Tomer Avraham

Fibrosis Is a Key Inhibitor of Lymphatic Regeneration

Background: Lymphedema is a common debilitating sequela of lymph node dissection. Although numerous clinical studies suggest that factors that lead to fibrosis are associated with the development of lymphedema, this relationship has not been proven. The purpose of these experiments was therefore to evaluate lymphatic regeneration in the setting of variable soft-tissue fibrosis. Methods: A section of mouse tail skin including the capillary and collecting lymphatics was excised. Experimental animals (n = 20) were treated with topical collagen type I gel and a moist dressing, whereas control animals (n = 20) underwent excision followed by moist dressing alone. Fibrosis, acute lymphedema, lymphatic function, gene expression, lymphatic endothelial cell proliferation, and lymphatic fibrosis were evaluated at various time points. Results: Collagen gel treatment significantly decreased fibrosis, with an attendant decrease in acute lymphedema and improved lymphatic function. Tails treated with collagen gel demonstrated greater numbers of lymphatic vessels, more normal lymphatic architecture, and more proliferating lymphatic endothelial cells. These findings appeared to be independent of vascular endothelial growth factor C expression. Decreased fibrosis was associated with a significant decrease in the expression of extracellular matrix components. Finally, decreased soft-tissue fibrosis was associated with a significant decrease in lymphatic fibrosis as evidenced by the number of lymphatic endothelial cells that coexpressed lymphatic and fibroblast markers. Conclusions: Soft-tissue fibrosis is associated with impairment in lymphatic regeneration and lymphatic function. These defects occur as a consequence of impaired lymphatic endothelial cell proliferation, abnormal lymphatic microarchitecture, and lymphatic fibrosis. Inhibition of fibrosis using a simple topical dressing can markedly accelerate lymphatic repair and promote regeneration of normal capillary lymphatics.

Radiation therapy causes loss of dermal lymphatic vessels and interferes with lymphatic function by TGF-β1-mediated tissue fibrosis

Although radiation therapy is a major risk factor for the development of lymphedema following lymphadenectomy, the mechanisms responsible for this effect remain unknown. The purpose of this study was therefore to determine the effects of radiation on lymphatic endothelial cells (LECs) and lymphatic function. The tails of wild-type or acid sphingomyelinase (ASM)-deficient mice were treated with 0, 15, or 30 Gy of radiation and then analyzed for LEC apoptosis and lymphatic function at various time points. To analyze the effects of radiation fibrosis on lymphatic function, we determined the effects of transforming growth factor (TGF)-beta1 blockade after radiation in vivo. Finally, we determined the effects of radiation and exogenous TGF-beta1 on LECs in vitro. Radiation caused mild edema that resolved after 12-24 wk. Interestingly, despite resolution of tail edema, irradiated animals displayed persistent lymphatic dysfunction. Radiation caused loss of capillary lymphatics and was associated with a dose-dependent increase in LEC apoptosis. ASM-/- mice had significantly less LEC apoptosis; however, this finding did not translate to improved lymphatic function at later time points. Short-term blockade of TGF-beta1 function after radiation markedly decreased tissue fibrosis and significantly improved lymphatic function but did not alter LEC apoptosis. Radiation therapy decreases lymphatic reserve by causing depletion of lymphatic vessels and LECs as well as promoting soft tissue fibrosis. Short-term inhibition of TGF-beta1 activity following radiation improves lymphatic function and is associated with decreased soft tissue fibrosis. ASM deficiency confers LEC protection from radiation-induced apoptosis but does not prevent lymphatic dysfunction.

Lymphatic function is regulated by a coordinated expression of lymphangiogenic and anti-lymphangiogenic cytokines

Lymphangiogenic cytokines such as vascular endothelial growth factor-C (VEGF-C) are critically required for lymphatic regeneration; however, in some circumstances, lymphatic function is impaired despite normal or elevated levels of these cytokines. The recent identification of anti-lymphangiogenic molecules such as interferon-γ (IFN-γ), transforming growth factor-β1, and endostatin has led us to hypothesize that impaired lymphatic function may represent a dysregulated balance in the expression of pro/anti-lymphangiogenic stimuli. We observed that nude mice have significantly improved lymphatic function compared with wild-type mice in a tail model of lymphedema. We show that gradients of lymphatic fluid stasis regulate the expression of lymphangiogenic cytokines (VEGF-A, VEGF-C, and hepatocyte growth factor) and that paradoxically the expression of these molecules is increased in wild-type mice. More importantly, we show that as a consequence of T-cell-mediated inflammation, these same gradients also regulate expression patterns of anti-lymphangiogenic molecules corresponding temporally and spatially with impaired lymphatic function in wild-type mice. We show that neutralization of IFN-γ significantly increases inflammatory lymph node lymphangiogenesis independently of changes in VEGF-A or VEGF-C expression, suggesting that alterations in the balance of pro- and anti-lymphangiogenic cytokine expression can regulate lymphatic vessel formation. In conclusion, we show that gradients of lymphatic fluid stasis regulate not only the expression of pro-lymphangiogenic cytokines but also potent suppressors of lymphangiogenesis as a consequence of T-cell inflammation and that modulation of the balance between these stimuli can regulate lymphatic function.

Functional outcomes of virtually planned free fibula flap reconstruction of the mandible.

Background: The free fibula osteocutaneous flap has become the criterion standard for reconstruction of complex mandibular defects. The authors present their institutional experience with optimization of flap contouring and inset using virtual planning and prefabricated cutting jigs. Methods: All free fibula–based mandible reconstructions performed at the authors’ institution using virtual planning technology between 2009 and 2012 were retrospectively analyzed. The authors evaluated a variety of patient and procedural variables and outcomes. A series of cases performed before virtual planning was reviewed for comparison purposes. Results: Fifty-four reconstructions were performed in 52 patients. Patients were divided evenly between a private university-affiliated medical center and a large county hospital. The most common indications were malignancy (43 percent), ameloblastoma (26 percent), and osteonecrosis/osteomyelitis (23 percent). Thirty percent of patients had irradiation of the recipient site and 38 percent had previous surgery. Sixty-three percent of patients received dental implants, with 47 percent achieving functional dentition. Twenty-five percent of patients had immediate dental implant placement, and 9 percent had immediate dental restoration. Postoperative imaging demonstrated excellent precision and accuracy of flap positioning. Comparison with cases performed before virtual planning demonstrated increased complexity of flap design along with reduced operative time in the virtually planned group. Conclusions: Preoperative virtual planning along with use of prefabricated cutting jigs allows for precise contouring and positioning of microvascular fibula free flaps in mandibular reconstruction. Using this technique, the authors have achieved unprecedented rates of dental rehabilitation along with reduced operative times. The authors believe that virtual planning technologies are an emerging criterion standard in mandible reconstruction. CLINICAL QUESTION/LEVEL OF EVIDENCE: Therapeutic, IV.

Synthesis of a Tissue-Engineered Periosteum with Acellular Dermal Matrix and Cultured Mesenchymal Stem Cells

Periosteal grafts can aid in bone repair by providing bone progenitor cells and acting as a barrier to scar tissue. Unfortunately, these grafts have many of the same disadvantages as bone grafts (donor site morbidity and limited donor sites). In this article, we describe a method of synthesizing a periosteum-like material using acellular human dermis and osteoblasts or mesenchymal stem cells (MSC). We show that osteoblasts readily attach to and proliferate on the acellular human dermis in vitro. In addition, osteoblasts retained the potential for differentiation in response to bone morphogenetic protein stimulation. Cells grown on the acellular human dermis were efficiently transfected with adenoviruses with no evidence of cellular toxicity. To assess for in vivo cell delivery and bone-forming potential, the acellular human dermis was seeded with green fluorescent protein (GFP)-positive MSCs, transfected with bone morphogenetic protein 2, wrapped around the adductor muscle in syngeneic mice, and used to treat critical-sized mandibular defects in nude rats. After 3 weeks, GFP-positive cells were still present, and bone had replaced the interface between the muscle and the constructs. After 6 weeks, critical-sized bone defects had been successfully healed. In conclusion, we show that an acellular human dermis can be used to synthesize a tissue-engineered periosteum capable of delivering cells and osteoinductive proteins.

Adipose-derived stem cells promote lymphangiogenesis in response to VEGF-C stimulation or TGF-β1 inhibition

AIMS Recent studies have demonstrated that augmentation of lymphangiogenesis and tissue engineering hold promise as a treatment for lymphedema. The purpose of this study was to determine whether adipose-derived stem cells (ASCs) can be used in lymphatic tissue-engineering by altering the balance between pro- and anti-lymphangiogenic cytokines. MATERIALS & METHODS ASCs were harvested and cultured in media with or without recombinant VEGF-C for 48 h. ASCs were then implanted in mice using Matrigel plugs. Additional groups of animals were implanted with ASCs transfected with a dominant-negative TGF-β1 receptor-II adenovirus with or without VEGF-C stimulation, since TGF-β1 has been shown to have potent antilymphangiogenic effects. Lymphangiogenesis, lymphatic differentiation and cellular proliferation were assessed. RESULTS Stimulation of ASCs with VEGF-C in vitro significantly increased expression of VEGF-A, VEGF-C and Prox-1. ASCs stimulated with VEGF-C prior to implantation induced a significant (threefold increase) lymphangiogenic response as compared with control groups (unstimulated ASCs or empty Matrigel plugs; p < 0.01). This effect was significantly potentiated when TGF-β1 signaling was inhibited using the dominant-negative TGF-β1 receptor-II virus (4.5-fold increase; p < 0.01). Stimulation of ASCs with VEGF-C resulted in a marked increase in the number of donor ASCs (twofold; p < 0.01) and increased the number of proliferating cells (sevenfold; p < 0.01) surrounding the Matrigel. ASCs stimulated with VEGF-C expressed podoplanin, a lymphangiogenic cell marker, whereas unstimulated cells did not. CONCLUSION Short-term stimulation of ASCs with VEGF-C results in increased expression of VEGF-A, VEGF-C and Prox-1 in vitro and is associated with a marked increase lymphangiogenic response after in vivo implantation. This lymphangiogenic response is significantly potentiated by blocking TGF-β1 function. Furthermore, stimulation of ASCs with VEGF-C markedly increases cellular proliferation and cellular survival after in vivo implantation and stimulated cells express podoplanin, a lymphangiogenic cell marker.

Toll-like receptor deficiency worsens inflammation and lymphedema after lymphatic injury

Mechanisms regulating lymphedema pathogenesis remain unknown. Recently, we have shown that lymphatic fluid stasis increases endogenous danger signal expression, and these molecules influence lymphatic repair (Zampbell JC, et al. Am J Physiol Cell Physiol 300: C1107-C1121, 2011). Endogenous danger signals activate Toll-like receptors (TLR) 2, 4, and 9 and induce homeostatic or harmful responses, depending on physiological context. The purpose of this study was to determine the role of TLRs in regulating tissue responses to lymphatic fluid stasis. A surgical model of lymphedema was used in which wild-type or TLR2, 4, or 9 knockout (KO) mice underwent tail lymphatic excision. Six weeks postoperatively, TLR KOs demonstrated markedly increased tail edema compared with wild-type animals (50-200% increase; P < 0.01), and this effect was most pronounced in TLR4 KOs (P < 0.01). TLR deficiency resulted in decreased interstitial and lymphatic transport, abnormal lymphatic architecture, and fewer capillary lymphatics (40-50% decrease; P < 0.001). Lymphedematous tissues of TLR KOs demonstrated increased leukocyte infiltration (P < 0.001 for TLR4 KOs), including higher numbers of infiltrating CD3+ cells (P < 0.05, TLR4 and TLR9 KO), yet decreased infiltrating F4/80+ macrophages (P < 0.05, all groups). Furthermore, analysis of isolated macrophages revealed twofold reductions in VEGF-C (P < 0.01) and LYVE-1 (P < 0.05) mRNA from TLR2-deficient animals. Finally, TLR deficiency was associated with increased collagen type I deposition and increased transforming growth factor-β1 expression (P < 0.01, TLR4 and TLR9 KO), contributing to dermal fibrosis. In conclusion, TLR deficiency worsens tissue responses to lymphatic fluid stasis and is associated with decreased lymphangiogenesis, increased fibrosis, and reduced macrophage infiltration. These findings suggest a role for innate immune responses, including TLR signaling, in lymphatic repair and lymphedema pathogenesis.

p21cip/WAF is a key regulator of long-term radiation damage in mesenchyme-derived tissues

This study aimed to determine the mechanisms responsible for long-term tissue damage following radiation injury. We irradiated p21-knockout (p21(-/-)) and wild-type (WT) mice and determined the long-term deleterious effects of this intervention on mesenchyme-derived tissues. In addition, we explored the mechanisms of radiation-induced mesenchymal stem cell (MSC) dysfunction in isolated bone marrow-derived cells. p21 expression was chronically elevated >200-fold in irradiated tissues. Loss of p21 function resulted in a >4-fold increase in the number of skin MSCs remaining after radiation. p21(-/-) mice had significantly less radiation damage, including 6-fold less scarring, 40% increased growth potential, and 4-fold more hypertrophic chondrocytes in the epiphyseal plate (P<0.01). Irradiated p21(-/-) MSCs had 4-fold increased potential for bone or fat differentiation, 4-fold greater proliferation rate, and nearly 7-fold lower senescence as compared to WT MSCs (P<0.01). Ectopic expression of p21 in knockout cells decreased proliferation and differentiation potential and recapitulated the WT phenotype. Loss of p21 function markedly decreases the deleterious effects of radiation injury in mesenchyme-derived tissues and preserves tissue-derived MSCs. In addition, p21 is a critical regulator of MSC proliferation, differentiation, and senescence both at baseline and in response to radiation.

Evolution of surgical techniques for mandibular reconstruction using free fibula flaps: The next generation.

Virtual surgical planning (VSP) has contributed to a number of technical innovations in mandible reconstruction. We report on these innovations and the overall evolution of mandible reconstruction using free fibula flaps at our institution.

A 35-Year Evolution of Free Flap-Based Breast Reconstruction at a Large Urban Academic Center.

BACKGROUND This study aims to characterize the evolution and trends in free flap breast reconstruction at our institution. METHODS The authors reviewed and analyzed a registry of free flap breast reconstructions performed at a large urban academic center. RESULTS Between 1979 and mid-2014, a total of 920 patients underwent breast reconstruction with 1,254 flaps. The mean age was 47.7 years (range, 16-79 years). Over the past 10 years, patients were older than all patients seen in the prior decade (average age 48.9 vs. 46.1 years, p = 0.002). Overall, 82% of flaps were performed at our university hospital, 17% at a major urban county hospital, and < 1% at other sites. A total of 99% patients received postmastectomy reconstruction for an existing cancer diagnosis or prophylaxis. There has been a significant increase in reconstructions, with 579 flaps performed over the past 5 years alone. There has been a fundamental shift in the predominant flap of choice over time. Perforator flaps have increased in popularity at our institution, with 74% of all reconstructions over this past 5 years being perforator based. Perforator flaps were more likely to be chosen over nonperforator flaps in older versus younger patients (p = 0.0008). There has been a steady increase in bilateral reconstructions since the first one was performed in 1987 (p = 0.002). CONCLUSIONS Over the past 35 years, our institution has seen a significant evolution in free flap-based breast reconstruction. Besides a massive increase in flap numbers we have seen a significant trend toward bilateral reconstructions and perforator-based flaps.