New healing agent for colonic anastomosis.

This study evaluated the healing property, on colonic anastomoses, of a new compound termed RGTA11 (standing for "regenerating agent," a dextran derivative with heparin-like properties). Colonic anastomoses were performed in 183 adult rats after dipping both ends of the colon in solutions containing RGTA11. The anastomoses were made end-to-end on a single plan, 8 sutures. Healing was evaluated mechanically by the bursting pressure in 108 animals and histological analysis in 75. Results indicated that after 48 h RGTA11-treated animals presented a twofold increased resistance to anastomoses breakage (P < 0.01) over nontreated (saline buffer) animals. After 96 h and until day 7 there was no longer a difference between study and control animals. Although this difference in breakage was not readily observed at histological level, results suggest that RGTA11 could be used to aid colonic anastomosis healing. RGTA11 is of potential clinical interest in this regard since complications that are known to occur postoperatively result from early leakage.

New concepts in tissue repair: skin as an example.

Several heparin-binding growth factors (HBGFs) are thought to play a key role in natural processes of tissue regeneration or repair after release from inflammatory or circulating cells and extracellular matrix-associated heparan sulfate proteoglycosaminoglycans. To clarify how the bioavailability of these HBGFs can help regulate wound-healing processes, we studied the healing effect of various chemically substituted dextrans (RGTA) selected for their affinity for HBGFs. One member of the RGTA family, RGTA11, obtained by substitution of carboxymethyl (CM), benzylamide (B) and benzylamide sulfonate (S) groups in a proportion of 110% (CM), 2.6% (B) and 36.5% (S) respectively was used in these studies. RGTA11 may potentiate the biological activity of fibroblast growth factors 1 and 2 and protect them against heat or pH inactivation and proteolytic degradation. RGTA11 was tested in a rat punch-biopsy skin-healing model for its ability to enhance wound repair. Wounds were filled with collagen plaster alone or soaked with RGTA, and skin regeneration was studied by histological analysis. In collagen plaster, RGTA11 affected both the kinetics and quality of restored skin. It seems likely that endogenous growth factors naturally released during the regeneration process are trapped and protected against natural proteases, thereby preserving their ability to stimulate tissue repair. Since most known growth factors have a nearly ubiquitous distribution and blind to heparin, our hypothesis was verified by studying the ability of RGTA to induce repair in damaged tissue. We demonstrated the RGTA could stimulate wound repair in various models, including bone, muscle, nerve, cornea and colonic anastomosis. The data presented here concern wound-healing in a deep skin model and suggest that heparan-like biopolymers constitute a new family of tissue-repair agents with a wide variety of potential uses. The efficiency of this approach in cases in which impaired healing is associated with a pathology, as in diabetes, remains to be determined.

Heparin-like polymers derived from dextran enhance colonic anastomosis resistance to leakage.

A new tissue repair agent, RGTA11, is described for its ability to enhance colonic anastomosis repair and resistance to leakage. RGTA11 is a dextran derivative containing 110% carboxymethyl groups, 2.6% carboxymethyl benzylamide groups, and 36.6% carboxymethyl benzylamide sulfonate groups. RGTA11 was deemed efficient to protect the heparin-binding growth factors FGF2 against trypsin digestion. By this property RGTA11 mimicked heparin or heparan sulfate. We have also found that RGTA11 protected TGF beta 1 against trypsin digestion while heparin did not. RGTA11 was then tested in an in vivo wound-healing model of colonic anastomosis. Our results indicate that after 48 h, RGTA11- or RGTA11/FGF-2-treated animals presented a resistance of the anastomosis to leakage which was increased twofold (p < 0.05) over untreated controls. After 96 h and until day 7 there was no more difference with control animals. Our results suggest that RGTA11 presents potential clinical interest by preventing earlier leakage of colonic anastomosis.

FGF protection and inhibition of human neutrophil elastase by carboxymethyl benzylamide sulfonate dextran derivatives.

Several derivatized dextrans (DxD) containing defined percentage of carboxymethyl, carboxymethyl benzylamide and carboxymethyl benzylamide sulfonate groups have been shown to stimulate tissue repair in various in vivo models including skin, bone, muscle and cornea. These selected DxD were also shown to mimic heparin or heparan sulfate by their ability to interact with, stabilise and protect the heparin-binding growth factor of the fibroblast growth factor family against trypsin digestion (Tardieu et al., J. Cell. Physiol. 1992; 150: 94). The wound healing action of these DxD was explained by postulating that the endogenously released heparin-binding growth factors could be protected within the wound. To further understand the action of these DxD on tissue repair, we have studied their effect on the human neutrophil elastase (HNE) activity, one of the proteases involved in wound repair. These DxD inhibited HNE in an hyperbolic non-competitive manner. Extent of HNE inhibition by DxD increased with their molecular weight and benzylamide sulfonate substitution levels. One DxD, RGT11, was the best inhibitor (Ki 40 pM) and efficiently inhibited FGF-2 proteolysis by HNE, restoring its growth-promoting activity towards human skin fibroblasts. The data contribute to a better understanding of the wound-healing property and anti-inflammatory activity of these polymers.

Modulation of cranial bone healing with a heparin-like dextran derivative.

Substituted dextran polymers have been shown to bind growth factors and protect them from enzymatic degradation. Using this information, other researchers have been able to use these substituted dextrans to enhance the healing of bone in an environment where bone would otherwise not regenerate. We used substituted dextran polymers to evaluate their ability to accelerate the healing of cranial bone in a rabbit model. We were able to document a more rapid rate of healing and demonstrate micrographic evidence to support that conclusion. Possible mechanisms are postulated.

Inhibition by dextran derivatives of FGF-2 plasmin-mediated degradation.

In previous work we have shown that some new regenerating agents (RGTAs), molecules which correspond to some dextran derivatives (DxD) containing defined amounts of carboxymethyl (CM), benzylamide (B) and benzylamide sulfonate (BS) groups, were able to stimulate tissue repair when applied at the site of injury. Based on in vitro studies showing that these DxD could interact and protect heparin binding growth factors (HBGFs), we postulated that DxD could also act in vivo by protecting endogenously released HBGFs against protease degradation. We now present data demonstrating that human plasmin (HP1), one of the known proteases involved in extracellular matrix remodelling and in the local activation of some growth factors is specifically inhibited by some specific DxD. The most efficient compounds for inhibiting the amidolytic activity were substituted by all functions with IC50 at 0.26 microM for RGTA11 (a DxD obtained from a 40,000 Da dextran containing 110% of CM, 2.5% of B and 36.5% of BS units and with IC50 at 1.1 microM for RGTA10 (derived from 10,000 Da dextran and containing 110% of CM, 0% of B and 27.3% of BS). Compounds which were substituted with only one or two functions were less effective. The degradation of FGF-2 by HP1 was analyzed by SDS-PAGE and by measuring its residual growth promoting activity using a bioassay on human skin fibroblasts. In this assay, RGTA11 at a concentration of 1 microM could inhibit by 80-100% FGF-2 degradation induced by HP1 treatment. In conclusion, the inhibitory activity of some DxD towards HP1 as well as the ability of these DxD to protect FGF-2 against this proteinase could partially explain its beneficial influence on extracellular matrix remodelling following tissue injury.

New approaches to tissue regeneration and repair.

Several Heparin Binding Growth Factors (HBGFs) are thought to play a key role in the natural processes of tissue regeneration or repair after being released by neighbouring, inflammatory or circulating cells as well as from extracellular matrix associated heparan sulfate proteoglycosaminoglycans. In order to better understand how the bioavailability of these HBGFs can take part in the regulation of the wound healing processes, we have studied the healing effect of various chemically substituted dextrans (CMDBS) selected for their affinity for HBGFs, alone and in association with HBGFs. The CMDBS was obtained by substitution of methylcarboxylic (CM), benzylamide (B) and benzylamine sulfonate (S) groups in proportion of 83%, 23% and 13% respectively for CMDBS K that we have further used (Mauzac et al., 1985 Biomaterials. 6: 61-63). CMDBS K could 1: potentiate the biological activity of 1 or 2 FGFs, 2: protect 1 and 2 FGFs against thermal or pH inactivation, 3: protect a and b FGFs against proteolytic degradation (Tardieu et al., 1992 J. Cell. Physiol. 150: 194-203). CMDBS K was tested alone in cutaneous and bone wound healing models and for its ability to stabilize FGFs. Rats were punched and skin regeneration was studied by morphometric and histological analysis. The wounds (6 mm diameter) were filled with collagen plaster alone or soaked with CMDBS. CMDBS K in collagen plaster was able to induce a remarkable effect both on the kinetics and on the quality of the restored skin. These results suggest that endogenous growth factors naturally released during the regeneration process could be trapped, protected and released by CMDBS.(ABSTRACT TRUNCATED AT 250 WORDS)

FGFs and their receptors, in vitro and in vivo studies: new FGF receptor in the brain, FGF-1 in muscle, and the use of functional analogues of low-affinity heparin-binding growth factor receptors in tissue repair.

Several heparin-binding growth factors (HBGFs) are thought to play a key role in the natural processes of tissue homeostasis, regeneration or repair. The HBGFs are active upon release from neighbouring inflammatory or circulating cells, as well as upon release from heparan sulfate proteoglycosaminoglycans that are associated with the extracellular matrix (ECM). To better understand the physiological role of these HBGFs, we have focused our effort on studying a subset of HBGFs, namely FGF-1 and FGF-2 and their receptors. We present the purification and characterisation of a new form of heparin-binding FGF receptor from adult bovine brain (Perderiset et al., 1992). This receptor has now been purified to homogeneity. Ligand blot and cross-linking experiments performed with labeled FGF-1 or FGF-2 revealed 80-kd and 130-kd bands. Preliminary sequence information indicates that receptor is different from the receptors, FGFR-1 to -4, but it may be related the cysteine-rich-FGF receptor (CFR). We have previously shown that FGF-1, but not FGF-2, is specifically expressed in myoblastic satellite cells during the proliferating phase preceding myoblast alignment and fusion. We have now transfected primary cultures of rat myoblastic satellite cells with FGF-1 cDNA and expressed this growth factor constitutively. The transfected cells were no longer able to form myotubes. Transfection with antisense FGF-1 induced myotube formation suggesting that endogenous expression of FGF-1 is associated with myoblastic cell differentiation. Numerous studies have concluded that the ECM represents a natural reservoir for various HBGFs.(ABSTRACT TRUNCATED AT 250 WORDS)

CMDBS, functional analogue of heparin sulfate as a new class of corneal ulcer healing agents.

Soluble dextran polymer derivatives (CMDBSs) are originally synthesized as heparin-like plasma substitutes. Some of them mimic heparin in its interactions and stabilize, protect and facilitate actions of heparin binding growth factors. The wound healing activity of one specific CMDBS was studied in a model of corneal ulcer on the rabbit eye and compared with the activity of basic fibroblast growth factors (bFGF) added alone or in association with CMDBS. Total reepithelization was observed with bFGF + CMDBS, bFGF alone and CMDBS alone after, respectively, 3.8 +/- 0.78, 4.3 +/- 0.67 and 4.4 +/- 0.51 days. All treatments were efficient if compared with eyes treated with saline (p < 0.0001). The grade of significance of the applied treatments was as follows: bFGF + CMDBS > bFGF > CMDBS > saline. Our study pinpoints that some specific CMDBS are as potent agents as bFGF for corneal ulcer healing, and can therefore be proposed for therapeutic use.