Kinetic study of early regenerative effects of RGTA11, a heparan sulfate mimetic, in rat craniotomy defects.

We previously reported that RGTA, a synthetic heparan sulfate mimetic, induces almost complete closure of craniotomy defects one month after surgery in adult rats. RGTA-treated wounds showed features suggesting unusual cell and matrix interactions reminiscent of developmental events. As healing success or failure is determined shortly after wounding, we examined early events in RGTA-treated wounds. Collagen plasters soaked in a solution of RGTA11 (1.5 Microg per piece) or saline (control) were implanted in rat craniotomy defects. Seven control and seven treated rats were killed daily from days 1 to 7 after surgery. The lesions and adjacent tissues were sampled and processed for morphometry. A layer of type III collagen along the dura mater (DM) thickened up to day 5 in RGTA-treated wounds (p < 0.05 vs day 1), but became thinner in control wounds. Alkaline phosphatase-positive osteoprogenitor cells were detected on day 1 in this layer. Their number increased, and they migrated toward the mid-sagittal sinus and to connective tissue adjacent to the sinus, where they aggregated and differentiated into osteoblasts, forming bone nodules on day 6. These features were not seen in control wounds. Angiogenesis was significantly enhanced in RGTA-treated wounds, especially near the sinus. In vitro, bovine bone endothelial (BBE) cell proliferation was inhibited by RGTA11 in a concentration-dependent manner. In contrast, RGTA11 strongly enhanced the effect of fibroblast growth factor-2 on BBE cell proliferation. These results show that RGTA11, possibly by interacting with heparin-binding growth factors, elicits vascular reactions accompanying the recruitment of a large pool of committed osteoprogenitors from the DM. The DM and the sinus appear to be important centers of organization for craniotomy defect healing. RGTA probably creates an environment that starts a program of directing healing towards bone formation and defect closure.

Cortisol inhibits hepatocyte growth factor/scatter factor expression and induces c-met transcripts in osteoblasts.

Hepatocyte growth factor/scatter factor (HGF/SF) is expressed by osteoblasts and has important effects on repair and bone remodeling. Because glucocorticoids regulate these two functions, we tested the effects of cortisol on the expression of HGF/SF and c-met, the protooncogene encoding the HGF/SF receptor, in cultures of osteoblast-enriched cells from 22-day fetal rat calvariae (Ob cells). Cortisol decreased HGF/SF mRNA levels and diminished the induction of HGF/SF transcripts by fibroblast growth factor-2 (FGF-2) and platelet-derived growth factor BB (PDGF BB). Cortisol also decreased FGF-2 and PDGF BB-induced HGF/SF mRNA and polypeptide levels in MC3T3 cells. In contrast, cortisol enhanced the expression of c-met transcripts in Ob cells. Cortisol did not modify the half-life of HGF/SF or of c-met mRNA in transcriptionally arrested cells, and it increased the rate of transcription of c-met. In conclusion, cortisol decreases HGF/SF transcripts in Ob cells and enhances c-met expression transcriptionally. The effects of cortisol on HGF/SF could be relevant to its inhibitory actions on bone formation and repair.

Effects of heparan-like polymers associated with growth factors on osteoblast proliferation and phenotype expression.

Heparan-like polymers derived from dextran, named RGTA, were shown to stimulate bone repair in different bone defect models. Like heparin and heparan sulfates, RGTA potentiate in vitro the biological activities of heparin-binding growth factors (HBGFs), such as fibroblast growth factor (FGF), by stabilizing them against denaturations and by enhancing their binding with cellular receptors. RGTA were postulated to stimulate bone healing by interacting with HBGFs released in the wound site and, subsequently, by promoting the proliferation and/or differentiation of cells implicated in this process. We examined the effects of RGTA alone and associated with HBGFs on MC3T3-E1 osteoblastic cell proliferation and differentiation. RGTA inhibited cell proliferation, as measured by [3H]-thymidine incorporation into DNA. They enhanced the inhibition of DNA synthesis caused by transforming growth factor-beta (TGF-beta1) and bone morphogenetic protein-2 (BMP-2). RGTA alone increased the alkaline phosphatase and parathyroid hormone-responsive adenylate cyclase activities in MC3T3. RGTA enhanced the stimulation of the alkaline phosphatase activity induced by BMP-2 and decreased or suppressed the inhibition caused by TGF-beta1 and FGF-2. Furthermore, RGTA increased the response to parathyroid hormone stimulated by BMP-2. In conclusion, RGTA stimulate the expression of osteoblast phenotype features alone or in association with HBGFs. The ability to promote the differentiation of bone-forming cells is a potential explanation of the stimulating effect of RGTA on bone repair.

A single low dose of RGTA, a new healing agent, hastens wound maturation and enhances bone deposition in rat craniotomy defects.

RGTA, a new family of dextran-derived healing agents, promotes the repair of various tissues, including bone. In this study, we examined whether a dose of RGTA lower than in our previous studies could still modify the healing pattern in craniotomy defects. In 24 rats, two defects (3 mm diameter) were drilled on either side of the calvaria sagittal suture. The right defect was filled with a piece of collagen soaked with RGTA in phosphate-buffered saline (PBS; 4 microg/ml), and the left one with collagen soaked in PBS only. After 7, 14 and 21 days, the calvaria were removed and processed for histometry. On day 7, in contrast with the control defects, the treated sites were inflammation-free and centripetal bone plates had started to grow. By day 14, the bone filling was significantly enhanced in the treated defects (+290%, p<0.05), and isolated bone nodules had formed within the fibrous connective tissue (= fibrous hammock) joining the defect edges. The hammock had already differentiated by day 7 in all the RGTA-treated defects, and it was significantly thicker on days 14 (+190%, p<0.05) and 21 (+139%, p<0.05). The colonization of the hammock by mast cells was increased in the treated sites (+320%, p<0.05 on day 21). On day 7, most of the bony edges of the treated defects had been resorbed by osteoclasts, while the process only started in the controls. These data indicate that a low dose of RGTA modified the cascade of events occurring at the initial stages of repair, so that the tissular maturation of the treated defects was more rapid. In fact the use of RGTA in the wounds provoked a shift from a fibrous repair as seen in the controls, to a bone reconstruction favoring defect closure.

Fibroblast growth factor-2 induces hepatocyte growth factor/scatter factor expression in osteoblasts.

Hepatocyte growth factor/scatter factor (HGF/SF) is a multifunctional growth factor with a major role in tissue morphogenesis and repair. It stimulates the proliferation of cells of the osteoblast and osteoclast lineages. Mitogenic factors playing a role in fracture repair may act by regulating HGF/SF expression or activity in bone-forming cells. We investigated the effect of fibroblast growth factor-2 (FGF-2) on the expression of HGF/SF and its receptor, encoded by c-met, in the MC3T3-E1 osteoblastic cell line. MC3T3-E1 cells expressed low levels of HGF/SF messenger RNA (mRNA), which were markedly increased by FGF-2 in a dose- and time-dependent manner. FGF-2 also induced HGF/SF polypeptide synthesis. The stimulation of HGF/SF mRNA expression by FGF-2 was blocked by cycloheximide, a protein synthesis inhibitor, but not by DNA or prostaglandin synthesis inhibitors. FGF-2 increased the rate of HGF/SF gene transcription by approximately 2-fold, as determined by nuclear run-on assays, and did not modify the decay of HGF/SF mRNA in transcriptionally arrested cells. FGF-2 also caused a dose- and time-dependent stimulation of c-met mRNA. In conclusion, FGF-2 induces HGF/SF expression in osteoblasts and may promote HGF/SF activity by increasing the expression of its receptor. Through these mechanisms, HGF/SF could mediate FGF actions on bone repair.

Cortisol enhances the expression of mac25/insulin-like growth factor-binding protein-related protein-1 in cultured osteoblasts.

Glucocorticoids inhibit the synthesis of insulin-like growth factor I (IGF-I) and regulate the expression of IGF-binding proteins (IGFBPs) in osteoblast cultures. IGFBP-related protein-1 (IGFBP-rP1), the product of the mac25 gene, binds IGF-I, IGF-II, and insulin, and we postulated that glucocorticoids regulate IGFBP-rP1 synthesis in osteoblasts. We tested the expression of mac25/IGFBP-rP1 in cultures of osteoblast-enriched cells from 22-day-old fetal rat calvariae (Ob cells). Cortisol treatment at 10 nM to 1 microM for 24-48 h caused a time- and dose-dependent increase in mac25/IGFBP-rP1 messenger RNA (mRNA) levels in Ob cells. Cycloheximide at 3.6 microM did not alter mac25/IGFBP-rP1 transcripts in control or cortisol-treated cells. Cortisol did not modify the decay of mac25/IGFBP-rP1 mRNA in transcriptionally arrested Ob cells and increased the rate of IGFBP-rP1 transcription as determined by nuclear run-on assays. Retinoic acid also increased mac25/IGFBP-rP1 mRNA levels, but 17beta-estradiol, testosterone, 5alpha-dihydrotestosterone, progesterone, and 1,25-dihydroxyvitamin D3 did not. In conclusion, cortisol stimulates mac25/IGFBP-rP1 expression in Ob cells by transcriptional mechanisms. As IGFBP-rP1 binds and possibly modifies the effects of IGFs and insulin, its increased expression could be relevant to the inhibitory actions of cortisol in bone.

Heparan-like molecules induce the repair of skull defects.

Heparin-binding growth factors (HBGFs) are known to stimulate bone repair when applied to bone lesions. Nevertheless, successful treatments are obtained with high protein doses since HBGFs are rapidly degraded in situ by multiple proteolytic activities associated with the inflammatory period of tissue healing. Like heparin or heparan sulfates, heparan-like molecules, named carboxymethyl-benzylamide-sulfonated dextrans (CMDBS), are known to potentiate fibroblast growth factor activities by stabilizing them against pH, thermal or proteolytic denaturations, and by enhancing their binding with cell surface receptors. We have postulated that CMDBS stimulate in vivo bone healing by interacting with endogenous HBGFs, spontaneously released in the wounded site. The effect of CMDBS on bone repair was studied in a skull defect model in rats by computer-assisted radio-morphometry and histomorphometry. Single application of CMDBS in a collagen vehicle to skull defects induced a dose-dependent increase in bone defect closure and new bone formation after 35 days. Complete bony bridging occurred in defects treated with 3 micrograms CMDBS, whereas bone formation was not observed in vehicle-treated defects which contained only dense fibrous connective tissue between the defect margins. These results indicate that heparan-like molecules, such as CMDBS, are able to induce bone regeneration of skull defects. This action is possibly mediated by potentiation of endogenous growth factor activities and/or by neutralization of proteolytic activities.

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 analogs of sulfate heparanes, used as osseous cicatrizing agents]. / Les CMDBS, analogues fonctionnels des héparanes sulfates, utilisés comme agents de la cicatrisation osseuse.

Several Heparin-Binding Growth Factors (HBGFs) are known to play an important role in bone repair. When osseous tissue is injured, an important increase of protease activities and a massive release of HBGFs occur. The local increase in HBGFs content at the wounded site, produced by a release of this factors from cells implicated in haemostasis and inflammatory reaction and from extracellular matrix associated heparan sulfate proteoglycans (HSPGs), seems to be a crucial step in bone healing. The proteolysis associated with the tissue injury probably limits the growth factors activities at the wound site. In order to define the bone healing potential of molecules that would be able to protect HBGFs against proteolytic activation, we studied the effect of derived dextrans, named carboxymethyl-benzylamide-sulfonated dextrans (CMDBS), behaving as heparan like molecules, in 5 mm in diameter skull trepaned defects in young adult rats. In this model CMDBS induced an important bone regeneration in a dose dependent manner while controls were not repaired. In CMDBS treated animals the defects were repaired and contained a tissue of normal appearance; in several treated animals the sagittal suture, initially removed by the trephination, was restored. This remarkable bone healing potential of CMDBS may result from the capacity to protect the endogenous HBGFs from proteolysis and to modulate their biological activities, in a similar manner to that observed for fibroblast growth factors and HSPGs. CMDBS represent a new form of bone healing agents, which have the advantage of being produced by a controlled chemical synthesis, and of avoiding the use of exogenous growth factors because of their capacity to enhance the bone healing potential of the endogenous growth factors.