infected sponges in rats showed a remarkable increase in the vascular component in granulation tissue as compared to Ad-LacZ controls. This result was confirmed by CD34 immunostaining. By 7 days post-infection of sponge implants in Flk-1(LacZ) knock-in mice, granulation tissue showed many more LacZ-positive cells in Ad-CARP-infected sponges than in virus controls. Ad-CARP treatment also induced neovascularization and increased blood perfusion in rabbit excisional wounds in and ischemic rat wounds. These findings indicate that CARP could play a unique role in therapeutic angiogenesis during wound healing.

038 Adenoviral overexpression of MCP‐2 induces macrophages and improves wound healing in diabetic rats

The recruitment of leukocyte subsets is mainly driven by chemokines. Monocyte chemotactic protein‐2 (MCP‐2) belongs to the CC subfamily of chemokines, and it is especially a chemoattractant for monocytes. Of all CC subfamily members only MCP‐2 interacts with multiple receptors, and its N‐terminal truncated isoform is a natural chemokine inhibitor. Gene expression profiling in excisional wounds showed MCP‐2 was down‐regulated 40% in wild type mice and 69.2% in diabetic mice at 24 h after wounding. To determine the biological response to MCP‐2 during wound healing, we constructed an adenoviral vector (Ad‐MCP‐2). Ad‐MCP‐2 infected cell conditioned medium contained 460 ng/ml secreted MCP‐2 (ELISA) and had chemotactic activity similar to recombinant full‐length MCP‐2 but not to MCP‐2 N‐terminal 8–13aa peptides in a THP‐1cell migration assay. In an incisional wound model in STZ induced diabetic rats, the breaking strength of Ad‐MCP‐2 infected skin wounds increased by 43%(108 PFU, p < 0.05) and 30%(107 PFU, p < 0.05) at 7d after Ad‐MCP‐2 injection compared with Ad‐LacZ control. The wound closure strength of 107 PFU Ad‐MCP‐2 infected wounds still increased by 21%(p < 0.05) at 10d after injection. In normal rats, the breaking strength of Ad‐MCP‐2 and Ad‐Lacz infected wounds was not significantly different. In rats, we injected either Ad‐MCP‐2 or Ad‐LacZ (108 PFU) into each PVA sponge at 3d after implantation. Histological analyses revealed numerous ED‐1 positive macrophages infiltrating in the experimental granulation tissue at 7d after Ad‐MCP‐2 injection. Our data provide evidence that MCP‐2 improved wound healing in diabetic rats, and the recruitment of macrophages into the wound granulation tissues was the one of the mechanisms.

Connective Tissue Growth Factor and Its Correlation to Other Growth Factors in Experimental Granulation Tissue

Connective tissue growth factor (CTGF) is upregulated in a variety of fibrotic disorders, probably secondary to the activation and production of transforming growth factor-beta (TGF- g ). We have studied the expression of CTGF in a rat wound-healing model using Northern blot, in situ hybridization, and immunohistochemistry. The expression of CTGF mRNA in Northern blot and immunohistochemistry were correlated to the expression of TGF- g 1 and platelet-derived growth factor (PDGF). Northern hybridization showed the maximum expression of CTGF mRNA on day 14, whereas TGF- g 1 expression was maximal on days 7 and 14 and the time-related changes were smaller than for CTGF. PDGF A and PDGF B mRNA expressions were at maximum on day 14 and on day 21, respectively. In situ hybridization showed that fibroblast-like cells expressed CTGF most intensively, expression declining rapidly after day 14. CTGF mRNA and protein were found in blood vessel cells during the first week. In immunohistochemistry, all growth factors were expressed by fibroblast-like cells, macrophage-like cells, and blood vessels but CTGF-positive cells were fewer and were more restricted on days 5 and 7. These results demonstrate that CTGF expression together with TGF- g and PDGF are upregulated in wound healing, and CTGF expression in blood vessels suggests that CTGF is involved in angiogenesis.

Connective Tissue Growth Factor and Its Correlation to Other Growth Factors in Experimental Granulation Tissue

Connective Tissue Growth Factor and Its Correlation to Other Growth Factors in Experimental Granulation Tissue

Expression and activity of matrix metalloproteinase‐2 and ‐9 in experimental granulation tissue

The restoration of functional connective tissue is a major goal of the wound healing process which is probably affected by matrix-modifying enzymes. To evaluate the spatial and temporal expression of matrix metalloproteinases (MMP) MMP-2 and MMP-9 and to study the regulation of MMP-2 in wound healing, subcutaneously implanted viscose cellulose sponges in rats were used to induce granulation tissue formation for up to 3 months. MMP-2 mRNA expression was seen throughout the experiment and it was highest after 2 months. MMP-9 gene expression was low between days 8-21 and increased after 4 weeks of granulation tissue formation. Membrane-type 1 MMP (MT1-MMP) mRNA was upregulated early and tissue inhibitor 2 of MMP (TIMP-2) mRNA later during wound healing. In in situ hybridization the expression of MMP-2 mRNA was seen mostly in fibroblast-like cells and MMP-9 mRNA in macrophage-like cells. MMP-9 immunoreactivity was detected in the polymorphonuclear leukocytes and macrophage-like cells on days 3-8. MMP-9 proteolytic activity was observed only on days 3-8. The active form of the MMP-2 increased up to day 14, whereafter it remained at a constant level, whereas latent MMP-2 did not show any apparent changes during the experimental period. We conclude that MMP-2 is important during the prolonged remodelling phase, whereas the gelatinolytic activity of MMP-9 was demonstrated only in early wound healing, and the MMP-9 gene is upregulated when the granulation tissue matures.

Absence of integrin alpha1beta1 in the mouse causes loss of feedback regulation of collagen synthesis in normal and wounded dermis.

Integrin alpha1beta1 is a collagen receptor predominantly found in mesenchymal tissues. Mice lacking this receptor are viable. We have previously suggested that alpha1beta1 might participate in the down-regulation of collagen gene expression observed in cells suspended inside collagen gels. The results presented here demonstrate that integrin alpha1beta1 acts as a feedback regulator of collagen synthesis both in vitro and in vivo. Firstly, alpha1 null animals show a higher rate of collagen synthesis in the dermis in vivo. Secondly, fibroblasts derived from alpha1 null cutaneous wounds show a reduced sensitivity to collagen gel induced downregulation of collagen mRNA synthesis, as compared to their wild-type counterparts. An increase in collagenase synthesis is also seen in the alpha1 null dermis and in collagen gel suspended fibroblasts. While dermal thickness is normal in the alpha1 null animals, an increase is seen in skin thickness of alpha1 null but not alpha1 heterozygote animals on a background of collagenase resistant collagen. Increased expression of both collagen and collagenase mRNA are seen in experimental granulation tissue in alpha1 null animals, but their ultimate accumulation of collagen is normal, probably due to non alpha1 dependent paracrine regulators of collagen turnover.

Cloning of cDNA for Rat Pro α1(V) Collagen mRNA. Expression Patterns of Type I, Type III and Type V Collagen Genes in Experimental Granulation Tissue

A cDNA clone for rat pro α1(V) collagen mRNA was constructed using PCR amplification, with primers based on human and hamster COL5A1 gene sequences. The clone pRCVA1 is 560 nucleotides long and it encodes for the carboxy propeptide of type V procollagen. Homology shared with type I collagen sequence was 64%, with type II collagen 65% and with type III collagen 61%. To evaluate the spatial and temporal expression of type V collagen mRNA in wound healing model, subcutaneously implanted viscose cellulose sponges in rats were used to induce granulation tissue formation. Analyses on granulation tissue were carried out on days 5, 8,14, 21, 30, 59 and 84. Specific cDNA probes to pro α1(I), pro α1(III) and pro α1(V) collagen mRNA were used in slot blot, Northern and in situ hybridization. Type I collagen gene expression was upregulated at the initial stage of wound healing, type III collagen gene expression was constant and from the day 14 onwards type I and III collagen gene expressions were at the same level. Type V collagen gene expression was seen at every time point studied but at a considerably lower level than type I and III collagens. In situ hybridization showed that type V collagen was expressed in two different cell types. In conclusion, type V collagen was expressed in the wound healing model from at least day 5 onwards and it was synthesized by fibroblast-like and rounded cells.

Type V Collagen in Experimental Granulation Tissue

To evaluate the spatial and temporal expression of type V collagen in a wound healing model, subcutaneously implanted viscose cellulose sponges in rats were used to induce granulation tissue formation. Analyses on granulation tissue were carried out on days 3, 5, 8, 14, 21, 30, 59 and 84. Acid soluble collagens were extracted and the relative amount of type V collagen was quantified by SDS-PAGE. Specific antibodies to type I, III and V collagens were used in immunohistochemistry and specific RNA probes to proα 1(1), proα1(IH) and proα1(V) collagen in in situ hybridization. Type V collagen content increased relative to type I and III collagens up to day 8 and remained at the same level for up to the three months. Type V collagen was expressed strongly in blood vessel walls as seen in immunohistochemistry. In situ hybridization showed that all of the three types of collagen were expressed mostly in fibroblast-like cells and also in rounded cells, especially type V collagen. In conclusion, type V collagen was seen in the wound healing model in increasing amounts from day 3 onwards, its localization being highly associated with blood vessels in granulation tissue and it was synthesized by fibroblast-like and rounded cells.

The effects of interleukin-1 and prostaglandin E2 on accumulation of collagen and steady-state levels of pro alpha 1(I) collagen messenger RNA in experimental granulation tissue in rats.

The effects of human interleukin 1 beta (IL-1 beta) and prostaglandin E2 (PGE2) on experimental granulation tissue in rats and on granulation tissue cells in culture were studied. IL-1 beta and PGE2 were injected into subcutaneously implanted sponges during the first 3 days after implantation. The rate of collagen synthesis in fibroblasts was measured as synthesis of protein-bound 3H-hydroxyproline. The steady-state levels of pro alpha 1(I) and pro alpha 1(III) collagen chain mRNAs were estimated by Northern transfer analyses. By 7 days postoperatively IL-1 beta had decreased the hydroxyproline content of granulation tissue. PGE2 decreased non-significantly the amounts of hydroxyproline, but the steady-state levels of pro alpha 1(I) and pro alpha 1(III) collagen chain mRNAs were slightly elevated. In IL-1 beta-treated fibroblast cultures collagen production decreased by 15% and following PGE2 treatment by 34% compared with the controls. The latter effect could be abolished by indomethacin. Indomethacin alone stimulated collagen production by 40%. In vivo IL-1 decreases the formation of normal granulation tissue. This effect may be partly due to stimulation of secretion of PGE2.

Cloning of cDNA for rat proα1(III) collagen mRNA: Different expression patterns of type I and type III collagen and fibronectin genes in experimental granulation tissue

A cDNA clone for rat proα1(III) collagen mRNA was isolated from a cDNA library constructed for poly(A) + RNA from 15-day experimental granulation tissue. Two clones, pRGR1 and pRGR5, were characterized by restriction mapping and sequencing. Comparison with human type III collagen sequences revealed 92% identity at the level of translated amino acids, and 88% identity at nucleotide level in the coding region. In the 3′-untranslated sequence the identity was even higher (90%). The clones were used together with cDNA clones for type I collagen chains, fibronectin and γ-actin to study the expression of the corresponding mRNAs during the development of experimental sponge-induced granulation tissue in rats. These studies revealed a marked activation of type I and type III collagen genes during the second week of granuloma development followed by a transient reduction in their levels during the third week. The mRNA levels for both collagen types remained relatively unchanged from day 25. The molar ratio of proα1(III) and proα1(I) collagen mRNAs was at a maximum on day 6, and then decreased to reach a plateau by the end of the third week. Fibronectin mRNA levels were found to increase slower; the maximum value was reached during the fifth week of granuloma development. The mRNA levels of γ-actin increased continuously up to the end of the fourth week, thus following the cellular maturation of the tissue.

Reduction of collagen degradation in experimental granulation tissue by vitamin E and selenium

The importance of antioxidants in inflammatory tissues has been studied in vivo. Degradation of homologous 3H-collagen powder by experimental granulation tissue induced by cellulose sponges in the rat was monitored as the radioactivity excreted in urine. By administering pharmacological doses of both vitamin E and selenium subcutaneously and by injection into sponges implanted subcutaneously, this breakdown of collagen was reduced. Injections in the sponges also arrested the maturation of the granulation tissue. Vitamin E and selenium are potential inhibitors of the free oxygen radicals from phagocytic inflammatory cells. It is therefore suggested that these radicals may play a rôle in the collagen destruction by granulation tissues, as in periodontitis.

Epidermal growth factor increases granulation tissue formation dose dependently

Recent studies have shown that epidermal growth factor (EGF) stimulated the rate of formation of granulation tissue in a model of wound repair (A. Buckley, et al., Proc. Nat. Acad. Sci. USA 82: 7340, 1985). Because pharmacologic doses of EGF were used previously, the relationship of EGF concentration to physiologic effects was determined in this study. Rats were implanted with subcutaneous polyvinyl alcohol sponges containing slow-release pellets formulated to release 0, 0.1, 1.0, or 10 μg of EGF/day. Tissue response was judged by the degree of histologic organization and vascularity, as well as several quantitative parameters: wet weight, hydroxyproline content, protein content, and DNA concentration. Each of these parameters showed consistent increases by Day 5 after implantation, when inflammation and edema had subsided. Compared with placebo controls, hydroxyproline (collagen) content was significantly increased by as little as 1 μg/day of EGF, and DNA content was significantly increased by all dose levels of EGF. Endogenous EGF concentration in experimental granulation tissue was found to be fairly constant (30–40 ng/g wet wt); however, the increasing cellularity of the sponges may have reduced the local concentration of free EGF to low levels. Pellets releasing as little as 4 ng/hr of EGF into the surrounding tissue were able to accelerate wound healing, suggesting that the availability of this growth factor may be a rate-limiting step in wound repair.

Progesterone metabolism by rat oral mucosa. III. Participation of granulation tissue and fibroblasts.

The metabolism of progesterone by rat granulation tissue was studied. Experimental granulation tissue was produced by implanting viscose‐cellulose sponges beneath the dorsal skin of female and male rats for 21 days. The granuloma capsules and fibroblasts in the sponges were homogenized, and mitochondrial, microsomal and soluble fractions were prepared with differential centrifugation. The subcellular Preparations were incubated with 4‐14C‐progesterone and NADPH for 30 min at PH 7.4 and 37°C. The metabolites were identified with column and multiple thinlayer chromatography and radioautography and quantitated with liquid scintillation counting. The granulation tissue and fibroblasts showed much less activity in metabolizing progesterone than the gingival tissue of either sex. As reported earlier, gingival tissues contain Δ4‐5α‐ and,Δ4‐5β‐steroid hydrogenases and 3α‐, 3β‐, 20α‐ and 20β‐hydroxysteroid dehydrogenases. The granulation tissue and fibroblasts lack 3 β‐hydroxysteroid dehydrogenase activity. In addition, the fibroblasts show no 20 β‐hydroxysteroid dehydrogenase activity. The enhanced metabolism of progesterone during gingival inflammation is thus probably not due to the formation of granulation tissue.

Role of granulation tissue and fibroblasts in gingival testosterone metabolism in the rat in vitro

The metabolism of testosterone by experimental granulation tissue, fibroblasts and the oral mucosa of rats of both sexes was studied. The experimental granulation tissue was produced by implanting viscose-cellulose sponges beneath the dorsal skin of female and male rats for 21 days. The granuloma capsules, fibroblasts in the sponges and the oral mucosae were homogenated. Mitochondrial, microsomal and soluble fractions were incubated with [4- 14C]testosterone and NADPH for 30 min at pH 7.4 and 37°C. The metabolites were identified with column and TLC and radioautography and quantified with liquid scintillation counting. The experimental granulation tissue and fibroblasts of both sexes showed less activity in metabolizing testosterone than the gingival tissue. The tissues were shown to contain 3α-, 3β- and 17β-hydroxysteroid dehydrogenase and 5α- and 5β-steroid hydrogenase activities. The activities of the enzymes in the oral mucosae were higher than in the experimental granulation tissue and fibroblasts.

Effect of epidermal growth factor (EGF) on experimental granulation tissue

Subcutaneously implanted cylindrical hollow viscose cellulose sponges were used to study the effects of locally applied epidermal growth factor (EGF) on developing granulation tissue in rats. In the first set of experiments the test implants were treated with a single injection of a solution containing 5 μg of EGF in 0.5% albumin while the control implants were treated correspondingly with the carrier solution only. In the second set of experiments the injections of both test and control implants were repeated daily. Analyses of wound fluid and granulation tissue in the sponge cylinders were carried out 10 days after implantation. After single application of EGF no essential differences were detected in wound fluid prostaglandin E 2 levels or various components of granulation tissue between the groups. After daily application, however, a stimulatory effect of EGF on granulation tissue formation was observed: cellularity increased, as evidenced by the elevated amounts of nucleic acids, and accumulation of collagen and glycosaminoglycans was enhanced.