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Keloids represent a dysregulated response to cutaneous wounding that results in an excessive deposition of extracellular matrix, especially collagen. However, the molecular mechanisms regulating this pathologic collagen deposition still remain to be elucidated. A previous study by this group demonstrated that transforming growth factor (TGF)-beta1 and -beta2 ligands were expressed at greater levels in keloid fibroblasts when compared with normal human dermal fibroblasts (NHDFs), suggesting that TGF-beta may play a fibrosis-promoting role in keloid pathogenesis.To explore the biomolecular mechanisms of TGF-beta in keloid formation, the authors first compared the expression levels of the type I and type II TGF-beta receptors in keloid fibroblasts and NHDFs. Next, they investigated the phosphorylation of Smad 3, an intracellular TGF-beta signaling molecule, in keloid fibroblasts and NHDFs. Finally, they examined the regulation of TGF-beta receptor II by TGF-beta1, TGF-beta2, and TGF-beta3 ligands. Our findings demonstrated an increased expression of TGF-beta receptors (types I and II) and increased phosphorylation of Smad 3 in keloid fibroblasts relative to NHDFs. These data support a possible role of TGF-beta and its receptors as fibrosis-inducing growth factors in keloids. In addition, all three isoforms of recombinant human TGF-beta proteins could further stimulate the expression of TGF-beta receptor II in both keloids and NHDFs. Taken together, these results substantiate the hypothesis that the elevated levels of TGF-beta ligands and receptors present in keloids may support increased signaling and a potential role for TGF-beta in keloid pathogenesis.

Fetal cutaneous wounds that occur in early gestation heal without scar formation. Although much work has been done to characterize the role of transforming growth factor-beta (TGF-beta) isoforms in the adult wound repair process, their function in fetal scarless wound repair is not well understood. The authors hypothesized that the pattern of expression for TGF-beta isoforms and their receptors may influence the phenotypic transition from scarless to scar-forming repair observed during fetal gestation. Using time-dated fetal Sprague-Dawley rat fibroblasts and unwounded skin at gestational ages 14, 16, 18, and 21 days postcoitum of the scarless (or =16 days) and scar-forming (16 days) periods of gestation (term = 21.5 days), the authors analyzed the endogenous messenger RNA (mRNA) levels of TGF-beta 1 and TGF-beta 3 and their signaling receptors TGF-beta-RI and TGF-beta-RII. Northern blot analyses in both fibroblasts and unwounded skin revealed that levels of TGF-beta 1 were not differentially expressed, whereas more TGF-beta 3 mRNA transcript was found in early than in late gestation. Fibroblast expression of TGF-beta-RI showed no substantial differences, whereas expression of TGF-beta-RII increased during gestation. In contrast, expression of both TGF-beta-RI and TGF-beta-RII in unwounded skin showed decreasing levels as a function of gestational age. The differential levels of TGF-beta 1 and TGF-beta 3 suggest that the ratio of these cytokines may provide a predominantly antiscarring or profibrotic signal upon wounding during the scar-free or scar-forming periods of gestation, respectively. Furthermore, lower amounts of the ligand-binding TGF-beta-RII seen in early gestation fibroblasts suggest a decreased ability to perceive ligand during the period of scarless repair.

Cutaneous injury, whether by laser, chemical, or scalpel, results in scar formation. The normal response to such an insult occurs in the middle of a continuum of wound repair processes. On one end of the continuum are the overhealed responses (i.e., keloids). On the opposite end are tissue regeneration and scarless healing as seen in fetal wounds. This article reviews the molecular biology and mechanisms leading to these various clinical phenotypes and discusses future potential modalities that may replicate the scarless wound healing mechanism in adults.

Normal bone growth and repair is dependent on angiogenesis. Fibroblast growth factor-2 (FGF-2), vascular endothelial growth factor (VEGF), and transforming growth factor-beta (TGFbeta) have all been implicated in the related processes of angiogenesis, growth, development, and repair. The purpose of this study was to investigate the relationships between FGF-2 and both VEGF and TGFbeta in nonimmortalized and clonal osteoblastic cells. Northern blot analysis revealed 6-fold peak increases in VEGF mRNA at 6 h in fetal rat calvarial cells and MC3T3-E1 osteoblastic cells after stimulation with FGF-2. Actinomycin D inhibited these increases in VEGF mRNA, whereas cycloheximide did not. The stability ofVEGF mRNA was not increased after FGF-2 treatment. Furthermore, FGF-2 induced dose-dependent increases in VEGF protein levels (P < 0.01). Although in MC3T3-E1 cells, TGFbeta1 stimulates a 6-fold peak increase in VEGF mRNA after 3 h of stimulation, we found that both TGFbeta2 and TGFbeta3 yielded 2- to 3-fold peak increases in VEGF mRNA levels noted after 6 h of stimulation. Similarly, both TGFbeta2 and TGFbeta3 dose dependently increased VEGF protein production. To determine whether FGF-2-induced increases in VEGF mRNA may have occurred independently of TGFbeta, we disrupted TGFbeta signal transduction (using adenovirus encoding a truncated form of TGFbeta receptor II), which attenuated TGFbeta1 induction of VEGF mRNA, but did not impede FGF-2 induction ofVEGF mRNA. In summary, FGF-2-induced VEGF expression by osteoblastic cells is a dose-dependent event that may be independent of concomitant FGF-2-induced modulation of TGFbeta activity.

An attractive experimental method to elucidate the role of growth factors and cytokines in cutaneous wound healing would be to overexpress or "knock out" a molecule using a gene delivery vector and observe the impact on the wound repair process. As a first step toward developing an adenoviral gene delivery procedure to study wound repair, the authors injected beta-galactosidase (beta-gal) adenoviruses either subcutaneously or intradermally into the dorsal skin of 10-day-old postnatal Sprague-Dawley rats. Histological analysis and beta-gal staining were used to determine the expression and localization of the transferred gene. Beta-gal expression was observed as early as day 1 and up to day 7 postintradermal injection and day 9 postsubcutaneous injection, with no obvious inflammatory reaction detected at the injection sites. Furthermore, as expected, greater beta-gal expression was observed in the dermis of intradermally injected rats compared with the dermis of subcutaneously injected rats. Next, the authors sought to determine whether cutaneous wounds would heal before dissipation of the transferred gene. They created incisional and excisional wounds on the backs of similar-age rats. They found that incisional wounds closed by day 5 postwounding, whereas excisional wounds closed by day 14 postwounding. Their study demonstrated that an adenoviral vector delivered a gene efficiently into newborn rat skin and maintained the gene expression for at least as long as it would take for an incisional wound to heal. The combined use of newborn rat wound models and an adenoviral vector may provide a useful in vivo system to define the biological roles of growth factors and cytokines involved in the wound repair process. These discoveries may lead to the development of gene therapy approaches for abnormal wound healing.

The remarkable ability of the fetus to heal early gestation skin wounds without scarring remains poorly understood. Taking advantage of recent advances in signal transduction, the tyrosine phosphorylation patterns of fetal rat fibroblasts, representing the scarless cutaneous repair phenotype, and adult rat fibroblasts, representing scarforming phenotype, were examined whether there were inherent differences in cellular signaling. Specifically, correlation of the phosphorylation patterns with the expression levels of the signaling molecules that transmit information from the plasma membrane receptor to the nucleus was sought. By using three different cell lines of explanted fibroblasts from gestational day 13 fetal rat skin ( n = 24) and 1-month-old postnatal adult rat skin ( n = 3), immunoblotting was performed to compare tyrosine phosphorylation patterns. The results revealed five major protein bands of interest in fetal rat fibroblasts, but not in the adult rat fibroblasts. These phosphorylated protein bands are of interest because of their possible role in wound repair and may have the potential to regulate cellular responses to the extracellular matrix and their secondary signaling molecules. It was hypothesized that these bands represented receptor tyrosine kinases, epidermal growth factor receptor, and discoidin domain receptor 1, and their downstream adaptor protein Shc that binds receptor tyrosine kinases to transduce signals intracellularly. Furthermore, elevated expression of platelet-derived growth factor receptor-beta in adult compared with fetal fibroblasts was demonstrated, suggesting that decreased expression of certain growth factors may also be important for the scarless phenomenon to occur.

A number of studies have demonstrated the critical role of angiogenesis for successful wound repair in the surgical patient. Vascular disruption from tissue injury due to trauma or surgery leads to a hypoxic zone in the healing wound. In this dynamic process, angiogenesis is vital for the delivery of oxygen, nutrients, and growth factors necessary to initiate the synthetic processes of wound healing. Fibroblasts, invading the wound early in the healing process, are involved in extracellular matrix (ECM) deposition as well as wound contraction. However, the exact mechanisms by which important genes are regulated remain unknown. In order to examine these processes, we studied the effects of hypoxia on fibroblasts for the expression of VEGF, type IalphaI collagen, and matrix-metalloproteinase-3, three genes essential for the regulation of angiogenesis, ECM deposition, and ECM degradation in wound healing. Primary cell cultures of normal human dermal fibroblasts (NHDFs) were placed in hypoxia for varying periods of time. Northern blot hybridization was performed with [alpha32P]dCTP-labeled cDNA probes for VEGF, type IalphaI collagen, and MMP-3. The results demonstrated a time-dependent VEGF mRNA upregulation (470% of baseline) under hypoxia. Type IalphaI collagen increased (170% of baseline) at 24 h, but was then abruptly downregulated to 3.8% of baseline at 48 h. MMP-3 was incrementally downregulated to 2.2% of baseline at 48 h. These experiments focused on the effect of hypoxia on genes thought to play a role in wound repair. VEGF upregulation in the hypoxic microenvironment of the early wound may serve to stimulate angiogenesis. Type IalphaI collagen, though upregulated early on, was abruptly downregulated at 48 h. This downregulation may reflect the in vivo requirement for angiogenesis to deliver oxygen for successful hydroxylation and collagen synthesis in the wound. MMP-3, also downregulated at 48 h, may also implicate the need for angiogenesis. These data support the theory that hypoxia-driven angiogenesis is critical for ECM formation and remodeling in successful soft tissue repair. Furthermore, they may represent the role of hypoxia as an important regulator to efficiently balance these complex processes in the healing wound.

The etiology of keloid formation is diverse. They are characterized grossly as thick scar tissue that extends beyond the boundaries of the original wound. Histologically, keloids are composed of excessive collagen with an abnormally large number of partially or totally occluded microvessels. This occlusion of keloid microvessels has been hypothesized to contribute to a hypoxic microenvironment within these pathological scars. Vascular endothelial growth factor (VEGF), a potent endothelial cell mitogen, and proangiogenic cytokine have been implicated in normal and pathological wound healing. The purpose of this study was to evaluate the amount of VEGF protein production by fibroblast cell lines derived from keloids and normal human dermal skin in hypoxic compared with normoxic culture conditions. By enzyme-linked immunosorbent protein assay, VEGF was increased in both keloid and normal human dermal fibroblasts in hypoxia over normoxic controls. There was not, however, a significant difference between upregulation of VEGF protein when comparing the keloid and normal fibroblast groups. As the result of the data, alternative hypotheses for hypoxia-induced keloid formation were explored: (1) downstream modulation or signal transduction of VEGF, (2) VEGF production from cells other than fibroblasts, (3) the importance of matrix accumulation stimulated by hypoxia, or (4) increased migration of cells (other than fibroblasts) specific to keloid biology. These hypotheses may help explain the possible role of hypoxia in the pathogenesis of keloid formation. Future studies involving in situ hybridization or immunohistochemical analysis may offer greater insight into the mechanisms underlying keloid formation. Ultimately, our therapeutic goal is the utilization of biomolecular approaches for the suppression of keloid formation.

Gene therapy is becoming a clinical reality. This has become possible due to the increased understanding of the biomolecular pathogenesis of certain diseases and the advances in gene transfer technology. Plastic surgery researchers have embraced this concept, which may revolutionize the broad spectrum of intricate surgical procedures that are performed, such as skin grafting, nerve and muscle repair, and microvascular tissue transfer, to name a few. Gene therapy has the potential for significant improvement in clinical outcome in these areas by selectively expressing genes of interest at targeted sites. Furthermore, this approach may lead to improvements in the rate and efficiency of the wound repair process, and thus will have a significant impact on the quality of healthcare and the management of healthcare costs. Growth factors play a major role in wound healing. Thus, their potential use as gene therapeutic agents is apparent. By using advanced gene transfer techniques, one can transfect a growth factor gene of interest into desired cells, which will ultimately result in the localized expression of their recombinant proteins. In theory, localized over-expression may be ideal, because systemically applied growth factors, which require large doses to produce a biologic effect, may elicit serious side effects. There are several applications of gene therapy for cutaneous wound healing. One approach may involve the use of genetically engineered keratinocytes or fibroblasts to over express growth factor genes, which may accelerate healing and regenerate injured tissue. Once the DNA of interest is inserted, genetically modified cells can then be transplanted to the wound area, and will express recombinant proteins for a prolonged time.

Keloids represent a dysregulated response to cutaneous wounding that results in disfiguring scars. Unique to humans, keloids are characterized by an accumulation of extracellular matrix components. The underlying molecular mechanisms of keloid pathogenesis, however, remain largely uncharacterized. Similarly, cellular signaling mechanisms, which may indicate inherent differences in the way keloid fibroblasts and normal human dermal fibroblasts interact with extracellular matrix or other cells, have not been investigated. As part of a fundamental assessment of cellular response to injury in keloid fibroblasts, phosphorylation studies were performed using three different keloid (n = 3) and normal human dermal (n = 3) fibroblast cell lines. These studies were undertaken to elucidate whether keloid and normal human dermal fibroblasts exhibit different tyrosine kinase activity. Initially, distinct tyrosine phosphorylation patterns of keloid and normal human dermal fibroblasts were demonstrated. Next, the phosphorylation patterns were correlated with known molecules that may be important to keloid pathogenesis. On the basis of molecular weight, it was hypothesized that the highly phosphorylated bands seen in keloid fibroblasts represented epidermal growth factor receptor (EGFR); discoidin domain receptor 1 (DDR1); and She, an adaptor protein known to bind many tyrosine kinases, including EGFR and DDR1. Individual immunoblotting using EGFR, DDR1, and She antibodies revealed greater expression in keloid fibroblasts compared with normal human dermal fibroblasts. These data substantiate for the first time the finding of greater phosphorylation by the above-mentioned molecules, which may be important in keloid pathogenesis. (Plast. Reconstr. Surg. 106: 1532, 2000.)

The ability of newborns and immature animals to reossify calvarial defects has been well described. This capacity is generally lost in children greater than 2 years of age and in mature animals. The dura mater has been implicated as a regulator of calvarial reossification. To date, however, few studies have attempted to identify biomolecular differences in the dura mater that enable immature, but not mature, dura to induce osteogenesis. The purpose of these studies was to analyze metabolic characteristics, protein/gene expression, and capacity to form mineralized bone nodules of cells derived from immature and mature dura mater. Transforming growth factor beta-1, basic fibroblast growth factor, collagen type IalphaI, osteocalcin, and alkaline phosphatase are critical growth factors and extracellular matrix proteins essential for successful osteogenesis. In this study, we have characterized the proliferation rates of immature (6-day-old rats, n = 40) and mature (adult rats, n = 10) dura cell cultures. In addition, we analyzed the expression of transforming growth factor beta-1, basic fibroblast growth factor-2, proliferating cell nuclear antigen, and alkaline phosphatase. Our in vitro findings were corroborated with Northern blot analysis of mRNA expression in total cellular RNA isolated from snap-frozen age-matched dural tissues (6-day-old rats, n = 60; adult rats, n = 10). Finally, the capacity of cultured dural cells to form mineralized bone nodules was assessed. We demonstrated that immature dural cells proliferate significantly faster and produce significantly more proliferating cell nuclear antigen than mature dural cells (p < 0.01). Additionally, immature dural cells produce significantly greater amounts of transforming growth factor beta-1, basic fibroblast growth factor-2, and alkaline phosphatase (p < 0.01). Furthermore, Northern blot analysis of RNA isolated from immature and mature dural tissues demonstrated a greater than 9-fold, 8-fold, and 21-fold increase in transforming growth factor beta-1, osteocalcin, and collagen IalphaI gene expression, respectively, in immature as compared with mature dura mater. Finally, in keeping with their in vivo phenotype, immature dural cells formed large calcified bone nodules in vitro, whereas mature dural cells failed to form bone nodules even with extended culture. These studies suggest that differential expression of growth factors and extracellular matrix molecules may be a critical difference between the osteoinductive capacity of immature and mature dura mater. Finally, we believe that the biomolecular bone- and matrix-inducing phenotype of immature dura mater regulates the ability of young children and immature animals to heal calvarial defects.

Normal and abnormal extracellular matrix turnover is thought to result, in part, from the balance in the expression of metalloproteinases and tissue inhibitors of metalloproteinases (TIMPs). The clinical manifestations of an imbalance in these relationships are evident in a variety of pathologic states, including osteoarthritis, deficient long-bone growth, rheumatoid arthritis, tumor invasion, and inadequate cartilage repair. Articular cartilage defects commonly heal as fibrocartilage, which is structurally inferior to the normal hyaline architecture of articular cartilage. Transforming growth factor-beta 1 (TGF-beta1), a cytokine central to growth, repair, and inflammation, has been shown to upregulate TIMP-1 expression in human and bovine articular cartilage. Additionally, members of the TGF-beta superfamily are thought to play key roles in chondrocyte growth and differentiation. Bone morphogenetic protein-2 (BMP-2), a member of this superfamily, has been shown to regulate chondrocyte differentiation states and extracellular matrix composition. It was proposed that, by optimizing extracellular matrix composition, BMP-2 would enhance articular cartilage healing. After determining the release kinetics of BMP-2 from a collagen type I implant (Long-Evans male rats; two implants/rat, n = 14), it was found that, in a tissue engineering application, BMP-2 induced a hyaline-like repair of New Zealand White rabbit knee articular cartilage defects (3-mm full-thickness defects in the femoral trochlea; 2 defects/rabbit, n = 36). The quality of cartilage repair with BMP-2 (with or without chondrocytes) was significantly better than defects treated with BMP-2, as assessed by a quantitative scoring scale. Immunohistochemical staining revealed TIMP-1 production in the cartilage defects treated with BMP-2. When studied in vitro, it was found that BMP-2 markedly increased TIMP-1 mRNA by both bovine articular and human rib chondrocytes. Additionally, increased TIMP-1 mRNA was translated into increased TIMP-1 protein production by bovine chondrocytes. Taken together, these data suggest that BMP-2 may be a useful cytokine to improve healing of cartilaginous defects. Furthermore, these data suggest that the beneficial effects of BMP-2 may be, in part, related to alterations in extracellular matrix turnover.

In contrast to adult wound healing, early-gestation fetal skin wound healing occurs rapidly, in a regenerative fashion, and without scar formation. The accelerated rate of healing, relative lack of an acute inflammatory response, and an absence of neovascularization distinguishes fetal from adult wound healing. However, this remarkable ability of the fetus to heal without scarring still remains poorly understood. The uncertainties include the role of cytokines, extracellular matrix components, homeobox genes, and certain cell types in the scarless wound repair process. Nevertheless, some strides have been made within the last two decades. This report, discusses the current knowledge of the mechanisms and characteristics of scarless fetal wound healing. Furthermore, to shy away from being just another all inclusive review, the authors point out deficiencies in the knowledge base on this important topic. Last, the future direction of research is discussed that may elucidate the mechanisms regulating the scarless repair phenomena.

Results: Gene expression levels of TGF-b1, TGF-b RI and TGF-b RII were enhanced (by 47%, 45% and 24% respectively vs control) at 3h strain and returned to control levels by 6h. Alpha-1 procollagen mRNA levels were elevated at both 3h and 6h (by 125% and 57% respectively vs control) with the peak at 3h. The transcription of collagen receptor DDR1 was enhanced at 3h, peaked at 6h, and remained elevated at 24h (36%, 60%, and 37% respectively vs control). Interestingly, the gene expression of the VEGF was unchanged with cellular strain.