ECM concentration and cell-mediated traction forces play a role in vascular network assembly in 3D bioprinted tissue.

Tissue vascularization is critical to enable oxygen and nutrient supply. Therefore, establishing expedient vasculature is necessary for the survival of tissue after transplantation. The use of biomechanical forces, such as cell-induced traction forces, may be a promising method to encourage growth of the vascular network. Three-dimensional (3D) bioprinting, which offers unprecedented versatility through precise control over spatial distribution and structure of tissue constructs, can be used to generate capillary-like structures in vitro that would mimic microvessels. This study aimed to develop an in vitro, 3D bioprinted tissue model to study the effect of cellular forces on the spatial organization of vascular structures and tissue maturation. The developed in vitro model consists of a 3D bioprinted polycaprolactone (PCL) frame with a gelatin spacer hydrogel layer and a gelatin-fibrin-hyaluronic acid hydrogel layer containing normal human dermal fibroblasts and human umbilical vein endothelial cells printed as vessel lines on top. The formation of vessel-like networks and vessel lumens in the 3D bioprinted in vitro model was assessed at different fibrinogen concentrations with and without inhibitors of cell-mediated traction forces. Constructs containing 5 mg/ml fibrinogen had longer vessels compared to the other concentrations of fibrinogen used. Also, for all concentrations of fibrinogen used, most of the vessel-like structures grew parallel to the direction the PCL frame-mediated tensile forces, with very few branching structures observed. Treatment of the 3D bioprinted constructs with traction inhibitors resulted in a significant reduction in length of vessel-like networks. The 3D bioprinted constructs also had better lumen formation, increased collagen deposition, more elaborate actin networks, and well-aligned matrix fibers due to the increased cell-mediated traction forces present compared to the non-anchored, floating control constructs. This study showed that cell traction forces from the actomyosin complex are critical for vascular network assembly in 3D bioprinted tissue. Strategies involving the use of cell-mediated traction forces may be promising for the development of bioprinting approaches for fabrication of vascularized tissue constructs.

Multicellular bioprinted skin facilitates human-like skin architecture in vivo.

Bioprinting is a promising alternative method to generate skin substitutes because it can replicate the structural organization of the skin into biomimetic layers in vitro. In this study, six primary human skin cell types were used to bioprint a trilayer skin construct consisting of epidermis, dermis, and hypodermis. Transplantation of the bioprinted skin with human cells onto full-thickness wounds of nu/nu mice promoted rapid vascularization and formation of epidermal rete ridges analogous to the native human epidermis, with a normal-looking extracellular matrix. Cell-specific staining confirmed the integration of the implanted cells into the regenerated skin. Using a similar approach, a 5 centimeter-by-5 centimeter bioprinted autologous porcine skin graft was transplanted onto full-thickness wounds in a porcine excisional wound model. The bioprinted skin graft improved epithelialization, reduced skin contraction, and supported normal collagen organization with reduced fibrosis. Differential gene expression demonstrated pro-remodeling protease activity in wounds transplanted with bioprinted autologous skin grafts. These results demonstrate that bioprinted skin can support skin regeneration to allow for nonfibrotic wound healing and suggest that the skin bioprinting technology may be applicable for human clinical use.

Small leucine-rich proteoglycans, decorin and fibromodulin, are reduced in postburn hypertrophic scar.

Small leucine-rich proteoglycans (SLRPs) are extracellular matrix molecules that regulate collagen fibrillogenesis and inhibit transforming growth factor-ß activity; thus, they may play a critical role in wound healing and scar formation. Hypertrophic scarring is a dermal form of fibroproliferative disorders, which occurs in over 70% of burn patients and leads to disfigurement and limitations in function. By understanding the cellular and molecular mechanisms that lead to scarring after injury, new clinical therapeutic approaches can by developed to minimize abnormal scar formation in hypertrophic scarring and other fibroproliferative disorders. To study the expression and localization of SLRPs with connective tissue cells in tissue immunohistochemistry, immunofluorescence staining, immunoblotting, and reverse-transcription polymerase chain reaction were used in normal skin and hypertrophic scar (HTS). In normal skin, there was more decorin and fibromodulin accumulation in the superficial layers than in the deeper dermal layers. The levels of decorin and fibromodulin were significantly lower in HTS, whereas biglycan was increased when compared with normal skin. There was an increased expression of biglycan, fibromodulin, and lumican in the basement membrane and around basal epithelial cells. In contrast, these proteoglycans were absent or weakly expressed in HTS. The findings suggest that down-regulation of SLRPs after wound healing in deep injuries to the skin plays an important role in the development of fibrosis and HTS.

Bioprinting small diameter blood vessel constructs with an endothelial and smooth muscle cell bilayer in a single step.

Bioengineered artificial blood vessels have been a major area of interest over the last decade. Of particular interest are small diameter vessels, as surgical options are currently limited. This study aimed to fabricate a small diameter, heterogeneous bilayer blood vessel-like construct in a single step with gelatin methacryloyl (GelMA) bioink using a 3D micro-extrusion bioprinter on a solid platform. GelMA was supplemented with Hyaluronic acid (HA), glycerol and gelatin to form a GelMA bioink with good printability, mechanical strength, and biocompatibility. Two separate concentrations of GelMA bioink with unique pore sizes were selected to fabricate a heterogeneous bilayer. A higher concentration of GelMA bioink (6% w/v GelMA, 2% gelatin, 0.3% w/v HA, 10% v/v glycerol) was used to load human umbilical vein endothelial cells (HUVECs) and form an inner, endothelial tissue layer. A lower concentration of GelMA bioink (4% w/v GelMA, 4% gelatin, 0.3% w/v HA, 10% v/v glycerol) was used to load smooth muscle cells (SMCs) and form an outer, muscular tissue layer. Bioprinted blood vessel-like grafts were then assessed for mechanical properties with Instron mechanical testing, and suture-ability, and for biological properties including viability, proliferation, and histological analysis. The resulting 20 mm long, 4.0 mm diameter lumen heterogeneous bilayer blood vessel-like construct closely mimics a native blood vessel and maintains high cell viability and proliferation. Our results represent a novel strategy for small diameter blood vessel biofabrication.

Bioprinted Skin Recapitulates Normal Collagen Remodeling in Full-Thickness Wounds.

Over 1 million burn injuries are treated annually in the United States, and current tissue engineered skin fails to meet the need for full-thickness replacement. Bioprinting technology has allowed fabrication of full-thickness skin and has demonstrated the ability to close full-thickness wounds. However, analysis of collagen remodeling in wounds treated with bioprinted skin has not been reported. The purpose of this study is to demonstrate the utility of bioprinted skin for epidermal barrier formation and normal collagen remodeling in full-thickness wounds. Human keratinocytes, melanocytes, fibroblasts, dermal microvascular endothelial cells, follicle dermal papilla cells, and adipocytes were suspended in fibrinogen bioink and bioprinted to form a tri-layer skin structure. Bioprinted skin was implanted onto 2.5 × 2.5 cm full-thickness excisional wounds on athymic mice, compared with wounds treated with hydrogel only or untreated wounds. Total wound closure, epithelialization, and contraction were quantified, and skin samples were harvested at 21 days for histology. Picrosirius red staining was used to quantify collagen fiber orientation, length, and width. Immunohistochemical (IHC) staining was performed to confirm epidermal barrier formation, dermal maturation, vascularity, and human cell integration. All bioprinted skin treated wounds closed by day 21, compared with open control wounds. Wound closure in bioprinted skin treated wounds was primarily due to epithelialization. In contrast, control hydrogel and untreated groups had sparse wound coverage and incomplete closure driven primarily by contraction. Picrosirius red staining confirmed a normal basket weave collagen organization in bioprinted skin-treated wounds compared with parallel collagen fibers in hydrogel only and untreated wounds. IHC staining at day 21 demonstrated the presence of human cells in the regenerated dermis, the formation of a stratified epidermis, dermal maturation, and blood vessel formation in bioprinted skin, none of which was present in control hydrogel treated wounds. Bioprinted skin accelerated full-thickness wound closure by promoting epidermal barrier formation, without increasing contraction. This healing process is associated with human cells from the bioprinted skin laying down a healthy, basket-weave collagen network. The remodeled skin is phenotypically similar to human skin and composed of a composite of graft and infiltrating host cells. Impact statement We have demonstrated the ability of bioprinted skin to enhance closure of full-thickness wounds through epithelialization and normal collagen remodeling. To our knowledge, this article is the first to quantify collagen remodeling by bioprinted skin in full-thickness wounds. Our methods and results can be used to guide further investigation of collagen remodeling by tissue engineered skin products to improve ongoing and future bioprinting skin studies. Ultimately, our skin bioprinting technology could translate into a new treatment for full-thickness wounds in human patients with the ability to recapitulate normal collagen remodeling in full-thickness wounds.

Skin bioprinting: the future of burn wound reconstruction?

Burns are a significant cause of trauma, and over the years, the focus of patient care has shifted from just survival to facilitation of improved functional outcomes. Typically, burn treatment, especially in the case of extensive burn injuries, involves surgical excision of injured skin and reconstruction of the burn injury with the aid of skin substitutes. Conventional skin substitutes do not contain all skin cell types and do not facilitate recapitulation of native skin physiology. Three-dimensional (3D) bioprinting for reconstruction of burn injuries involves layer-by-layer deposition of cells along with scaffolding materials over the injured areas. Skin bioprinting can be done either in situ or in vitro. Both these approaches are similar except for the site of printing and tissue maturation. There are technological and regulatory challenges that need to be overcome for clinical translation of bioprinted skin for burn reconstruction. However, the use of bioprinting for skin reconstruction following burns is promising; bioprinting will enable accurate placement of cell types and precise and reproducible fabrication of constructs to replace the injured or damaged sites. Overall, 3D bioprinting is a very transformative technology, and its use for wound reconstruction will lead to a paradigm shift in patient outcomes. In this review, we aim to introduce bioprinting, the different stages involved, in vitro and in vivo skin bioprinting, and the various clinical and regulatory challenges in adoption of this technology.

Osteogenic response of bone marrow stromal cells from normal and ovariectomized rats treated with a low dose of basic fibroblast growth factor.

Basic fibroblast growth factor (bFGF) is a potent mitogen that exhibits stimulatory effects on bone tissue regeneration. To gain further insight into the potential of bFGF for systemic therapy in osteoporosis, we investigated the responsiveness of bone marrow stromal cells (BMSCs) explanted from 7-month-old normal and ovariectomized (OVX) rats that were intravenously treated with a low dose of bFGF (25 microg/kg) for 2 weeks. The BMSCs were obtained using femoral aspiration and maintained in an osteogenic medium. The amount of cells recovered from bFGF-treated rats was lower than that from saline-treated rats, and proliferation of the cells was markedly less for the bFGF-treated rats. The BMSCs from the bFGF-treated rats also showed lower levels of specific alkaline phosphatase (ALP) activity (ALP/deoxyribonucleic acid) and mineralization. Expression of the extracellular matrix proteins critical for mineralization, in particular osteopontin, was greater for bFGF-treated cells from both types of animals in the first week of culture, after which the expression of all markers significantly declined. Dual energy x-ray absorptiometry analyses of the tibiae showed an increase in bone mineral density after bFGF treatment only for OVX rats. We conclude that osteoprogenitor cells were depleted from the marrow of bFGF-treated rats, most likely because of the stimulatory effect of bFGF on bone formation.

Advances in Skin Substitutes-Potential of Tissue Engineered Skin for Facilitating Anti-Fibrotic Healing.

Skin protects the body from exogenous substances and functions as a barrier to fluid loss and trauma. The skin comprises of epidermal, dermal and hypodermal layers, which mainly contain keratinocytes, fibroblasts and adipocytes, respectively, typically embedded on extracellular matrix made up of glycosaminoglycans and fibrous proteins. When the integrity of skin is compromised due to injury as in burns the coverage of skin has to be restored to facilitate repair and regeneration. Skin substitutes are preferred for wound coverage when the loss of skin is extensive especially in the case of second or third degree burns. Different kinds of skin substitutes with different features are commercially available; they can be classified into acellular skin substitutes, those with cultured epidermal cells and no dermal components, those with only dermal components, and tissue engineered substitutes that contain both epidermal and dermal components. Typically, adult wounds heal by fibrosis. Most organs are affected by fibrosis, with chronic fibrotic diseases estimated to be a leading cause of morbidity and mortality. In the skin, fibroproliferative disorders such as hypertrophic scars and keloid formation cause cosmetic and functional problems. Dermal fibroblasts are understood to be heterogeneous; this may have implications on post-burn wound healing since studies have shown that superficial and deep dermal fibroblasts are anti-fibrotic and pro-fibrotic, respectively. Selective use of superficial dermal fibroblasts rather than the conventional heterogeneous dermal fibroblasts may prove beneficial for post-burn wound healing.

Fibrocytes participate in the development of heterotopic ossification.

Heterotopic ossification (HO) is a complication of musculoskeletal injury characterized by the formation of mature bone in soft tissues. The etiology of HO is unknown. We investigated the role of bone marrow derived progenitor cells in HO pathophysiology. We isolated the cells from HO specimens by cell explantation. Using flow cytometry and immunofluorescence microscopy, we found that 35 to 65% of the HO cells exhibit a bone marrow derived fibrocyte profile consisting in spindle-shaped morphology associated with type 1 pro-collagen and LSP1 expression. When cultured in osteogenic differentiation medium, active machinery for bone mineralization (high gene expression of Anx2, TNAP, and Pit-1), and calcium/phosphate deposits were found. Interestingly, interferon-alpha 2b significantly reduced the proliferation rate and COL1 gene expression in HO cells. We have characterized a novel subset of bone marrow derived progenitor cells in the HO specimens. The findings from this research study will provide new insights into the development of HO in burn patients.

Growth factor delivery for bone tissue repair: an update.

Growth factors (GFs) are endogenous proteins capable of acting on cell-surface receptors and directing cellular activities involved in the regeneration of new bone tissue. The specific actions and long-term effects of GFs on bone-forming cells have resulted in exploration of their potential for clinical bone repair. The concerted efforts have led to the recent approval of two GFs, bone morphogenetic protein-2 and osteogenic protein-1, for clinical bone repair, and human parathryroid hormone (1-34) for augmentation of systemic bone mass. This review provides a selective summary of recent (2001-2004) attempts for GF delivery in bone tissue regeneration. First, a summary of non-human primate studies involving local regeneration and repair is provided, with special emphasis on the range of biomaterials used for GF delivery. Next, efforts to administer GFs for systemic augmentation of bone tissue are summarised. Finally, an alternative means of GF delivery, namely the delivery of genes coding for osteogenic proteins, rather than the delivery of the proteins, is summarised from rodent models. To conclude, future avenues of research considered promising to enhance the clinical application of GFs are discussed.

Transcatheter, Endovascular Repair of Abdominal Aortic Aneurysm: Feasibility, Noninvasive Diagnosis, Problems, and Follow-Up.

Until recently, the repair of an abdominal aortic aneurysm (AAA) required major surgery. Recently, the transcatheter technique has allowed minimally invasive endovascular stenting of infrarenal AAAs. This procedure is less traumatic and is associated with a shorter hospital stay than conservative surgery. With the stent placement, the effective aortic lumen diameter decreases and the aneurysmal space is excluded from the circulation. Ultrasonographic studies have allowed imaging of the abdominal aorta, its main branches, and the endovascular stent. The aortic blood flow after the repair is ideally limited to the stent lumen. Follow-up studies have permitted reevaluation of the aorta and the stent, with special emphasis on the aortic expansion and blood flow within the excluded space. These studies have correlated well with other imaging techniques such as intravascular ultrasound, computed tomography scanning, and aortography.

The effect of keratinocytes on the biomechanical characteristics and pore microstructure of tissue engineered skin using deep dermal fibroblasts.

Fibrosis affects most organs, it results in replacement of normal parenchymal tissue with collagen-rich extracellular matrix, which compromises tissue architecture and ultimately causes loss of function of the affected organ. Biochemical pathways that contribute to fibrosis have been extensively studied, but the role of biomechanical signaling in fibrosis is not clearly understood. In this study, we assessed the effect keratinocytes have on the biomechanical characteristics and pore microstructure of tissue engineered skin made with superficial or deep dermal fibroblasts in order to determine any biomaterial-mediated anti-fibrotic influences on tissue engineered skin. Tissue engineered skin with deep dermal fibroblasts and keratinocytes were found to be less stiff and contracted and had reduced number of myofibroblasts and lower expression of matrix crosslinking factors compared to matrices with deep fibroblasts alone. However, there were no such differences between tissue engineered skin with superficial fibroblasts and keratinocytes and matrices with superficial fibroblasts alone. Also, tissue engineered skin with deep fibroblasts and keratinocytes had smaller pores compared to those with superficial fibroblasts and keratinocytes; pore size of tissue engineered skin with deep fibroblasts and keratinocytes were not different from those matrices with deep fibroblasts alone. A better understanding of biomechanical characteristics and pore microstructure of tissue engineered skin may prove beneficial in promoting normal wound healing over pathologic healing.

Superficial dermal fibroblasts enhance basement membrane and epidermal barrier formation in tissue-engineered skin: implications for treatment of skin basement membrane disorders.

Basement membrane is a highly specialized structure that binds the dermis and the epidermis of the skin, and is mainly composed of laminins, nidogen, collagen types IV and VII, and the proteoglycans, collagen type XVIII and perlecan, all of which play critical roles in the function and resilience of skin. Both dermal fibroblasts and epidermal keratinocytes contribute to the development of the basement membrane, and in turn the basement membrane and underlying dermis influence the development and function of the epidermal barrier. Disruption of the basement membrane results in skin fragility, extensive painful blistering, and severe recurring wounds as seen in skin basement membrane disorders such as epidermolysis bullosa, a family of life-threatening congenital skin disorders. Currently, there are no successful strategies for treatment of these disorders; we propose the use of tissue-engineered skin as a promising approach for effective wound coverage and to enhance healing. Fibroblasts and keratinocytes isolated from superficial and deep dermis and epidermis, respectively, of tissue from abdominoplasty patients were independently cocultured on collagen-glycosaminoglycan matrices, and the resulting tissue-engineered skin was assessed for functional differences based on the underlying specific dermal fibroblast subpopulation. Tissue-engineered skin with superficial fibroblasts and keratinocytes formed a continuous epidermis with increased epidermal barrier function and expressed higher levels of epidermal proteins, keratin-5, and E-cadherin, compared to that with deep fibroblasts and keratinocytes, which had an intermittent epidermis. Further, tissue-engineered skin with superficial fibroblasts and keratinocytes formed better basement membrane, and produced more laminin-5, nidogen, collagen type VII, compared to that with deep fibroblasts and keratinocytes. Overall, our results demonstrate that tissue-engineered skin with superficial fibroblasts and keratinocytes forms significantly better basement membrane with higher expression of dermo-epidermal adhesive and anchoring proteins, and superior epidermis with enhanced barrier function compared to that with deep fibroblasts and keratinocytes, or with superficial fibroblasts, deep fibroblasts, and keratinocytes. The specific use of superficial fibroblasts in tissue-engineered skin may thus be more beneficial to promote adhesion of newly formed skin and wound healing, and is therefore promising for the treatment of patients with basement membrane disorders and other skin blistering diseases.

Fibrotic remodeling of tissue-engineered skin with deep dermal fibroblasts is reduced by keratinocytes.

Two-thirds of burn patients with deep dermal injuries are affected by hypertrophic scars, and currently, there are no clinically effective therapies. Tissue-engineered skin is a very promising model for the elucidation of the role of matrix microenvironment and biomechanical characteristics and could help in the identification of new therapeutic targets for hypertrophic scars. Conventionally, tissue-engineered skin is made of heterogeneous dermal fibroblasts and keratinocytes; however, recent work has shown that superficial and deep dermal fibroblasts are antifibrotic and profibrotic, respectively. Furthermore, keratinocytes are believed to regulate the development and remodeling of fibrosis in skin. This study aimed to assess the influence of keratinocytes and layered fibroblasts on the characteristics of tissue-engineered skin. Layered fibroblasts and keratinocytes isolated from superficial and deep dermis and epidermis, respectively, of the lower abdominal tissue were independently co-cultured on collagen-glycosaminoglycan scaffolds, and the resulting tissue-engineered skin was assessed for differences in tissue remodeling based on the underlying specific dermal fibroblast subpopulation. Collagen production by deep fibroblasts but not by superficial fibroblasts was significantly reduced upon co-culture with keratinocytes. Also, keratinocytes in the tissue-engineered skin resulted in significantly reduced expression of profibrotic connective tissue growth factor and fibronectin, and increased expression of the antifibrotic matrix metalloproteinase-1 by deep fibroblasts but not by superficial fibroblasts. Tissue-engineered skin made of deep fibroblasts and keratinocytes had lower levels of small proteoglycans, decorin, and fibromodulin, and higher levels of large proteoglycan, versican, compared to tissue-engineered skin made of superficial fibroblasts and keratinocytes. Tissue-engineered skin made of deep fibroblasts and keratinocytes had lower expression of transforming growth factor (TGF)-α, interleukin (IL)-1, and keratinocyte growth factor but higher expression of platelet-derived growth factor and IL-6, compared to tissue-engineered skin made of superficial fibroblasts and keratinocytes. Furthermore, co-culture with keratinocytes reduced TGF-ß1 production of deep but not superficial fibroblasts. Additionally, keratinocytes reduced the differentiation of deep fibroblasts to myofibroblasts in tissue-engineered skin constructs, but not that of superficial fibroblasts. Taken together, keratinocytes reduce fibrotic remodeling of the scaffolds by deep dermal fibroblasts. Our results therefore demonstrate that tissue-engineered skin made specifically with a homogeneous population of superficial fibroblasts and keratinocytes is less fibrotic than that with a heterogeneous population of fibroblasts and keratinocytes.

Reduced decorin, fibromodulin, and transforming growth factor-ß3 in deep dermis leads to hypertrophic scarring.

Hypertrophic scar (HTS) occurs after injuries involving the deep dermis, while superficial wounds (SWs) to the skin heal with minimal or no scarring. The levels of transforming growth factor (TGF)-ß1 and small leucine-rich proteoglycans (SLRPs) with fibroblast subtype and function may influence the development of HTS. The aim of this study was to characterize the expression and localization of factors that regulate wound healing including SLRPs, TGF-ß1, and TGF-ß3 in an experimental human SW and deep wound (DW) scar model including fibroblasts from superficial and deep layers of normal dermis. A 6-cm horizontal dermal scratch experimental wound was created, which consisted of progressively deeper wounds that were superficial at one end (0-0.75 mm deep) and deep (0.75-3 mm deep) at the other end, located on the anterior thigh of an adult male. Immunofluorescence staining, immunoblotting, reverse transcription polymerase chain reaction, and flow cytometry were performed to analyze the cellular and molecular differences between the SW scar and DW scar as well as fibroblasts isolated from superficial layer (L1) and deep layer (L5) of normal dermis. Comparing SWs and L1 fibroblasts, the expression of decorin, fibromodulin, and TGF-ß3 was considerably lower than in DWs and L5 fibroblasts; however, TGF-ß1 was higher in the deeper dermal wounds. When compared with L1 fibroblasts, L5 fibroblasts had lower Thy-1 immunoreactivity and significantly higher expression of TGF-ß receptor type II. Decreased antifibrotic molecules in matrix of deep dermis of the skin and the unique features of the associated fibroblasts including an increased sensitivity to TGF-ß1 stimulation contribute to the development of HTS after injuries involving the deep dermis.

Deep dermal fibroblasts refractory to migration and decorin-induced apoptosis contribute to hypertrophic scarring.

Hypertrophic scar (HTS) represents the dermal equivalent of fibroproliferative disorders. Fibroblasts from the deep dermis are implicated in the development of HTS after injuries that involve deeper areas of the skin. However, fibroblasts that reside in the superficial layer of the skin show antifibrotic properties, and injuries limited to this area heal with little or no scarring. Previously, cellular and molecular characteristics of superficial fibroblasts and deep dermal fibroblasts that may influence HTS formation were analyzed. In this study, differences in cellular behavior between superficial fibroblasts and deep dermal fibroblasts that may also affect the development of HTS or tissue fibrosis were further characterized. Immunostaining and migration, adhesion, apoptosis, and cell viability assays were performed in fibroblasts from the superficial and deep dermis. Reverse-transcription polymerase chain reaction was used to examine the gene expression of molecules involved in cell death after treatment of fibroblasts with decorin. When compared with superficial fibroblasts, deep dermal fibroblasts showed lower migration rates. Although all the fibroblasts tested showed no difference in adhesion to fibronectin, superficial fibroblasts demonstrated increased apoptotic and dead cells when treated with decorin. Decorin resulted in a significant increase in the expression of apoptosis markers, histone-1, caspase-1, caspase-8, and p53 in superficial fibroblasts when compared with deep dermal fibroblasts. Taken together, the findings suggest that reduced migration, lack of decorin, and resistance of deep dermal fibroblasts to decorin-induced apoptosis may result in hypercellularity in injuries involving the deep dermis, leading to deposition of excess extracellular matrix and HTS formation.

Differential collagen-glycosaminoglycan matrix remodeling by superficial and deep dermal fibroblasts: potential therapeutic targets for hypertrophic scar.

Skin substitutes are the preferred treatment option in the case of extensive skin loss following burns or other injuries. Among skin substitutes, cultured skin substitutes containing autologous fibroblasts and keratinocytes on collagen-glycosaminoglycan (C-GAG) matrix are most preferred for wound repair. A significant negative outcome of wound healing is hypertrophic scarring (HTS), a dermal fibroproliferative disorder, that leads to considerable morbidity. To examine the role of superficial and deep dermal fibroblasts in HTS, and determine if they differentially remodel C-GAG matrices, fibroblasts were isolated from superficial and deep dermis of lower abdominal tissue of abdominoplasty patients and cultured on C-GAG matrices for four weeks. Over time, deep fibroblasts contracted and stiffened the matrices significantly more and decreased their ultimate tensile strength compared to superficial fibroblasts. Differential remodeling of C-GAG matrices by fibroblasts obtained from different locations of the same organ has not been reported before. Deep fibroblasts were found to express significantly more osteopontin, angiotensin-II, peroxisome proliferator-activated receptor (PPAR)-α, and significantly less tumor necrosis factor-α, PPAR-ß/δ, PPAR-γ, and the proteoglycan, fibromodulin compared to superficial fibroblasts. These molecular targets could potentially be used in therapeutic strategies for treatment of HTS.

In vitro osteogenic induction of human gingival fibroblasts for bone regeneration.

BACKGROUND AND OBJECTIVE: Periodontitis is an inflammatory disease causing bone loss, and is a primary cause of tooth loss. Gingival fibroblasts are readily available with minimal donor site morbidity and may be ideal for tissue engineering efforts in regenerating lost alveolar bone. Dexamethasone (Dex) is commonly employed for in vitro osteogenic induction of a variety of cells, but its effect on human gingival fibroblasts (HGF) is still controversial. Therefore, the aim of our study was to investigate the osteogenic differentiation of HGF following Dex treatment. METHODS: Cultured HGFs were exposed to osteogenic medium containing a wide range of Dex concentrations (0.01-10 µM). The osteogenic phenotype was assessed based on changes in alkaline phosphatase (ALP) activity, the mRNA expression of selected extracellular matrix proteins critical for mineralization and the extent of extracellular mineralization (Von Kossa staining and Ca-content). RESULTS: All assays showed a consistent and maximal osteogenic effect of Dex on HGF at 0.1 and 0.5 µM (weeks 3 and 4), as evidenced by significant osteopontin and osteocalcin expression and mineralization. Longer cultures (week 4) also yielded positive osteogenic effect of Dex at 0.01 µM. Moreover, ALP activity was significantly stimulated at 0.1 and 0.5 µM Dex initially after one week, but ALP was subsequently reduced under Dex. Higher Dex concentrations caused down regulation of osteogenic effects observed at the optimal (0.1-0.5 µM) concentrations. CONCLUSION: Under appropriate osteogenic conditioning, Dex treated HGFs could be a potential source of cells for cell-based therapy for periodontal bone regeneration.

Use of real time three-dimensional transesophageal echocardiography in intracardiac catheter based interventions.

BACKGROUND: Real-time three-dimensional (RT3D) echocardiography is a recently developed technique that is being increasingly used in echocardiography laboratories. Over the past several years, improvements in transducer technologies have allowed development of a full matrix-array transducer that allows acquisition of pyramidal-shaped data sets. These data sets can be processed online and offline to allow accurate evaluation of cardiac structures, volumes, and mass. More recently, a transesophageal transducer with RT3D capabilities has been developed. This allows acquisition of high-quality RT3D images on transesophageal echocardiography (TEE). Percutaneous catheter-based procedures have gained growing acceptance in the cardiac procedural armamentarium. Advances in technology and technical skills allow increasingly complex procedures to be performed using a catheter-based approach, thus obviating the need for open-heart surgery. METHODS: The authors used RT3D TEE to guide 72 catheter-based cardiac interventions. The procedures included the occlusion of atrial septal defects or patent foramen ovales (n=25), percutaneous mitral valve repair (e-valve clipping; n=3), mitral balloon valvuloplasty for mitral stenosis (n=10), left atrial appendage obliteration (n=11), left atrial or pulmonary vein ablation for atrial fibrillation (n=5), percutaneous closures of prosthetic valve dehiscence (n=10), percutaneous aortic valve replacement (n=6), and percutaneous closures of ventricular septal defects (n=2). In this review, the authors describe their experience with this technique, the added value over multiplanar two-dimensional TEE, and the pitfalls that were encountered. RESULTS: The main advantages found for the use RT3D TEE during catheter-based interventions were (1) the ability to visualize the entire lengths of intracardiac catheters, including the tips of all catheters and the balloons or devices they carry, along with a clear depiction of their positions in relation to other cardiac structures, and (2) the ability to ability to demonstrate certain structures in an "en face" view, which is not offered by any other currently available real-time imaging technique, enabling appreciation of the exact nature of the lesion that is undergoing intervention. CONCLUSION: RT3D TEE is a powerful new imaging tool that may become the technique of choice and the standard of care for guidance of selected percutaneous catheter-based procedures.

Evaluation of the abdominal aorta and the renal arteries with an intracardiac echocardiography probe placed in the inferior vena cava: a feasibility study.

BACKGROUND: Ultrasound evaluation of the abdominal aorta and its branches is usually performed transabdominally. Not infrequently, the image quality is suboptimal. Recently, an intracardiac echocardiography probe has become commercially available. These probes are usually inserted intravenously and advanced to the right heart for diagnostic and monitoring purposes during procedures such as atrial septal defect closure and pulmonary vein isolation. Because of the close anatomic relation between the abdominal aorta and the inferior vena cava, we hypothesized that these probes would be useful in the evaluation of the abdominal aorta and the renal arteries. METHODS: Sixteen patients with normal renal function and no history of hypertension who were undergoing a pulmonary vein isolation procedure or atrial septal defect closure were studied. In each patient, the intracardiac echocardiography probe was inserted in the femoral vein and advanced to the right atrium for the evaluation of the left atrium and the pulmonary veins during the procedure. At the end of the therapeutic procedure, the probe was withdrawn into the inferior vena cava for the evaluation of the aorta and renal arteries. RESULTS: High-resolution images of the abdominal aorta from the diaphragm to its bifurcation were easily obtained in all patients. These images allowed for the evaluation of arterial size, shape, and blood flow. Both renal arteries were easily visualized in each patient. With the probe in the inferior vena cava, both renal arteries were parallel to the imaging plane and, therefore, accurate measurement of renal blood flow velocity and individual renal blood flow were measured.