Anthony J. Penington

New murine model of spontaneous autologous tissue engineering, combining an arteriovenous pedicle with matrix materials

The authors previously described a model of tissue engineering in rats that involves the insertion of a vascular pedicle and matrix material into a semirigid closed chamber, which is buried subcutaneously. The purpose of this study was to develop a comparable model in mice, which could enable genetic mutants to be used to more extensively study the mechanisms of the angiogenesis, matrix production, and cellular migration and differentiation that occur in these models. A model that involves placing a split silicone tube around blood vessels in the mouse groin was developed and was demonstrated to successfully induce the formation of new vascularized tissue. Two vessel configurations, namely, a flow-through pedicle (n = 18 for three time points) and a ligated vascular pedicle (n = 18), were compared. The suitability of chambers constructed from either polycarbonate or silicone and the effects of incorporating either Matrigel equivalent (n = 18) or poly(dl-lactic-co-glycolic acid) (n = 18) on angiogenesis and tissue production were also tested. Empty chambers, chambers with vessels only, and chambers with matrix only served as control chambers. The results demonstrated that a flow-through type of vascular pedicle, rather than a ligated pedicle, was more reliable in terms of patency, angiogenesis, and tissue production, as were silicone chambers, compared with polycarbonate chambers. Marked angiogenesis occurred with both types of extracellular matrix scaffolds, and there was evidence that native cells could migrate into and survive within the added matrix, generating a vascularized three-dimensional construct. When Matrigel was used as the matrix, the chambers filled with adipose tissue, creating a highly vascularized fat flap. In some cases, new breast-like acini and duct tissue appeared within the fat. When poly(dl-lactic-co-glycolic acid) was used, the chambers filled with granulation and fibrous tissue but no fat or breast tissue was observed. No significant amount of tissue was generated in the control chambers. Operative times were short (25 minutes), and two chambers could be inserted into each mouse. In summary, the authors have developed an in vivo murine model for studying angiogenesis and tissue-engineering applications that is technically simple and quick to establish, has a high patency rate, and is well tolerated by the animals.

Formation of New Tissue from an Arteriovenous Loop in the Absence of Added Extracellular Matrix

A major requirement for the microsurgical repair of contour defects of the skin, for example, following removal of a skin cancer on the face, is a mass of vascularised subcutaneous tissue. Such tissue can be generated in vivo using basic tissue engineering principles. In previous studies in our laboratory, we have used a model comprising an arteriovenous (AV) shunt loop sandwiched in artificial dermis, placed in a cylindrical plastic growth chamber, and inserted subcutaneously to grow new connective tissue progressively up to 4 weeks. To learn more about the basic growth characteristics with this model, the same AV shunt loop within a chamber without added extracellular matrix was inserted subcutaneously into the groins of rats for 2, 4, or 12 weeks (n = 5 per group). There was a progressive increase in the mass and volume of tissue such that the chamber was two-thirds full after 12 weeks. Histological examination showed that at 2 weeks there was evidence of fibroblast and vascular outgrowth from the AV shunt, with the formation of granulation tissue, surrounded by a mass of coagulated exudate. At 4 weeks the connective tissue deposition was more extensive, with a mass of more mature granulation tissue containing considerable collagen. By 12 weeks there was an extensive, well vascularized mass of mature fibrous tissue. The blood vessels and residual adventitia of the AV shunt were the likely source of growth factors and of the cells which populated the chamber with new maturing connective tissue. A patent AV shunt in an isolated chamber appears to be the minimal requirement for the generation of new vascularized tissue that is potentially suitable for microsurgical transplantation.

The Influence of Extracellular Matrix on the Generation of Vascularized, Engineered, Transplantable Tissue

Abstract: In a recently described model for tissue engineering, an arteriovenous loop comprising the femoral artery and vein with interposed vein graft is fabricated in the groin of an adult male rat, placed inside a polycarbonate chamber, and incubated subcutaneously. New vascularized granulation tissue will generate on this loop for up to 12 weeks. In the study described in this paper three different extracellular matrices were investigated for their ability to accelerate the amount of tissue generated compared with a no‐matrix control. Poly‐d,l‐lactic‐co‐glycolic acid (PLGA) produced the maximal weight of new tissue and vascularization and this peaked at two weeks, but regressed by four weeks. Matrigel was next best. It peaked at four weeks but by eight weeks it also had regressed. Fibrin (20 and 80 mg/ml), by contrast, did not integrate with the generating vascularized tissue and produced less weight and volume of tissue than controls without matrix. The limiting factors to growth appear to be the chamber size and the capacity of the neotissue to integrate with the matrix. Once the sides of the chamber are reached or tissue fails to integrate, encapsulation and regression follow. The intrinsic position of the blood supply within the neotissue has many advantages for tissue and organ engineering, such as ability to seed the construct with stem cells and microsurgically transfer new tissue to another site within the individual. In conclusion, this study has found that PLGA and Matrigel are the best matrices for the rapid growth of new vascularized tissue suitable for replantation or transplantation.

Four common glomulin mutations cause two thirds of glomuvenous malformations (“familial glomangiomas”): evidence for a founder effect

Background: Glomuvenous malformation (GVM) (“familial glomangioma”) is a localised cutaneous vascular lesion histologically characterised by abnormal smooth muscle-like “glomus cells” in the walls of distended endothelium lined channels. Inheritable GVM has been linked to chromosome 1p21-22 and is caused by truncating mutations in glomulin. A double hit mutation was identified in one lesion. This finding suggests that GVM results from complete localised loss of function and explains the paradominant mode of inheritance. Objective: To report on the identification of a mutation in glomulin in 23 additional families with GVM. Results: Three mutations are new; the others have been described previously. Among the 17 different inherited mutations in glomulin known up to now in 43 families, the 157delAAGAA mutation is the most common and was present in 21 families (48.8%). Mutation 108C→A was found in five families (11.8%), and the mutations 554delA+556delCCT and 1179delCAA were present together in two families (4.7% each). Polymorphic markers suggested a founder effect for all four mutations. Conclusions: Screening for these mutations should lead to a genetic diagnosis in about 70% of patients with inherited GVM. So far, a mutation in glomulin has been found in all GVM families tested, thus demonstrating locus homogeneity.

Hereditary cutaneomucosal venous malformations are caused by TIE2 mutations with widely variable hyper-phosphorylating effects

Mutations in the angiopoietin receptor TIE2/TEK have been identified as the cause for autosomal dominantly inherited cutaneomucosal venous malformation (VMCM). Thus far, two specific germline substitutions (R849W and Y897S), located in the kinase domain of TIE2, have been reported in five families. The mutations result in a fourfold increase in ligand-independent phosphorylation of the receptor. Here, we report 12 new families with TEK mutations. Although the phenotype is primarily characterized by small multifocal cutaneous vascular malformations, many affected members also have mucosal lesions. In addition, cardiac malformations are observed in some families. Six of the identified mutations are new, with three located in the tyrosine kinase domain, two in the kinase insert domain, and another in the carboxy terminal tail. The remaining six are R849W substitutions. Overexpression of the new mutants resulted in ligand-independent hyperphosphorylation of the receptor, suggesting this is a general feature of VMCM-causative TIE2 mutations. Moreover, variation in the level of activation demonstrates, to the best of our knowledge for the first time, that widely differing levels of chronic TIE2 hyperphosphorylation are tolerated in the heterozygous state, and are compatible with normal endothelial cell function except in the context of highly localized areas of lesion pathogenesis.

Bone Regeneration in a Rabbit Critical-Sized Skull Defect Using Autologous Adipose-Derived Cells

Repair of substantial cranial defects in adults and children may be compromised due to limitations in donor bone stocks for autologous grafts. We evaluated the capability of autologous adipose-derived mesenchymal cells (ADCs) in combination with polylactic acid (PLA) scaffolds to regenerate bone in a critical-sized skull defect. Thirty adult New Zealand White rabbits were divided into six groups of five animals each: (1) PLA alone (control), (2) fibronectin-coated PLA, (3) PLA with ADCs, (4) fibronectin-coated PLA with ADCs, (5) PLA with osteogenically induced ADCs (osADCs), and (6) fibronectin-coated PLA with osADCs. All the animals were humanely killed after 6 weeks. X-ray, histology, and histomorphometric analysis were performed to evaluate the new bone formation inside the PLA scaffold. Radiographically and histomorphometrically, the groups in which the PLA was not fibronectin coated showed no bone formation in contrast to the fibronectin-coated groups (Gp1 vs. Gp2, p < 0.0005); the group treated with osteo-induced ADCs and fibronectin (Gp6) showed significantly more bone formation than the group treated with undifferentiated ADCs (Gp4) and the group treated without cells (Gp5, p < 0.0005, in both cases). These data indicate that the surface treatment with fibronectin promotes bone formation within the scaffold, and that autologous, osteo-induced adipose-derived cells enhance bone formation if seeded into a fibronectin-treated PLA scaffold.

Increasing the volume of vascularized tissue formation in engineered constructs: an experimental study in rats.

The authors have previously described a model of in vivo tissue generation based on an implanted, microsurgically created vessel loop in a plastic chamber (volume, 0.45 ml) containing a poly(DL-lactic-co-glycolic acid) (PLGA) scaffold. Tissue grew spontaneously in association with an intense angiogenic sprouting from the loop and almost filled the chamber, resulting in a mean amount of tissue in chambers of 0.23 g with no added matrix scaffold and 0.33 g of tissue in PLGA-filled chambers after 4 weeks of incubation. The aim of the present study was to investigate whether a greater volume of tissue could be generated when the same-size vessel loop was inserted into a larger (1.9 ml) chamber. In four groups of five rats, an arteriovenous shunt sandwiched between two disks of PLGA, used as a scaffold for structural support, was placed inside a large polycarbonate growth chamber. Tissue and PLGA weight and volume, as well as histological characteristics of the newly formed tissue, were assessed at 2, 4, 6, and 8 weeks. Tissue weight and volume showed a strong linear correlation. Tissue weight increased progressively from 0.13 +/- 0.04 g at 2 weeks to 0.57 +/- 0.06 g at 6 weeks (p < 0.0005). PLGA weight decreased progressively from 0.89 +/- 0.07 g at 2 weeks to 0.20 +/- 0.09 g at 8 weeks (p < 0.0005). Histological examination of the specimens confirmed increased tissue growth and maturation over time. It is concluded that larger quantities of tissue can be grown over a longer period of time by using larger-size growth chambers.

Monocyte Chemoattractant Protein‐1 and Nitric Oxide Promote Adipogenesis in a Model That Mimics Obesity

Objective: An increasing body of evidence is emerging linking adipogenesis and inflammation. Obesity, alone or as a part of the metabolic syndrome, is characterized by a state of chronic low‐level inflammation as revealed by raised plasma levels of inflammatory cytokines and acute‐phase proteins. If inflammation can, in turn, increase adipose tissue growth, this may be the basis for a positive feedback loop in obesity. We have developed a tissue engineering model for growing adipose tissue in the mouse that allows quantification of increases in adipogenesis. In this study, we evaluated the adipogenic potential of the inflammogens monocyte chemoattractant protein (MCP)‐1 and zymosan‐A (Zy) in a murine tissue engineering model.

Clinical applications and technical limitations of prefabricated flaps

&NA; Vascular pedicle implantation into the subdermal level of the skin induces an angiogenic response that can be sufficient to artificially create or “prefabricate” an axialpattern flap suitable for island pedicle or free‐flap transfer. This technique allows the creation of large, thin skin flaps that retain the qualities of the donor‐site skin. Three cases are presented in which such thin flaps have been created to cover defects, one on the knee and two on the face. While the reconstructions are satisfactory, difficulties were encountered with venous insufficiency in the flaps after the second‐stage transfer. We believe that the complexity and staged nature of these procedures limit their application to highly selected patients, but their role probably lies in the transfer of supraclavicular skin for the reconstruction of facial defects.

Zymosan-induced inflammation stimulates neo-adipogenesis.

Objective:To investigate the potential of inflammation to induce new adipose tissue formation in the in vivo environment.Methods and results:Using an established model of in vivo adipogenesis, a silicone chamber containing a Matrigel and fibroblast growth factor 2 (1 μg/ml) matrix was implanted into each groin of an adult male C57Bl6 mouse and vascularized with the inferior epigastric vessels. Sterile inflammation was induced in one of the two chambers by suspending Zymosan-A (ZA) (200–0.02 μg/ml) in the matrix at implantation. Adipose tissue formation was assessed at 6, 8, 12 and 24 weeks. ZA induced significant adipogenesis in an inverse dose-dependent manner (P<0.001). At 6 weeks adipose tissue formation was greatest with the lowest concentrations of ZA and least with the highest. Adipogenesis occurred both locally in the chamber containing ZA and in the ZA-free chamber in the contralateral groin of the same animal. ZA induced a systemic inflammatory response characterized by elevated serum tumour necrosis factor-α levels at early time points. Aminoguanidine (40 μg/ml) inhibited the adipogenic response to ZA-induced inflammation. Adipose tissue formed in response to ZA remained stable for 24 weeks, even when exposed to the normal tissue environment.Conclusions:These results demonstrate that inflammation can drive neo-adipogenesis in vivo. This suggests the existence of a positive feedback mechanism in obesity, whereby the state of chronic, low-grade inflammation, characteristic of the condition, may promote further adipogenesis. The mobilization and recruitment of a circulating population of adipose precursor cells is likely to be implicated in this mechanism.