Enhanced boundary lubrication properties of engineered menisci by lubricin localization with insulin-like growth factor I treatment

In this study we analyzed the effects of IGF-I on the boundary lubricating ability of engineered meniscal tissue using a high density collagen gel seeded with meniscal fibrochondrocytes. Biochemical, histological, immunohistochemical, and tribological analyses were carried out to determine a construct's ability to functionally localize lubricin. Our study revealed that supplementation with IGF-I enhanced both the proliferation of cells within the construct as well as enhanced the anabolic activity of the seeded cells. Growth factor supplementation also facilitated the localization of ECM constituents (i.e. fibronectin and type II collagen) near the tissue surface that are important for the localization of lubricin, a boundary lubricant. Consequently, we found localized lubricin in the constructs supplemented with IGF-I. Tribologically, we demonstrated that lubricin serves as a boundary lubricant adsorbed to native meniscal surfaces. Lubricin removal from the native meniscus surface increased boundary friction coefficient by 40%. For the engineered constructs, the lubricin localization facilitated by growth factor supplementation also reduced friction coefficient by a similar margin, but similar results were not evident in control constructs. This study demonstrates that the use of growth factors in meniscal tissue engineering can enhance tribological properties by facilitating the localization of boundary lubricants at the surface of engineered tissue.     

Platelet-derived growth factor and transforming growth factor-beta enhance tissue repair activities by unique mechanisms

Platelet-derived growth factor (PDGF) and transforming growth factor-beta (TGF-beta) markedly potentiate tissue repair in vivo. In the present experiments, both in vitro and in vivo responses to PDGF and TGF-beta were tested to identify mechanisms whereby these growth factors might each enhance the wound-healing response. Recombinant human PDGF B-chain homodimers (PDGF-BB) and TGF-beta 1 had identical dose-response curves in chemotactic assays with monocytes and fibroblasts as the natural proteins from platelets. Single applications of PDGF-BB (2 micrograms, 80 pmol) and TGF-beta 1 (20 micrograms, 600 pmol) were next applied to linear incisions in rats and each enhanced the strength required to disrupt the wounds at 5 d up to 212% of paired control wounds. Histological analysis of treated wounds demonstrated an in vivo chemotactic response of macrophages and fibroblasts to both PDGF-BB and to TGF-beta 1 but the response to TGF-beta 1 was significantly less than that observed with PDGF-BB. Marked increases of procollagen type I were observed by immunohistochemical staining in fibroblasts in treated wounds during the first week. The augmented breaking strength of TGF-beta 1 was not observed 2 and 3 wk after wounding. However, the positive influence of PDGF-BB on wound breaking strength persisted through the 7 wk of testing. Furthermore, PDGF-BB-treated wounds had persistently increased numbers of fibroblasts and granulation tissue through day 21, whereas the enhanced cellular influx in TGF-beta 1-treated wounds was not detectable beyond day 7. Wound macrophages and fibroblasts from PDGF-BB-treated wounds contained sharply increased levels of immunohistochemically detectable intracellular TGF-beta. Furthermore, PDGF-BB in vitro induced a marked, time-dependent stimulation of TGF-beta mRNA levels in cultured normal rat kidney fibroblasts. The results suggest that TGF-beta transiently attracts fibroblasts into the wound and may stimulate collagen synthesis directly. In contrast, PDGF is a more potent chemoattractant for wound macrophages and fibroblasts and may stimulate these cells to express endogenous growth factors, including TGF-beta, which, in turn, directly stimulate new collagen synthesis and sustained enhancement of wound healing over a more prolonged period of time.     

Role of platelet-derived growth factor in wound healing

Platelet-derived growth factor (PDGF) is a potent activator for cells of mesenchymal origin. PDGF stimulates chemotaxis, proliferation, and new gene expression in monocytes-macrophages and fibroblasts in vitro, cell types considered essential for tissue repair. Therefore, we analyzed the influence of exogenously administered recombinant B chain homodimers of PDGF (PDGF-BB) on two experimental tissue repair paradigms, incisional and excisional wounds. In both types of wounds, as little as 20-200 picomoles applied a single time to wounds significantly augmented the time dependent influx of inflammatory cells and fibroblasts and accelerated provisional extracellular matrix deposition and subsequent collagen formation. In incisional wounds, PDGF-BB augmented wound breaking strength 50-70% over the first 3 weeks; in excisional wounds, PDGF-BB accelerated time to closure by 30%. PDGF-BB exaggerated, but did not alter, the normal course of soft tissue repair, resulting in a significant acceleration of healing. Long term observations established no apparent differences between PDGF-BB treated and non-treated wounds. Thus, the vulnerary effects of PDGF-BB were transient and fully reversible in both wound healing models. Furthermore, analysis of PDGF-treated and non-treated wounds has provided important insights into mechanisms of normal and deficient tissue repair processes. PDGF appears to transduce its signal through wound macrophages and may trigger the induction of positive autocrine feedback loops and synthesis of endogenous wound PDGF and other growth factors, thereby enhancing the cascade of tissue repair processes required for a fully-healed wound. Thus, PDGF and other wound produced polypeptide growth factors may be the critical regulators of extracellular matrix deposition within healing wounds.     

Transforming growth factor-beta and platelet-derived growth factor synergistically stimulate contraction by testicular peritubular cells in culture in serum-free medium

We report investigations on factors influencing contractility by testicular peritubular cells (PC) maintained in culture in a three-dimensional collagen gel system, and the behavior of PC in culture on a two-dimensional system. At low and moderate cell densities, PC embedded in collagen gels in serum-free Eagle's minimal essential medium (MEM) have a lesser degree of contractility than PC in culture in MEM containing calf serum. The contractility by PC, measured by determining changes in diameter of the collagen gel, was increased by addition of transforming growth factor-beta (TGF-beta) to serum-free MEM, and this was further enhanced by supplementing the medium with platelet-derived growth factor (PDGF). In the absence of TGF-beta, however, PDGF had no detectable effects on PC contractility. Other growth factors examined (epidermal growth factor, insulin, and fibroblast growth factor) did not influence the degree of contractility of PC in serum-free MEM in the presence or absence of TGF-beta. PC maintained in MEM supplemented with platelet-poor serum (PPS) have a lesser degree of contractility than their counterparts in MEM containing 2.5% calf serum. The addition of TGF-beta and PDGF to PPS-supplemented MEM restored contractility by PC to a level comparable to that observed by PC in MEM containing complete serum. The addition of nonpurified bovine serum albumin (BSA) to MEM greatly increased PC contractility. By contrast, highly purified BSA had no such effect, suggesting that one or more components adsorbed to the impure BSA was implicated. Polyclonal antibody against fibronectin did not influence the contractility of PC in collagen gels in the presence or absence of serum. Antiserum against TGF-beta partially blocked the enhancement of contractility of PC in MEM containing non-purified BSA. In PC plated on top of a collagen gel lattice, the attachment, spreading, and cell shape were greatly influenced by the presence of TGF-beta and PDGF, both singly and together. Data presented are interpreted to indicate that effects elicited by serum on the properties of PC in culture, and on the contractility of PC, can be attributed in part to the combined influences of TGF-beta and PDGF in serum.     

Antiinflammatory effects of polypeptide growth factors. Platelet-derived growth factor, epidermal growth factor, and fibroblast growth factor inhibit the cytokine-induced expression of the alternative complement pathway activator factor B in human fibroblasts.

The synthesis of complement components in human fibroblasts is modulated by mediators of inflammation such as cytokines. In particular, interleukin-1 (IL-1) and tumor necrosis factor (TNF) induce time- and dose-dependent increases in the synthesis of complement proteins factor B (FB), C3, and factor H (FH). Polypeptide growth factors are also soluble mediators released during inflammation and able to modulate many fibroblast functions. We have studied the effects of polypeptide growth factors platelet-derived growth factor (PDGF), epidermal growth factor (EGF), and fibroblast growth factor (FGF) on the synthesis of complement proteins in cultured human fibroblasts. PDGF, EGF, and FGF alone did not affect the level of synthesis of any of the complement proteins analyzed, but simultaneous incubation of PDGF, EGF, or FGF with IL-1 and TNF resulted in a dose-dependent inhibition of the cytokine-enhanced expression of FB. Inhibition of FB synthesis was observed between 4 and 8 h of exposure to PDGF and persisted for 4 h after the removal of the growth factor. Analysis of steady-state levels of specific FB mRNA suggested that PDGF-induced inhibition of FB synthesis is mediated at a pretranslational level and that it requires new protein synthesis. The effect of the growth factors was limited to FB, with marginal or no inhibition on the cytokine-enhanced synthesis of C3 and FH, excluding the possibility that the inhibitory effects of PDGF, EGF, and FGF on FB synthesis were due to a negative modulation of the growth factors on cytokine cell membrane receptors. Specific inhibition of cytokine-induced increases in FB synthesis by the growth factors may represent down regulation of the acute inflammatory process, further permitting progression to processes of tissue repair and remodeling. Study of the interactions between cytokines and growth factors in the regulation of synthesis of complement proteins may also provide a system for investigating mechanisms of signal transduction of both polypeptide growth factors and cytokines.     

Promigratory and procontractile growth factor environments differentially regulate cell morphogenesis

Three-dimensional (3D) cell-matrix cultures provide a useful model to analyze and dissect the structural, functional, and mechanical aspects of cell-matrix interactions and motile behavior important for cell and tissue morphogenesis. In the current studies we tested the effects of serum and physiological growth factors on the morphogenetic behavior of human fibroblasts cultured on the surfaces of 3D collagen matrices. Fibroblasts in medium containing serum contracted into clusters, whereas cells in medium containing platelet-derived growth factor (PDGF) were observed to migrate as individuals. The clustering activity of serum appeared to depend on lysophosphatidic acid, required cell contraction based on inhibition by blocking Rho kinase or myosin II, and was reversed upon switching to PDGF. Oncogenic Ras transformed human fibroblasts did not exhibit serum-stimulated cell clustering. Our findings emphasize the importance of cell-specific promigratory and procontractile growth factor environments in the differential regulation of cell motile function and cell morphogenesis.     

Integrative repair of the meniscus: lessons from in vitro studies

Current therapies for meniscal injury seek to preserve and repair damaged tissue since loss of meniscal tissue is associated with degenerative changes in the joint, ultimately leading to osteoarthritis (OA). After a meniscal tear, the difficulty of integrating juxtaposed meniscal surfaces continues to be an obstacle. In order to determine the local factors that are necessary for successful tissue repair, previous studies have developed in vitro model systems that allow both biological and quantitative biomechanical measures of meniscus repair. Many studies have shown the importance of individual factors in meniscus metabolism, but there is a complex interplay among a variety of factors that influence meniscal healing, including inflammatory cytokines, growth factors, mechanical loading, and zonal differences in cell and tissue properties. In particular, the upregulation of inflammatory cytokines following joint injury appears to have significant catabolic influences on meniscal cell metabolic activity that must be overcome in order to promote repair. In the presence of inflammatory cytokines, such as interleukin-1 (IL-1) or tumor necrosis factor alpha (TNF-alpha), intrinsic meniscal repair in vitro is significantly inhibited. While anabolic growth factors, such as transforming growth factor-beta1 (TGF-beta1), enhance meniscal repair, they cannot completely overcome the IL-1-mediated inhibition of repair. The mechanisms by which these mediators influence meniscal repair, and their interactions with other factors in the microenvironment, such as mechanical loading, remain to be determined. Future studies must address these complex interactions during meniscal healing to ultimately enhance meniscal repair.     

Epigallocatechin-3-O-gallate inhibits fibroblast contraction of floating collagen gel: interaction between epigallocatechin-3-O-gallate and platelet derived growth factor

The effects of (-)-epigallocatechin gallate (EGCG) on the contraction of floating collagen gel by fibroblasts were investigated. EGCG inhibited collagen gel contraction dose-dependently. On the basis of the fact that platelet-derived growth factor (PDGF) is one of the serum components with stimulatory activity in collagen gel contraction, we examined the possibility that interaction between EGCG and PDGF may be involved in this inhibition mechanism. We confirmed this by recombinant PDGF-BB in the present system and we found that EGCG inhibited PDGF-stimulated collagen gel contraction. The results of affinity chromatography indicated that PDGF was bound by EGCG immobilized on agarose gel as detected by enzyme-linked immunoassay and Western blotting. These findings suggest that binding of EGCG to PDGF is at least partly involved in the mechanism of inhibition of collagen gel contraction by EGCG.     

Platelet-derived growth factor in combination with collagen promotes the migration of human skin fibroblasts into a denuded area of a cell monolayer.

Since we have found previously that adult donor skin fibroblasts (TIG-114) migrated more slowly in serum-depleted medium than in medium supplemented with 10% FBS, we tried to identify a factor(s) which promotes fibroblast migration from the edge of a denuded area in a monolayer. In medium supplemented with 10% FBS, the effects of both suramin, a competitor of growth factors at the receptor level, and monensin, an inhibitor of the secretion of extracellular matrix, were examined. Both substances suppressed cell migration, suggesting that growth factors and matrix substances are important for cell migration. Then, we examined the effects of growth factors and extracellular matrix on fibroblast migration in serum-free medium. Platelet-derived growth factor (PDGF), basic fibroblast growth factor, acidic fibroblast growth factor, and transforming growth factor-beta did not stimulate cell migration. Type I collagen, plasma fibronectin, and heparin also did not promote cell migration. However, the combination of PDGF and type I collagen did promote cell migration. Addition of anti-PDGF antibody reduced the stimulatory effect induced by the combination of PDGF and type I collagen. These results suggest that the copresence of growth factors and extracellular matrix regulates fibroblast migration into a denuded area in a monolayer.     

Effects of platelet-derived growth factor on bone formation in vitro

Platelet-derived growth factor (PDGF) is a polypeptide found in a variety of tissues, including bone, where it could act as an autologous regulator of skeletal remodeling. Therefore, a recombinant B chain homodimer of human PDGF was studied for its effects on bone formation in cultured rat calvariae. PDGF at 10-100 ng/ml stimulated [3H]thymidine incorporation into DNA by up to sixfold and increased the DNA content and the number of colcemid-induced metaphase arrested cells. This effect was observed in the fibroblast and precursor cell-rich periosteum. As a result of its mitogenic actions, PDGF enhanced [3H]proline incorporation into collagen, an effect that was observed primarily in the osteoblast-rich central bone. The effect of PDGF was not specific for collagen since it also increased noncollagen protein synthesis. In addition, PDGF increased bone collagen degradation. PDGF and insulin-like growth factor (IGF) I had additive effects on calvarial DNA synthesis, but PDGF opposed the stimulatory effect of IGF I on collagen synthesis and IGF I prevented the PDGF effect on collagen degradation. In conclusion, PDGF stimulates calvarial DNA synthesis which causes an increased number of collagen-synthesizing cells, but PDGF also enhances bone collagen degradation.     

Platelet-derived growth factor or basic fibroblast growth factor induce anchorage-independent growth of human fibroblasts

Anchorage-independent growth, i.e., growth in semi-solid medium is considered a marker of cellular transformation of fibroblast cells. Diploid human fibroblasts ordinarily do not exhibit such growth but can grow transiently when medium contains high concentrations of fetal bovine serum. This suggests that some growth factor(s) in serum is responsible for anchorage-independent growth. Much work has been done to characterize the peptide growth factor requirements of various rodent fibroblast cells for anchorage-independent growth; however, the requirements of human fibroblasts are not known. To determine the peptide growth factor requirements of human fibroblasts for anchorage-independent growth, we used medium containing serum that had had its peptide growth factors inactivated. We found that either platelet-derived growth factor (PDGF) or the basic form of fibroblast growth factor (bFGF) induced anchorage-independent growth. Epidermal growth factor (EGF) did not enhance the growth induced by PDGF, or did so only slightly. Transforming growth factor beta (TGF-beta) decreased the growth induced by PDGF. EGF combined with TGF-beta induced colony formation in semi-solid medium at concentrations at which neither growth factor by itself was effective, but the combination was much less effective in stimulating anchorage-independent growth than PDGF or bFGF. This work showed that PDGF, or bFGF, or EGF combined with TGF-beta can stimulate anchorage-independent growth of nontransformed human fibroblasts. The results support the idea that cellular transformation may reduce or eliminate the need for exogenous PDGF or bFGF.     

Human platelet-derived growth factor: radioimmunoassay and discovery of a specific plasma-binding protein

The platelet-derived growth factor (PDGF) is the principal mitogen in serum for cultured cells of mesenchymal origin. PDGF also is a potent chemotactic protein for inflammatory cells and for cells required for wound repair. Because activity levels of PDGF in biological fluids are difficult to measure, we attempted to develop a radioimmunoassay for PDGF. Rabbits were immunized with purified PDGF; the antiserum obtained was monospecific for PDGF in immunodiffusion analysis against concentrated platelet lysates, serum, and plasma. A radioimmunoassay for PDGF was developed with a sensitivity of congruent to 0.2 ng/ml. Levels of PDGF in plasma/serum were measured and compared with PDGF levels determined by a receptor-competition assay and by a standard biological assay measuring incorporation of [3H]thymidine into 3T3 cells. Radioimmunoassay showed apparent PDGF levels of 50 ng/ml in human plasma and 103 ng/ml in serum. The 50 ng/ml PDGF in plasma was unexpected because the plasma samples contained little or no platelet release products as determined by very low levels of platelet factor 4. We therefore sought an immunologically reactive PDGF molecule in human plasma. No immunologically reactive protein was detected by immunodiffusion analysis or when plasma was treated with an immunoaffinity gel. Subsequently, a 125I-PDGF-binding protein was identified; the 125I-PDGF-plasma-binding protein complex was not reactive with anti-PDGF immunoglobulin. Correction for 125I-PDGF bound by the plasma-binding protein established serum levels of PDGF of congruent to 50 ng/ml; congruent to 50 ng/ml PDGF was found in serum by radioreceptor-competition assays and by mitogenic assays as well. The plasma-binding protein may serve to clear PDGF released in the circulation, thereby limiting PDGF activity to its local interactions at the site of blood-vessel injury.     

Similarities between fibroblast-derived growth factor and platelet-derived growth factor

Platelet-derived growth factor (PDGF), one of the most potent mitogens in serum for non-transformed cells, shares many biological and physical properties with fibroblast-derived growth factor (FDGF), a polypeptide produced by BHK cells transformed by SV40. Thus FDGF and PDGF have biological activity which is recoverable from sodium dodecyl sulphate (SDS) polyacrylamide gel electrophoresis, at positions indicating similar molecular weights. Further, the biological activity of both factors is heat-stable but sensitive to mercaptoethanol. FDGF and PDGF have similar abilities to induce DNA synthesis synergistically in the presence of either insulin, epidermal growth factor (EGF), vasopressin or colchicine. In contrast to other growth factors, (i) either FDGF or PDGF can induce DNA synthesis in the absence of other mitogens in 3T3 cells maintained in serum-free medium and (ii) a transient exposure of cultures to FDGF or PDGF causes a persistent stimulation of DNA synthesis. Either FDGF or PDGF enhances colony formation of non-transformed cells cultured in suspension in the presence of EGF and serum. FDGF is not PDGF adsorbed by SV40-BHK cells from serum, since SV40-BHK cells plated and grown in the absence of serum still produce FDGF. In view of the similarities between PDGF and FDGF, we suggest that they may belong to the same family of growth factors.     

Platelet-derived growth factor in chemotactic for fibroblasts

Chemotaxis assays in modified Boyden chambers were used to detect fibroblast chemoattractants in materials released from early-stage inflammatory cells, namely, mast cells, platelets, and neutrophils. Strong attractant activity was found in substances released from platelets. This activity was accounted for mainly by the platelet- derived growth factor (PDGF), which is released from the platelets and which was active as a chemoattractant at 0.5-1.0 mitogenic units/ml. The mitogenic activity of purified PDGF, measured by [3H]thymidine incorporation, occurs at a similar concentration range. By varying the gradient of PDGF, we demonstrated that PDGF stimulates chemotaxis rather than random motility. Preincubation of suspensions of fibroblasts in the presence of PDGF decreased the subsequent migration of cells to a gradient of PDGF as well as to a gradient of fibronectin, which is also in attractant for fibroblasts. The chemotactic response of fibroblasts to PDGF was not inhibited by hydroxyurea or azidocytidine but was inhibited by actinomycin D and cycloheximide, suggesting that synthesis of RNA and proteins but not of DNA is required for the chemotactic response to occur. Fibroblast growth factor, epidermal growth factor, nerve growth factor, and insulin were not chemotactic for human skin fibroblasts, suggesting that the chemoattractant activity of PDGF for fibroblasts is not a general property of growth factors and mitogens. These results suggest that PDGF could have two functions in wound healing: to attract fibroblasts to migrate into the clot and then to induce their proliferation.     

Transplantation of autologous rabbit BM-derived mesenchymal stromal cells embedded in hyaluronic acid gel sponge into osteochondral defects of the knee

BACKGROUND: Mesenchymal stromal cells (MSC) have the potential to differentiate into distinct mesenchymal tissues including cartilage, suggesting that these cells are an attractive cell source for cartilage tissue engineering approaches. Various methods, such as using hyaluronan-based materials, have been employed to improve transplantation for repair. Our objective was to study the effects of autologous transplantation of rabbit MSC with hyaluronic acid gel sponges into full-thickness osteochondral defects of the knee. METHODS: Rabbit BM-derived MSC were cultured and expanded with fibroblast growth factor (FGF). Specimens were harvested at 4 and 12 weeks after implantation, examined histologically for morphologic features, and stained immunohistochemically for type II collagen and CD44. RESULTS: The regenerated area after autologous transplantation of hyaluronic acid gel sponge loaded with MSC into the osteochondral defect at 12 weeks after surgery showed well-repaired cartilage tissue, resembling the articular cartilage of the surrounding structure, of which the histologic score was significantly better than that of the untreated osteochondral defect. In the regenerated cartilage, type II collagen was found in the pericellular matrix of regenerative chondrocytes, while CD44 expression in the regenerative tissue could not be revealed. DISCUSSION: These data suggest that the autologous transplantation of MSC embedded in hyaluronan-based material may support chondrogenic differentiation and be useful for osteochondral defect repair.     

Three-dimensional culture of human meniscal cells: Extracellular matrix and proteoglycan production

Background  

The meniscus is a complex tissue whose cell biology has only recently begun to be explored. Published models rely upon initial culture in the presence of added growth factors. The aim of this study was to test a three-dimensional (3D) collagen sponge microenvironment (without added growth factors) for its ability to provide a microenvironment supportive for meniscal cell extracellular matrix (ECM) production, and to test the responsiveness of cells cultured in this manner to transforming growth factor-β (TGF-β).     

Influence of the extracellular matrix on the proliferative response of human skin fibroblasts to serum and purified platelet-derived growth factor

The culture of adult human skin fibroblasts on reconstituted bovine type 1 fibrillar collagen gels, ranging in concentration from 2.5-35.0 mg/ml, results in a reduction in proliferation rate by 40%-60% as measured by (3H) thymidine incorporation. The suppressive effect was noted when cells were cultured in both human and bovine serum. Drying the gels into thin films abolishes the suppressive effect of the fibrillar collagen on cell proliferation. Cell attachment studies showed that differences in the proliferation rate of cells on the various substrata were not simply due to differences in initial attachment. Studies with purified platelet-derived growth factor (PDGF) demonstrated that the reduced responsiveness of cells to this factor, when cultured on collagen gels as compared to plastic, was largely responsible for the reduced proliferative activity of the cells when cultured in the presence of serum. The reduced proliferative activity of fibroblasts in response to PDGF, when cultured on collagen gels, was confirmed by total DNA determination. It was shown that the reduced responsiveness of cells to PDGF was not simply because the factor bound to the fibrillar collagen gel or was inaccessible to the cells. The data indicate that the reduced proliferation rate of fibroblasts cultured on collagen gels is a direct result of the influence of the extracellular matrix on the cells' ability to respond to a soluble mitogenic mediator.     

Cell Population Kinetics of Collagen Scaffolds in Ex Vivo Oral Wound Repair

Biodegradable collagen scaffolds are used clinically for oral soft tissue augmentation to support wound healing. This study sought to provide a novel ex vivo model for analyzing healing kinetics and gene expression of primary human gingival fibroblasts (hGF) within collagen scaffolds. Sponge type and gel type scaffolds with and without platelet-derived growth factor-BB (PDGF) were assessed in an hGF containing matrix. Morphology was evaluated with scanning electron microscopy, and hGF metabolic activity using MTT. We quantitated the population kinetics within the scaffolds based on cell density and distance from the scaffold border of DiI-labled hGFs over a two-week observation period. Gene expression was evaluated with gene array and qPCR. The sponge type scaffolds showed a porous morphology. Absolute cell number and distance was higher in sponge type scaffolds when compared to gel type scaffolds, in particular during the first week of observation. PDGF incorporated scaffolds increased cell numbers, distance, and formazan formation in the MTT assay. Gene expression dynamics revealed the induction of key genes associated with the generation of oral tissue. DKK1, CYR61, CTGF, TGFBR1 levels were increased and integrin ITGA2 levels were decreased in the sponge type scaffolds compared to the gel type scaffold. The results suggest that this novel model of oral wound healing provides insights into population kinetics and gene expression dynamics of biodegradable scaffolds.     

The diabetic rat as an impaired wound healing model: stimulatory effects of transforming growth factor-beta and basic fibroblast growth factor

Two models of wound repair compared the effect of defined, recombinant growth factors on the rate of wound repair in both normal and streptozotocin-induced diabetic rats: subcutaneous implantation of polyvinyl alcohol sponges and incisional wounding. Transverse incisional wounds were made on the dorsal surface of rats and closed with steel sutures. Three days postwounding the rats received a single injection of either transforming growth factor-beta or vehicle alone directly into the wound site. Animals were sacrificed 7, 14, and 21 days postwounding, and fresh and formalin-fixed wound tensile strength were measured. Diabetic rats had expected defects in wound repair, including decreased granulation tissue and reduced amounts of collagen, protein, and DNA. Fresh tensile strength of the diabetic incisions was 53% of normal on Day 7 (p < or = .01) and 29% of normal on Day 21. Fixed tensile strength was 41% of normal on Day 7 (p < or = .01) and fell to 78% of normal by Day 21 (p < or = .01), suggesting that collagen concentrations of diabetic wounds increased towards normal but did not undergo maturation. TGF beta produced a moderate increase in tensile strength of fresh and fixed wounds of diabetic rats, but not to the levels of wounds in untreated normal rats. Sponges fill with granulation tissue, their reproducible rate of organization being measured by histological and biochemical methods. A single injection into sponges 3 days postimplantation of basic fibroblast growth factor, transforming growth factor-beta, or vehicle only, was evaluated at 7 and 9 days postimplantation. In the sponge model, bFGF and TGF beta were each able to induce significant increases in the accumulation of granulation tissue in both diabetic and normal rats. TGF beta increased the collagen content of sponges by 136% in sponges from diabetic animals (p < or = .001), thereby raising the collagen content to that of normal control wounds, while stimulating a 49% (p < or = .02) increase in sponges from normal animals on Day 9. By contrast, the response to bFGF was predominantly an increase in the protein and DNA content of the sponges. These results emphasize the differential effects of the two cytokines in accelerating healing under conditions of defective wound repair.     

Highly purified fibroblast-derived growth factor, an SV40-transformed fibroblast-secreted mitogen, is closely related to platelet-derived growth factor.

Fibroblast-derived growth factor (FDGF), a polypeptide secreted by an SV40-transformed baby hamster kidney cell line (SV28), was purified approximately 1000-fold from SV28-conditioned medium. FDGF, which gave a single band on SDS-polyacrylamide gel electrophoresis, is a hydrophobic and cationic protein of apparent mol. wt. 31 000 containing disulphide-linked polypeptides. This factor is positive in Western blots using human platelet-derived growth factor (PDGF) anti-serum. FDGF is a potent mitogen for Swiss 3T3 cells; half-maximal stimulation of DNA synthesis was achieved at a concentration of approximately 1 nM, comparable with those for human and porcine PDGF. FDGF inhibits EGF binding to Swiss 3T3 cells, as does PDGF. The coincidence of the physical, biological and immunological characteristics of FDGF and PDGF strongly suggests that they are closely related in structure.