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In this chapter, we point out distinct anatomical features of the male buttocks, as well as the surgical landmarks the senior author utilizes for staying safe. Gluteal augmentation in males must deliver a muscular appearance, which consequently will also provide some projection and contour; nevertheless, the targeted visual impression must be an athletic one.

Liposuction of the male abdomen can be a very challenging. If undertreated, any of several techniques can leave a less than satisfying result showing little if any improvement. With more aggressive generalized suction of the area, the patient can be left with irregularities, loose residual lower, and excess infraumbilical soft tissue and skin. In the modern age of fitness, we find that patients are increasingly interested in a fitter, more sculpted look with greater definition to the abdominal musculature. Currently we are experiencing a “golden age” of plastic surgery body contouring with new innovations by thought leaders, including fat grafting, which are completely raising the bar of postoperative results that can now be delivered to the patients in terms of appearing youthful and sculpted yet natural.1 Previous techniques involving abdominal contour including selective fat reduction or “sculpting” have been limited in terms of inability to achieve muscular appearance in patients with very little muscular bulk to the rectus abdominis.2,3 Furthermore, abdominal muscle enhancement with silicone has been equally disappointing due visible incisions, an unnatural appearance, technical difficultly, lack of implant availability, and increased infection rate. Here we present the senior author's (D.S.S) technique for achieving superior results in abdominal contouring using selective lipo-contouring with fat grafting. The technique may be used with general anesthesia, local anesthesia with sedation, or simply local anesthesia. All markings are done with the patient standing prior to any sedative administration. Anatomic asymmetry is noted. The patient is brought in the operating room placed in the supine position after circumferential betadine prep. Stab incisions are made after injection of 2% lidocaine with epinephrine and tumescent solution (0.9% saline with lidocaine 0.1% and epinephrine 1:1,100,000) is introduced into the areas to be suctioned. Three stab incisions are made in the underwear line to …

Background The number of total cosmetic procedures performed yearly has increased by more than 274% between 1997 and 2014, according to the American Society for Aesthetic Plastic Surgery. However, the vast majority of plastic surgery procedures are still targeted toward women, with little attention toward men. Objectives This study sought to quantify the extent of gender discrepancies observed in online plastic surgery marketing in this country. Methods For the 48 contiguous United States, a systematic Google (Mountain View, CA) search was performed for “[state] plastic surgeon.” The first 10 solo or group practice websites in each state were analyzed for the gender of the first 10 images featured, presence of a male services section, and which procedures were offered to men. The results were statistically analyzed using SPSS Software (IBM Corporation, Armonk, NY). Results A total of 453 websites were analyzed, as 5 states did not have 10 unique solo or group practice websites. Of the 4239 images reviewed, 94.1% were of females, 5.0% were of males, and 0.9% were of a male and female together. A male services page was present in 22% of websites. The most common procedures marketed toward men were gynecomastia reduction (58%), liposuction (17%), blepharoplasty (13%), and facelift (10%). Less than 10% of all websites offered other procedures to males, with a total of 15 other aesthetic procedures identified. Conclusions Many plastic surgeons choose to ignore or minimize male patients in their online marketing efforts. However, as the number of men seeking cosmetic procedures continues to grow, plastic surgeons will benefit from incorporating male patients into their practice model. [10.1093/asj/sjv211][1] [1]: /lookup/doi/10.1093/asj/sjv211

Background Lateral canthal procedures are often indicated to correct or prevent lower eyelid malposition. When determining an appropriate lateral canthal procedure, planning is essential and includes proper analysis and identification of any contributory anatomical factors. Methods A 12-month retrospective review was performed on patients undergoing lateral canthal procedures. Important components of the preoperative examination were studied to relate patient anatomy and results. Outcomes were followed for a minimum of 5 years. Results Of 288 consecutive lower eyelid canthal procedures, a total of 146 met the inclusion criteria. Common designated abnormal preoperative findings included a negative vector (62 percent), lid margin eversion (12 percent), scleral show (21 percent), neutral or negative canthal tilt (49 percent and 18 percent, respectively), and lateral canthus -to -orbital rim distance of more than 1 cm (11 percent). The distribution of lateral canthal procedures performed in our study population included inferior retinacular lateral canthopexy (n = 36), inferior retinacular lateral canthoplasty (n = 88), tarsal strip lateral canthoplasty (n = 15), and dermal-orbicular pennant lateral canthoplasty (n = 7). Successful outcomes were noted to be 86 percent and 91 percent according to surgeons and patients, respectively. Conclusions Specific findings on the preoperative physical examination identify when simple or more complex lateral canthal procedures should be performed. The authors report seven key physical findings that should be documented to effectively determine a lateral canthal procedure that is appropriate for prevention and management of lower eyelid malposition. Clinical question/level of evidence Therapeutic, IV.

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.

Angiogenesis, the formation of new blood vessels, is crucial to the process of fracture healing. Vascular disruption after osseous injury results in an acidic, hypoxic wound environment. We have previously shown that osteoblasts can produce vascular endothelial growth factor (VEGF) in response to a variety of stimuli. In this study we examined pH and lactate concentration, two components of the putative fracture extracellular microenvironment, and determined their relative contribution to regulation of rat calvarial osteoblast VEGF production under both normoxic and hypoxic conditions. Our results demonstrate that pH and lactate concentration do independently affect osteoblast VEGF mRNA and protein production. Acidic pH (7.0) significantly decreased VEGF production, under normoxic and hypoxic conditions ( P < 0.05), compared with neutral pH (7.4). This decrease was primarily transcriptionally regulated, because the rate of VEGF mRNA degradation was unchanged at pH 7.0 vs. 7.4. Similarly, an elevated lactate concentration (22 mM) also depressed osteoblast elaboration of VEGF at both neutral and acidic pH ( P < 0.001). Furthermore, the effects of increasing acidity and elevated lactate appeared to be additive.

A number of growth factors have been implicated in fracture repair. Transforming growth factor-beta 3 (TGF-beta 3) is believed to be involved in osteoblast proliferation, chemotaxis, and collagen synthesis. The collagens act as the scaffolding for new bone matrix formation, whereas tissue inhibitors of metalloproteinases (TIMPs) may help regulate matrix remodeling in bone repair. Despite their hypothesized integral role in fracture repair, the temporal expression of these molecules in membranous bone fracture healing remains unknown. The objective of this study was to assess the temporal pattern of TGF-beta 3 and TIMP type 1 (TIMP-1) expression in rat mandibular fracture healing. Twenty-eight adult male Sprague-Dawley rats underwent a mandibular osteotomy, and the healing regenerate was harvested on postoperative days 3, 5, 7, 9, 23, and 37. Total cellular ribonucleic acid was isolated, and Northern analysis was performed. TGF-beta 3 expression was downregulated dramatically 3 days after the osteotomy and remained less than 20% of control levels throughout repair. In marked contrast, TIMP-1 gene expression, low during early repair, increased more than twofold over control at later time points. Understanding the temporal pattern of gene expression during membranous fracture healing has important clinical implications because elucidating these mechanisms may lead to appropriate biomolecular approaches to augment membranous bone fracture healing.

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.

Vascular disruption secondary to fracture leads to a hypoxic zone of injury where the oxygen tension at the center of the wound is quite low. In this dynamic microenvironment, a number of growth factors are elaborated to stimulate the synthetic processes of fracture repair. Previously the authors have shown the hypoxia-induced increase of vascular endothelial growth factor expression in osteoblasts. The purpose of these experiments was to examine osteoblast expression of insulinlike growth factors (IGF) I and II-cytokines believed to play a role in increased collagen synthesis, chemotaxis, and proliferation of osteoblasts in response to hypoxia. Primary cell cultures of osteoblasts isolated from neonatal rat calvaria were subjected to hypoxia (PO 2 = 35 mmHg) for 0, 3, 6, 24, and 48 hours. Northern blot analysis of ribonucleic acid (RNA) from resulting cultures demonstrated a more than 60% increase in IGF-II messenger RNA (mRNA) expression after 3 hours of hypoxia. IGF-II mRNA expression continued to increase through later time points to 200% and 260% of baseline at 24 and 48 hours respectively. In contrast, IGF-I demonstrated no significant change in mRNA expression compared with baseline control (normoxia) cultures. In these experiments the authors have demonstrated a hypoxia-induced increase in IGF-II but not IGF-I in primary osteoblasts. The differential expression of these two growth factors may underscore important differences in the behavior of osteoblasts in the hypoxic fracture microenvironment. Taken together, these data add additional support to the theory that hypoxia induces gene-specific changes in expression of molecules important to extracellular matrix formation for successful bone healing.

Poorly healing mandibular fractures and osteotomies can be troublesome complications of craniomaxillofacial trauma and reconstructive surgery. Gene therapy may offer ways of enhancing bone formation by altering the expression of desired growth factors and extracellular matrix molecules. The elucidation of suitable candidate genes for therapeutic intervention necessitates investigation of the endogenously expressed patterns of growth factors during normal (i.e., successful) fracture repair. Transforming growth factor beta1 (TGF-beta1), its receptor (Tbeta-RII), and the extracellular matrix proteins osteocalcin and type I collagen are thought to be important in long-bone (endochondral) formation, fracture healing, and osteoblast proliferation. However, the spatial and temporal expression patterns of these molecules during membranous bone repair remain unknown. In this study, 24 adult rats underwent mandibular osteotomy with rigid external fixation. In addition, four identically treated rats that underwent sham operation (i.e., no osteotomy) were used as controls. Four experimental animals were then killed at each time point (3, 5, 7, 9, 23, and 37 days after the procedure) to examine gene expression of TGF-beta1 and Tbeta-RII, osteocalcin, and type I collagen. Northern blot analysis was used to compare gene expression of these molecules in experimental animals with that in control animals (i.e., nonosteotomized; n = 4). In addition, TGF-beta1 and T-RII proteins were immunolocalized in an additional group of nine animals killed on postoperative days 3, 7, and 37. The results of Northern blot analysis demonstrated a moderate increase (1.7 times) in TGF-beta1 expression 7 days postoperatively; TGF-beta1 expression returned thereafter to near baseline levels. Tbeta-RII mRNA expression was downregulated shortly after osteotomy but then increased, reaching a peak of 1.8 times the baseline level on postoperative day 9. Osteocalcin mRNA expression was dramatically downregulated shortly after osteotomy and remained low during the early phases of fracture repair. Osteocalcin expression trended slowly upward as healing continued, reaching peak expression by day 37 (1.7 times the control level). In contrast, collagen type IalphaI mRNA expression was acutely downregulated shortly after osteotomy, peaked on postoperative days 5, and then decreased at later time points. Histologic samples from animals killed 3 days after osteotomy demonstrated TGF-beta1 protein localized to inflammatory cells and extracellular matrix within the fracture gap, periosteum, and peripheral soft tissues. On postoperative day 7, TGF-beta1 staining was predominantly localized to the osteotomized bone edges, periosteum, surrounding soft tissues, and residual inflammatory cells. By postoperative day 37, complete bony healing was observed, and TGF-beta1 staining was localized to the newly formed bone matrix and areas of remodeling. On postoperative day 3, Tbeta-RII immunostaining localized to inflammatory cells within the fracture gap, periosteal cells, and surrounding soft tissues. By day 7, Tbeta-RII staining localized to osteoblasts of the fracture gap but was most intense within osteoblasts and mesenchymal cells of the osteotomized bone edges. On postoperative day 37, Tbeta-RII protein was seen in osteocytes, osteoblasts, and the newly formed periosteum in the remodeling bone. These observations agree with those of previous in vivo studies of endochondral bone formation, growth, and healing. In addition, these results implicate TGF-beta1 biological activity in the regulation of osteoblast migration, differentiation, and proliferation during mandibular fracture repair. Furthermore, comparison of these data with gene expression during mandibular distraction osteogenesis may provide useful insights into the treatment of poorly healing fractures because distraction osteogenesis has been shown to be effective in the management of these difficult clinical cases.

Angiogenesis is essential for the increased delivery of oxygen and nutrients required for the reparative processes of bone healing. Vascular endothelial growth factor (VEGF), a potent angiogenic growth factor, has been implicated in this process. We have previously shown that hypoxia specifically and potently regulates the expression of VEGF by osteoblasts. However, the molecular mechanisms governing this interaction remain unknown. In this study, we hypothesized that the hypoxic regulation of VEGF expression by osteoblasts occurs via an oxygen-sensing mechanism similar to the regulation of the erythropoietin gene (EPO). To test this hypothesis, we examined the kinetics of oxygen concentration on osteoblast VEGF expression. In addition, we analyzed the effects of nickel and cobalt on the expression of VEGF in osteoblastic cells because these metallic ions mimic hypoxia by binding to the heme portion of oxygen-sensing molecules. Our results indicated that hypoxia potently stimulates VEGF mRNA expression. In addition, we found that nickel and cobalt both stimulate VEGF gene expression in a similar time- and dose-dependent manner, suggesting the presence of a hemelike oxygen-sensing mechanism similar to that of the EPO gene. Moreover, actinomycin D, cycloheximide, dexamethasone, and mRNA stabilization studies collectively established that this regulation is predominantly transcriptional, does not require de novo protein synthesis, and is not likely mediated by the transcriptional activator AP-1. These studies demonstrate that hypoxia, nickel, and cobalt regulate VEGF expression in osteoblasts via a similar mechanism, implicating the involvement of a heme-containing oxygen-sensing molecule. This may represent an important mechanism of VEGF regulation leading to increased angiogenesis in the hypoxic microenvironment of healing bone.

The ability to avoid a subsequent bone graft makes the use of gingivoperiosteoplasty (GPP) at the time of cleft lip repair an attractive technique. The use of GPP, in combination with presurgical orthodontics, has been shown to result in successful bony union in the majority of patients. However, secondary bone grafting is still necessary in 30% to 40% of patients due to persistent alveolar bony defects. The elucidation of methods to improve the success rates of these procedures has been hampered by the lack of reproducible animal models. The purpose of this study was, therefore, to develop a rodent model of GPP that would facilitate the investigation of methods to improve osteogenesis in alveolar defects. We report a surgically produced rat model (9 x 5 x 3-mm alveolar defect) that is reproducible, inexpensive (relative to large-animal models), and simple technically. In addition, healing in this model occurs in a predictable manner during a 12-week period, thus enabling analysis of methods designed to accelerate or facilitate osseous regeneration.

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.

To date, many efforts to engineer cartilage have focused on matrix construction with the goal of producing a durable construct as cartilage replaces the resorbing matrix. However, the importance of matrix construction is at least matched by the challenge of efficient chondrocyte extraction, culture expansion, and prevention of dedifferentiation. This challenge is underscored by the large number of chondrocytes needed for a clinically significant construct such as an ear. Because human rib provides a large, readily available source of hyaline cartilage, the authors evaluated human rib chondrocyte extraction and found that maximum viable cell yield occurred after a 6-hour digestion. They also evaluated human microtic auricular remnant chondrocyte extraction and identified fibroblast contamination as a shortcoming of this potential source of chondrocytes. Initially, rib chondrocytes proliferated in vitro with a doubling time of approximately 1 week. As the cells were passaged, proliferation decreased such that the cells stopped proliferating and adopted a large, spindle-shaped morphology by passage 6. Interestingly, no increase in proliferation was noted when rib chondrocytes were stimulated with transforming growth factor beta 1, bone morphogenetic protein 2, and basic fibroblast growth factor. The major obstacles to the use of autologous rib chondrocytes in matrix construction are the low cell yield from a small piece of rib and the limited proliferation that these cells will undergo in vitro. Further investigation of culture systems and mitogenic cytokines may help resolve these limitations.

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.

A number of granulomatous lung diseases, including sarcoidosis, are associated with an increase in angiotensin-converting enzyme (ACE) activity. To evaluate whether the amount of ACE activity reflects the burden of granulomas, we used an animal model of granulomatous lung disease caused by injecting killed Mycobacterium butyricum intravenously into mice after a primary intracutaneous injection with Freund's complete adjuvant. In this model, essentially all of the granulomas are present in the lung. RNA was isolated from one lung and assayed for ACE mRNA. The other lung was evaluated histologically; the area of lung occupied by granulomas was determined by morphometry. We found that total lung RNA and RNA specific for ACE increased in close correlation with the area of granulomas. These findings provide direct evidence that the amount of ACE activity in granulomatous disease reflects the total burden of granulomas. This may be a useful model to further evaluate ACE regulation in granulomatous lung disease.

Neoangiogenesis is essential for successful wound repair. Platelets are among the earliest cells recruited to a site of skeletal injury and are thought to provide numerous factors critical to successful repair. The release of platelet-derived growth factor (PDGF) after skeletal injury increases osteoblast proliferation, chemotaxis, and collagen synthesis; however, its angiogenic effect on osteoblast biology remains unknown. The purpose of this study was to investigate the effect of recombinant human (rh)PDGF-BB on the synthesis of vascular endothelial growth factor (VEGF) by primary neonatal rat calvarial osteoblasts. Furthermore, the authors investigated whether PDGF works in concert with hypoxia, another component of the fracture microenvironment, to additively or synergistically induce VEGF production. Osteoblast cultures were stimulated with varying concentrations of rhPDGF-BB (1, 10, 50, and 100 ng/ml) in normoxic and hypoxic (

Mandibular fractures, resulting from either trauma or reconstructive surgery, can be challenging craniofacial problems. The morbidity of failed fracture healing is significant and may require bone grafting. Donor site morbidity and finite amounts of autogenous bone are major drawbacks of autogenous bone grafting. Similarly, the use of allografts and xenografts may be associated with an increased risk of rejection, infection, and nonunion. To circumvent the limitations of bone grafting, research efforts have focused on formulating a suitable bone substitute. The purpose of our study was to evaluate the efficacy of type I collagen implants in repairing critical sized mandibular defects in rats. Twelve male Sprague-Dawley rats (200-300g) were divided equally into control and experimental groups. Full thickness, round, four millimeter in diameter defects were created in the ramus of the right mandible of all rats using an electrical burr at low speed. The defects were irrigated of all bone chips, and either filled with a precisely fitted disk of allogenic collagen type I gel (experimental animals) or left empty (control animals). Animals were killed 6 weeks after surgery and healing of the bone defects was assessed in a blinded fashion using radiologic and histologic analysis. Radiologic analysis of the control group revealed a clear circular right mandibular defect in all animals, whereas the collagen disk implant group revealed an indistinct to nonexistent right mandibular defect in all animals. Densitometric analysis revealed a significant difference between these groups (* P = 0.01). Similarly, gross analysis of control mandibles revealed a 4mm round, soft-tissue filled defect, while implanted defects demonstrated gross bone spanning the defect. Finally, histologic analysis of all control mandibles revealed clearly demarcated bony edges at the defect border with connective tissue spanning the defect. In contrast, histological analysis of all implanted mandibles revealed indistinct bony edges at the defect border with a thin layer of osteoblasts and viable bone spanning the defects. We have demonstrated the ability of type I collagen to promote healing of a membranous bony defect that would not otherwise heal at 6 weeks. The suitability of type I collagen as a carrier matrix provides ample opportunity for tissue-engineered approaches to further facilitate bony defect healing. Promoting bone formation through tissue engineering matrices offers great promise for skeletal healing and reconstruction.

Vascular disruption secondary to fracture creates a hypoxic gradient of injury wherein the oxygen tension at the center of the wound is very low. In vivo this hypoxic microenvironment stimulates the expression of a variety of cytokines from inflammatory cells, fibroblasts, endothelial cells, and osteoblasts. In order to begin to dissect this complex system, we have examined the effects of hypoxia on isolated osteoblast gene expression in vitro. Understanding gene expression in this system may facilitate the development of targeted therapeutic modalities designed to accelerate fracture repair and reduce complications. Using an established model of in vitro hypoxia, we have analyzed the expression of genes involved in bone matrix production and turnover. Subconfluent neonatal rat calvarial osteoblasts were exposed to hypoxia (pO(2) = 35-40 mm Hg) and total cellular RNA was collected at 0, 3, 6, 24, and 48 h. Northern analysis was used to analyze the expression patterns of (1) transforming growth factors (TGFs)-beta1, -beta2, and -beta3 and their type I receptor; (2) collagens I and III; and (3) tissue inhibitor of metalloproteinase-1. We have demonstrated a marked elevation of TGF-beta1 gene expression within 3 h of hypoxia. Although neither TGF-beta2 nor TGF-beta3 expression was affected by hypoxia, the TGF-beta type I receptor was substantially upregulated within 6 h. In addition, extracellular matrix scaffolding molecules (collagens I and III) were markedly, but differentially, upregulated. Finally, we have demonstrated that the expression of an inhibitor of extracellular matrix turnover, the tissue inhibitor of metalloproteinase-1, was strikingly decreased in response to hypoxia. These results imply that hypoxia can affect osseous healing by altering the expression of cytokines, bone-specific extracellular matrix molecules, and their regulators.

Distraction osteogenesis is a well-established method of endogenous tissue engineering. This technique has significantly augmented our armamentarium of reconstructive craniofacial procedures. Although the histologic and ultrastructural changes associated with distraction osteogenesis have been extensively described, the molecular mechanisms governing successful membranous distraction remain unknown. Using an established rat model, the molecular differences between successful (i.e., osseous union with gradual distraction) and ineffective (i.e., fibrous union with acute lengthening) membranous bone lengthening was analyzed. Herein, the first insight into the molecular mechanisms of successful membranous bone distraction is provided. In addition, these data provide the foundation for future targeted therapeutic manipulations designed to improve osseous regeneration. Vertical mandibular osteotomies were created in 52 adult male Sprague-Dawley rats, and the animals were fitted with customized distraction devices. Twenty-six animals underwent immediate acute lengthening (3 mm; a length previously shown to result in fibrous union) and 26 animals were gradually distracted (after a 3-day latency period, animals were distracted 0.25 mm twice daily for 6 days; total = 3 mm). Four mandibular regenerates were harvested from each group for RNA analysis on 5, 7, 9, 23, and 37 days postoperatively (n = 40). Two mandibular regenerates were also harvested from each group and prepared for immunohistochemistry on postoperative days 5, 7, and 37 (n = 12). In addition to the 52 experimental animals, 4 control rats underwent sham operations (skin incision only) and mandibular RNA was immediately collected. Control and experimental specimens were analyzed for collagen I, osteocalcin, tissue inhibitor of metalloproteinase-1, and vascular endothelial growth factor mRNA and protein expression. In this study, marked elevation of critical extracellular matrix molecules (osteocalcin and collagen I) during the consolidation phase of gradual distraction compared with acute lengthening is demonstrated. In addition, the expression of an inhibitor of extracellular matrix turnover, tissue inhibitor of metalloproteinase-1, remained strikingly elevated in gradually distracted animals. Finally, this study demonstrated that neither gradual distraction nor acute lengthening appreciably alters vascular endothelial growth factor expression. These results suggest that gradual distraction osteogenesis promotes successful osseous bone repair by regulating the expression of bone-specific extracellular matrix molecules. In contrast, decreased production or increased turnover of bone scaffolding proteins (i.e., collagen) or regulators of mineralization (i.e., osteocalcin) may lead to fibrous union during acute lengthening.

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 well-described detrimental effects of ionizing radiation on the regeneration of bone within a fracture site include decreased osteocyte number, suppressed osteoblast activity, and diminished vascularity. However, the biologic mechanisms underlying osteoradionecrosis and the impaired fracture healing of irradiated bone remain undefined. Ionizing radiation may decrease successful osseous repair by altering cytokine expression profiles resulting from or leading to a change in the osteoblastic differentiation state. These changes may, in turn, cause alterations in osteoblast proliferation and extracellular matrix formation. The purpose of this study was to investigate the effects of ionizing radiation on the proliferation, maturation, and cytokine production of MC3T3-E1 osteoblast-like cells in vitro. Specifically, the authors examined the effects of varying doses of ionizing radiation (0, 40, 400, and 800 cGy) on the expression of transforming growth factor-beta1 (TGF-beta1), vascular endothelial growth factor (VEGF), and alkaline phosphatase. In addition, the authors studied the effects of ionizing radiation on MC3T3-E1 cellular proliferation and the ability of conditioned media obtained from control and irradiated cells to regulate the proliferation of bovine aortic endothelial cells. Finally, the authors evaluated the effects of adenovirus-mediated TGF-beta1 gene therapy in an effort to "rescue" irradiated osteoblasts. The exposure of osteoblast-like cells to ionizing radiation resulted in dose-dependent decreases in cellular proliferation and promoted cellular differentiation (i.e., increased alkaline phosphatase production). Additionally, ionizing radiation caused dose-dependent decreases in total TGF-beta1 and VEGF protein production. Decreases in total TGF-beta1 production were due to a decrease in TGF-beta1 production per cell. In contrast, decreased total VEGF production was secondary to decreases in cellular proliferation, because the cellular production of VEGF by irradiated osteoblasts was moderately increased when VEGF production was corrected for cell number. Additionally, in contrast to control cells (i.e., nonirradiated), conditioned media obtained from irradiated osteoblasts failed to stimulate the proliferation of bovine aortic endothelial cells. Finally, transfection of control and irradiated cells with a replication-deficient TGF-beta1 adenovirus before irradiation resulted in an increase in cellular production of TGF-beta1 protein and VEGF. Interestingly, this intervention did not alter the effects of irradiation on cellular proliferation, which implies that alterations in TGF-beta1 expression do not underlie the deficiencies noted in cellular proliferation. The authors hypothesize that ionizing radiation-induced alterations in the cytokine profiles and differentiation states of osteoblasts may provide insights into the cellular mechanisms underlying osteoradionecrosis and impaired fracture healing.

Gene therapy has moved from the promise of laboratory investigation to the reality of clinical practice in just the last decade. Various methods for delivery of genes to host cells have been developed and utilized both in vitro and in vivo. From the perspective of the plastic surgeon, gene therapy holds the promise to augment healing in clinical situations that remain difficult to treat, such as chronic wounds, osteoradionecrosis, or possibly to expedite current clinical practices, such as distraction osteogenesis. The authors chose to investigate the potential for gene therapy in osseous tissues using a replication-deficient adenovirus vector to deliver the marker transgene beta-galactosidase. An adenovirus vector is ideal for use in situations in which transgene expression is desired for only a relatively short period of time, such as wound and fracture healing. Utilizing a rat mandibular osteotomy model, they demonstrated that, using an adenoviral vector, foreign genes can be delivered in a simple fashion and can be expressed in a reliable manner within and around the osteotomy site for at least 10 days. Furthermore, there was no evidence of transfection of distant tissues associated with local application of the adenovirus vector. With this information, clinicians may now attempt to deliver osteogenic and angiogenic genes in a site-specific fashion to improve and expedite osseous healing.

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.

Modulation of biological pathways governing osteogenesis may accelerate osseous regeneration and reduce the incidence of complications associated with fracture healing. Transforming growth factor beta1 (TGF-beta1) is a potent growth factor implicated in the regulation of osteogenesis and fracture repair. The use of recombinant proteins, however, has significant disadvantages and has limited the clinical utility of these molecules. Targeted gene therapy using adenovirus vectors is a technique that may circumvent difficulties associated with growth factor delivery. In this study, we investigate the efficacy of replication-deficient adenoviruses containing the human TGF-beta1 and the bacterial lacZ genes in transfecting osteoblasts in vitro and osseous tissues in vivo. We demonstrate that adenovirus-mediated gene therapy efficiently transfects osteoblasts in vitro with the TGF-beta1 virus causing a marked up-regulation in TGF-beta1 mRNA expression even 7 days after transfection. Increased TGF-beta1 mRNA expression was efficiently translated into protein production and resulted in approximately a 46-fold increase in TGF-beta1 synthesis as compared with control cells (vehicle- or B-galactosidase-transfected). Moreover, virally produced TGF-beta1 was functionally active and regulated the expression of collagen IalphaI (5-fold increase) and the vascular endothelial growth factor (2.5-fold increase). Using an adenovirus vector encoding the Escherichia coli LacZ gene, we demonstrated that adenovirus-mediated gene transfer efficiently transfects osteoblasts and osteocytes in vivo and that transfection can be performed by a simple percutaneous injection. Finally, we show that delivery of the hTGF-beta1 gene to osseous tissues in vivo results in significant changes in the epiphyseal plate primarily as a result of increased thickness of the provisional calcification zone.

Recruitment of a blood supply is critical for successful bone induction and fracture healing. Despite the clinical success of distraction osteogenesis (DO), an analysis of angiogenesis during membranous bone DO has not been performed. The purpose of this study was to evaluate the temporal and spatial pattern of angiogenesis during mandibular DO. The right hemimandible of adult male rats was osteotomized, and a customized distraction device was applied. Following a 3-day latency period, distraction was begun at a rate of 0.25 mm twice daily for 6 days (3.0 mm total; 12% increase in mandibular length). Three animals each were sacrificed on days 2, 4, and 6 of distraction (D1, D2, and D3 respectively), or after 1, 2, or 4 weeks of consolidation (C1, C2, and C3 respectively). Two experienced pathologists reviewed the regenerate histology, and angiogenesis was assessed by counting the number of blood vessels per intermediate-power field (IPF). Statistical analysis was performed using analysis of variance, with p < or = 0.05 considered significant. Results demonstrate that mandibular DO was associated with an intense vascular response during the early stages of distraction (D1). On average, 31.5+/-7.9 vessels were noted in each IPF examined during this time point. The number of blood vessels in the distraction regenerate decreased significantly during the later distraction time points, with approximately 14.0+/-2.0 and 14.7+/-3.5 blood vessels per IPF in sections obtained after days 4 and 6 of distraction (D2, D3) respectively. However, blood vessels at these time points took on a more mature histological pattern. During the consolidation period, the number of blood vessels noted in the regenerate decreased with 8.0+/-2.6, 9.3+/-2.1, and 4.0+/-2.0 vessels per IPF in sections obtained after 1, 2, or 4 weeks of consolidation (C1, C2, C3) respectively (p < 0.05 compared with vessel counts during the earliest distraction time point). This study demonstrates for the first time that an intense vascular response associated with mandibular DO occurs primarily during the early stages of distraction. The authors hypothesize that as distraction continues, newly formed vessels likely undergo consolidation, thus forming more mature vessels capable of withstanding distraction forces. Future studies will assess the effects of therapeutic interventions designed to increase angiogenesis during DO on bony regenerate formation.

The etiology of craniosynostosis is unknown. The elucidation of the biological pathways responsible for this disorder has been hampered by an inability to evaluate cranial sutures before, during, and after cranial suture fusion. The programmed fusion of the rat posterofrontal (PF) suture postnatally provides an excellent model to study the molecular events that occur during cranial suture fusion. Previous experiments have implicated transforming growth factor beta (TGF-beta) growth factors in the regulation of PF suture fusion. The purpose of these experiments was to localize the expression of high-affinity receptors for these growth factors during cranial suture fusion. Four rats were sacrificed on postnatal days 8, 12, 17, and 40 (N = 16). The PF and sagittal sutures were harvested and prepared for immunohistochemical localization of TGF-beta receptor 1 and receptor 2 (Tbeta-RI, Tbeta-RII) protein. Results indicate that immunostaining for Tbeta-RI and Tbeta-RII is markedly increased in the dura mater and osteoblasts of the sutural margin of the PF suture during active suture fusion (on postnatal days 12, 17, and 40) compared with the osteoblasts and dura mater underlying the patent sagittal suture. These results, in combination with the authors' previous findings as well as studies supporting a role for TGF-beta molecules in the regulation of osteogenesis, implicate TGF-beta signaling in the regulation of suture fusion. The possible mechanisms of ligand-receptor interaction are discussed.

Distraction osteogenesis is a powerful technique capable of generating viable osseous tissue by the gradual separation of osteotomized bone edges. Although the histologic and ultrastructural changes associated with this process have been extensively delineated, the molecular events governing these changes remain essentially unknown. We have devised a rat model of mandibular distraction osteogenesis that facilitates molecular analysis of this process. Such information has significant clinical implications because it may enable targeted therapeutic manipulations designed to accelerate osseous regeneration. In this study, we have evaluated the expression of transforming growth factor beta-1, a major regulator of osteogenesis during endochondral bone formation and development, and osteocalcin, an abundant noncollagenous extracellular matrix protein implicated in the regulation of mineralization and bone turnover. The right hemimandible of 36 adult male rats was osteotomized, and a customized distraction device was applied. Animals were allowed to recover and, after a 3-day latency period, were distracted at a rate of 0.25 mm twice daily for 6 days followed by a 2- or 4-week consolidation period. Distraction regenerate was harvested after the latency period, days 2, 4, or 6 of distraction, and after 2 or 4 weeks of consolidation and processed for Northern analysis (n = 4 at each time point) and immunohistochemical localization of TGF-beta1 (n = 2 at each time point). Six sham-operated animals (i.e., skin incision without osteotomy) were also killed (immediately postoperatively), and the mandibles were harvested and prepared in a similar fashion. Equal loading and transfer of RNA for Northern analysis was ensured by stripping and probing membranes with a probe against GAPDH (a housekeeping gene). Our results demonstrate that the spatial and temporal patterns of TGF-beta1 mRNA expression and protein production coincide with osteoblast migration, differentiation, and extracellular matrix synthesis. In addition, we demonstrate that TGF-beta1 production may be an important regulator of vasculogenesis during mandibular distraction osteogenesis. Finally, we have shown that osteocalcin gene expression coincides temporally with mineralization during rat mandibular distraction osteogenesis.

Hemangiomas are benign vascular tumors of childhood that can lead to disfigurement and/or life-threatening consequences. The pathogenesis of hemangioma formation is likely to involve increased angiogenesis. Basic fibroblast growth factor and vascular endothelial growth factor are cytokines that stimulate angiogenesis in multiple in vivo and in vitro models. Proliferative hemangiomas have been found to have elevated levels of basic fibroblast growth factor and vascular endothelial growth factor protein, but the gene expression of these cytokines in human specimens has not been previously studied. We examined the gene expression and spatial distribution of basic fibroblast growth factor and vascular endothelial growth factor messenger RNA in proliferative versus involuted human hemangioma specimens using nonisotopic in situ hybridization techniques. Thirteen hemangioma specimens were harvested during initial surgical excision. In situ hybridization was performed on frozen sections of both proliferative and involuted hemangioma specimens using genetically engineered antisense probes specific for basic fibroblast growth factor and vascular endothelial growth factor messenger RNA. Controls were an interleukin-6 sense sequence and a transforming growth factor-beta 1 antisense sequence. A large number of cells within the specimens of proliferative hemangiomas revealed localized gene expression of basic fibroblast growth factor and vascular endothelial growth factor messenger RNA (626 +/- 129 and 1660 +/- 371 cells/mm2, respectively). The majority of the cells were endothelial in origin. In contrast, involuted hemangioma specimens revealed significantly lower numbers of cells staining positive for basic fibroblast growth factor and vascular endothelial growth factor messenger RNA (44 +/- 11 and 431 +/- 76 cells/mm2, respectively; p < 0.05). Transforming growth factor-beta 1 messenger RNA was slightly more expressed by involuted hemangiomas (117 +/- 30 cells/mm2). There were very low levels of transforming growth factor-beta 1 gene expression from proliferative hemangiomas (37 +/- 24 cells/mm2; p < 0.02). These data demonstrate that (1) in situ hybridization allows identification and relative quantitation of cells expressing messenger RNA for specific growth factors in human hemangioma specimens; (2) basic fibroblast growth factor and vascular endothelial growth factor messenger RNA are up-regulated in proliferative hemangiomas; and (3) transforming growth factor-beta 1 messenger RNA remains low in both proliferative and involuted hemangiomas. Because basic fibroblast growth factor and vascular endothelial growth factor messenger RNA have been implicated in the pathobiology of human hemangioma formation, biochemical modulation of these angiogenic cytokines may eventually help inhibit proliferation and promote regression of hemangiomas.