Characterization of isolated liver sinusoidal endothelial cells for liver bioengineering.

BACKGROUND: The liver sinusoidal capillaries play a pivotal role in liver regeneration, suggesting they may be beneficial in liver bioengineering. This study isolated mouse liver sinusoidal endothelial cells (LSECs) and determined their ability to form capillary networks in vitro and in vivo for liver tissue engineering purposes. METHODS AND RESULTS: In vitro LSECs were isolated from adult C57BL/6 mouse livers. Immunofluorescence labelling indicated they were LYVE-1+/CD32b+/FactorVIII+/CD31-. Scanning electron microscopy of LSECs revealed the presence of characteristic sieve plates at 2 days. LSECs formed tubes and sprouts in the tubulogenesis assay, similar to human microvascular endothelial cells (HMEC); and formed capillaries with lumens when implanted in a porous collagen scaffold in vitro. LSECs were able to form spheroids, and in the spheroid gel sandwich assay produced significantly increased numbers (p = 0.0011) of capillary-like sprouts at 24 h compared to HMEC spheroids. Supernatant from LSEC spheroids demonstrated significantly greater levels of vascular endothelial growth factor-A and C (VEGF-A, VEGF-C) and hepatocyte growth factor (HGF) compared to LSEC monolayers (p = 0.0167; p = 0.0017; and p < 0.0001, respectively), at 2 days, which was maintained to 4 days for HGF (p = 0.0017) and VEGF-A (p = 0.0051). In vivo isolated mouse LSECs were prepared as single cell suspensions of 500,000 cells, or as spheroids of 5000 cells (100 spheroids) and implanted in SCID mouse bilateral vascularized tissue engineering chambers for 2 weeks. Immunohistochemistry identified implanted LSECs forming LYVE-1+/CD31- vessels. In LSEC implanted constructs, overall lymphatic vessel growth was increased (not significantly), whilst host-derived CD31+ blood vessel growth increased significantly (p = 0.0127) compared to non-implanted controls. LSEC labelled with the fluorescent tag DiI prior to implantation formed capillaries in vivo and maintained LYVE-1 and CD32b markers to 2 weeks. CONCLUSION: Isolated mouse LSECs express a panel of vascular-related cell markers and demonstrate substantial vascular capillary-forming ability in vitro and in vivo. Their production of liver growth factors VEGF-A, VEGF-C and HGF enable these cells to exert a growth stimulus post-transplantation on the in vivo host-derived capillary bed, reinforcing their pro-regenerative capabilities for liver tissue engineering studies.

High and low mammographic density human breast tissues maintain histological differential in murine tissue engineering chambers.

Mammographic density (MD) is the area of breast tissue that appears radiologically white on mammography. Although high MD is a strong risk factor for breast cancer, independent of BRCA1/2 mutation status, the molecular basis of high MD and its associated breast cancer risk is poorly understood. MD studies will benefit from an animal model, where hormonal, gene and drug perturbations on MD can be measured in a preclinical context. High and low MD tissues were selectively sampled by stereotactic biopsy from operative specimens of high-risk women undergoing prophylactic mastectomy. The high and low MD tissues were transferred into separate vascularised biochambers in the groins of SCID mice. Chamber material was harvested after 6 weeks for histological analyses and immunohistochemistry for cytokeratins, vimentin and a human-specific mitochondrial antigen. Within-individual analysis was performed in replicate mice, eliminating confounding by age, body mass index and process-related factors, and comparisons were made to the parental human tissue. Maintenance of differential MD post-propagation was assessed radiographically. Immunohistochemical staining confirmed the preservation of human glandular and stromal components in the murine biochambers, with maintenance of radiographic MD differential. Propagated high MD regions had higher stromal (p = 0.0002) and lower adipose (p = 0.0006) composition, reflecting the findings in the original human breast tissue, although glands appeared small and non-complex in both high and low MD groups. No significant differences were observed in glandular area (p = 0.4) or count (p = 0.4) between high and low MD biochamber tissues. Human mammary glandular and stromal tissues were viably maintained in murine biochambers, with preservation of differential radiographic density and histological features. Our study provides a murine model for future studies into the biomolecular basis of MD as a risk factor for breast cancer.

The influence of nitric oxide synthase 2 on cutaneous wound angiogenesis.

BACKGROUND: Inducible nitric oxide synthase (nitric oxide synthase 2, NOS 2) inhibition significantly suppresses chronically ischaemic skin flap survival, possibly because of reduced angiogenesis. OBJECTIVES: To investigate the effect of genetic NOS 2 inhibition on cutaneous wound angiogenesis in two in vivo murine models. The impact of NOS 2 manipulation on vascular endothelial growth factor (VEGF)-A stimulated and fibroblast growth factor (FGF)-2 stimulated angiogenesis was also investigated in the Matrigel(®) plug assay. METHODS: (i) Matrigel plugs/incisional wounds: two groups of NOS 2-/- mice and two groups of wild-type (WT) mice had bilateral Matrigel plugs containing 500 ng mL(-1) VEGF-A or 1000 ng mL(-1) FGF-2 injected subcutaneously in the abdomen. A 2·5 cm long dorsal incisional skin wound was created and sutured closed in the same animals. Wounds and plugs were explored at 7 or 12 days. (ii) Excisional wounds: dorsal 0·5 × 1·0 cm excisional skin wounds were created in four groups (two NOS 2-/- and two WT) and explored at 7 or 14 days. Wounds and Matrigel plugs were examined histologically and morphometrically for determination of percentage vascular volume (PVV). RESULTS: The PVV in NOS 2-/- incisional wounds and excisional wounds was significantly less than in WT wounds (P = 0·05 and P < 0·001, respectively). The PVV was significantly less in VEGF-A stimulated Matrigel plugs compared with FGF-2 stimulated plugs in NOS 2-/- mice (P < 0·01), but not in WT mice. CONCLUSIONS: NOS 2 is significantly involved in angiogenic signalling in healing skin wounds, particularly within the first 7 days. However, Matrigel plug vascularization suggests that the role of NOS 2 in angiogenesis is related to VEGF-A but not FGF-2 stimulated angiogenesis.

HIV-1 selection by epidermal dendritic cells during transmission across human skin.

Macrophage tropic HIV-1 is predominant during the initial viremia after person to person transmission of HIV-1 (Zhu, T., H. Mo, N. Wang, D.S. Nam, Y. Cao, R.A. Koup, and D.D. Ho. 1993. Science. 261:1179-1181.), and this selection may occur during virus entry and carriage to the lymphoid tissue. Human skin explants were used to model HIV-1 selection that may occur at the skin or mucosal surface. Macrophage tropic, but not T cell line tropic strains of HIV-1 applied to the abraded epidermis were recovered from the cells emigrating from the skin explants. Dermis and epidermis were separated by dispase digestion after virus exposure to determine the site of viral selection within the skin. Uptake and transmission to T cells of all HIV-1 isolates was found with the dermal emigrant cells, but only macrophage tropic virus was transferred by emigrants from the epidermis exposed to HIV-1, indicating selection only within the epidermis. CD3+, CD4+ T cells were found in both the dermal and epidermal emigrant cells. After cell sorting to exclude contaminating T cells, macrophage tropic HIV-1 was found in both the dermal emigrant dendritic cells and in dendritic cells sorted from the epidermal emigrants. These observations suggest that selective infection of the immature epidermal dendritic cells represents the cellular mechanism that limits the initial viremia to HIV-1 that can use the CCR5 coreceptor.

Pancreatic islet transplantation using vascularised chambers containing nerve growth factor ameliorates hyperglycaemia in diabetic mice.

Intraportal islet transplantation has shown initial promise for the treatment of type 1 diabetes. However, the portal vein site is associated with complications such as thrombosis and hepatic steatosis, leading to transplant failure. The aims of this study were to (1) test the feasibility of an alternative islet transplantation method that utilises a FDA-approved gelatin sponge as a novel islet carrier and (2) assess if exogenous addition of nerve growth factor (NGF) has any additional beneficial effects on graft performance in diabetic mice. Mice were rendered diabetic by a single intraperitoneal injection of streptozotocin. Five hundred syngeneic islets were seeded onto a Gelitaspon((R)) disc in the presence or absence of NGF, and placed into a silicone chamber surrounding the femoral neurovascular pedicle. Islet function was assessed by weekly monitoring of blood glucose levels and an intraperitoneal glucose tolerance test performed at the end of the study. Chambers were harvested for further histological analysis. Four of five mice transplanted with islets seeded onto Gelitaspon with NGF showed a significant reduction in blood glucose levels by 4 weeks after transplantation, and demonstrated a response similar to non-diabetic mice when tested with an intraperitoneal glucose tolerance test. Chamber tissue from this group contained islets with insulin-producing beta cells adjacent to the vascular pedicle. Islets seeded onto Gelitaspon with NGF and sited on femoral vessels using a tissue-engineering chamber offer an alternative method for islet transplantation in diabetic mice. This may have potential as a method for clinical islet transplantation.

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 microg/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 microg/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-alpha levels at early time points. Aminoguanidine (40 microg/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.

Myogel, a novel, basement membrane-rich, extracellular matrix derived from skeletal muscle, is highly adipogenic in vivo and in vitro.

BACKGROUND/AIMS: Biological and synthetic scaffolds play important roles in tissue engineering and are being developed towards human clinical applications. Based on previous work from our laboratory, we propose that extracellular matrices from skeletal muscle could be developed for adipose tissue engineering. METHODS: Extracellular matrices (Myogels) extracted from skeletal muscle of various species were assessed using biochemical assays including ELISA and Western blotting. Biofunctionality was assessed using an in vitro differentiation assay and a tissue engineering construct model in the rat. RESULTS: Myogels were successfully extracted from mice, rats, pigs and humans. Myogels contained significant levels of laminin alpha4- and alpha2-subunits and collagen I compared to Matrigel, which contains laminin 1 (alpha1beta1gamma1) and collagen IV. Levels of growth factors such as fibroblast growth factor 2 were significantly higher than Matrigel, vascular endothelial growth factor-A levels were significantly lower and all other growth factors were comparable. Myogels reproducibly stimulated adipogenic differentiation of preadipocytes in vitro and the growth of adipose tissue in the rat. CONCLUSIONS: We found Myogel induces adipocyte differentiation in vitroand shows strong adipogenic potential in vivo, inducing the growth of well-vascularised adipose tissue. Myogel offers an alternative for current support scaffolds in adipose tissue engineering, allowing the scaling up of animal models towards clinical adipose tissue engineering applications.

Hypoxic preconditioning of myoblasts implanted in a tissue engineering chamber significantly increases local angiogenesis via upregulation of myoblast vascular endothelial growth factor-A expression and downregulation of miRNA-1, miRNA-206 and angiopoietin-1.

Vascularization is a major hurdle for growing three-dimensional tissue engineered constructs. This study investigated the mechanisms involved in hypoxic preconditioning of primary rat myoblasts in vitro and their influence on local angiogenesis postimplantation. Primary rat myoblast cultures were exposed to 90 min hypoxia at <1% oxygen followed by normoxia for 24 h. Real time (RT) polymerase chain reaction evaluation indicated that 90 min hypoxia resulted in significant downregulation of miR-1 and miR-206 (p < 0.05) and angiopoietin-1 (p < 0.05) with upregulation of vascular endothelial growth factor-A (VEGF-A; p < 0.05). The miR-1 and angiopoietin-1 responses remained significantly downregulated after a 24 h rest phase. In addition, direct inhibition of miR-206 in L6 myoblasts caused a significant increase in VEGF-A expression (p < 0.05), further establishing that changes in VEGF-A expression are influenced by miR-206. Of the myogenic genes examined, MyoD was significantly upregulated, only after 24 h rest (p < 0.05). Preconditioned or control myoblasts were implanted with Matrigel™ into isolated bilateral tissue engineering chambers incorporating a flow-through epigastric vascular pedicle in severe combined immunodeficiency mice and the chamber tissue harvested 14 days later. Chambers implanted with preconditioned myoblasts had a significantly increased percentage volume of blood vessels (p = 0.0325) compared with chambers implanted with control myoblasts. Hypoxic preconditioned myoblasts promote vascularization of constructs via VEGF upregulation and downregulation of angiopoietin-1, miR-1 and miR-206. The relatively simple strategy of hypoxic preconditioning of implanted cells - including non-stem cell types - has broad, future applications in tissue engineering of skeletal muscle and other tissues, as a technique to significantly increase implant site angiogenesis.

Vascularized tissue-engineered chambers promote survival and function of transplanted islets and improve glycemic control.

We have developed a chamber model of islet engraftment that optimizes islet survival by rapidly restoring islet-extracellular matrix relationships and vascularization. Our aim was to assess the ability of syngeneic adult islets seeded into blood vessel-containing chambers to correct streptozotocin-induced diabetes in mice. Approximately 350 syngeneic islets suspended in Matrigel extracellular matrix were inserted into chambers based on either the splenic or groin (epigastric) vascular beds, or, in the standard approach, injected under the renal capsule. Blood glucose was monitored weekly for 7 weeks, and an intraperitoneal glucose tolerance test performed at 6 weeks in the presence of the islet grafts. Relative to untreated diabetic animals, glycemic control significantly improved in all islet transplant groups, strongly correlating with islet counts in the graft (P<0.01), and with best results in the splenic chamber group. Glycemic control deteriorated after chambers were surgically removed at week 8. Immunohistochemistry revealed islets with abundant insulin content in grafts from all groups, but with significantly more islets in splenic chamber grafts than the other treatment groups (P<0.05). It is concluded that hyperglycemia in experimental type 1 diabetes can be effectively treated by islets seeded into a vascularized chamber functioning as a "pancreatic organoid."

Composite breast reconstruction: Implant-based breast reconstruction with adjunctive lipofilling.

INTRODUCTION: Options for breast reconstructions enclose autologous tissue transfers or implants. Fat grafting is gaining more interest in this specific field of breast surgery. This study concentrates on the technique and aesthetic results of breast reconstruction with fat grafts combined with implants, in women who have undergone total mastectomy. METHODS: Breast reconstructions (n = 23) was performed using a protocol of intratissular expansion with serial deflation-lipofilling. In order to achieve the best aesthetic outcome, an additional small implant was placed. A retrospective data analysis was performed. In all patients a tissue expander was placed at the time of mastectomy or after removal of a previous breast reconstruction. The mean of lipoaspirate material for the reconstruction was 333 mL (range 120-715 mL). To create an adequate volume of the reconstructed breast, a supplementary small implant was placed, with a mean volume of 222 mL (range 125-375 mL). The mean follow-up was 33 months (range 19-50 months). RESULTS: A MRI analysis was performed in eight patients at least 9 months after the last lipofilling procedure, demonstrating a mean of 171 mL (range 64-538 mL) of transferred fat, a mean fat survival of 53% and a volume ratio of fat graft/implant of 0.97 (range 0,3-3,8). CONCLUSION: This composite technique of using autologous fat tissue and implants shows aesthetic pleasant results and must be considered as a valid alternative in a subset of patients. Further investigations to optimize the fat graft take must be encouraged.

Rigid removable cover for dorsal wound protection and tube fixation in pigs.

OBJECTIVE: To report the design and benefits of a rigid polyethylene cover 'shell' for the protection of dorsal torso wounds and tube fixation in pigs. METHODS: Open C-shaped polyethylene shells were designed to protect wounds and dressings on the dorsum of pigs used in research into negative pressure dressing-assisted wound healing. The shells were designed to resist trauma and contamination, to be comfortable and expansible, and to facilitate tube fixation and management. Strap fixation was optimised during experimentation. Efficacy was assessed by direct observation of dressing and wound protection, tube integrity and by macroscopic and microscopic assessments of wound healing. RESULTS: The shells effectively protected the wounds against blunt and sharp trauma, were simple to remove and reapply, were well tolerated and allowed for growth of the pigs. Circumferential neck straps attached by lateral straps to the shells proved critical. There was no wound infection or inflammation underlying the shells. Porting tubing via mid-dorsal holes in the shells and affixing the tubing just cranial to these holes prevented tube damage and traction, permitted tube management from outside the cages and allowed the pigs to move freely without becoming entangled. CONCLUSION: These shells effectively protected dorsal skin wounds and dressings, prevented tube damage and facilitated tube management in pigs. Similar systems may be useful for other production animals for wound management and for tube management with negative pressure wound healing, drain tubes or the delivery of nutrition, fluids or medications.

Diamond encapsulated photovoltaics for transdermal power delivery.

A safe, compact and robust means of wireless energy transfer across the skin barrier is a key requirement for implantable electronic devices. One possible approach is photovoltaic (PV) energy delivery using optical illumination at near infrared (NIR) wavelengths, to which the skin is highly transparent. In the work presented here, a subcutaneously implantable silicon PV cell, operated in conjunction with an external NIR laser diode, is developed as a power delivery system. The biocompatibility and long-term biostability of the implantable PV is ensured through the use of an hermetic container, comprising a transparent diamond capsule and platinum wire feedthroughs. A wavelength of 980 nm is identified as the optimum operating point based on the PV cell's external quantum efficiency, the skin's transmission spectrum, and the wavelength dependent safe exposure limit of the skin. In bench-top experiments using an external illumination intensity of 0.7 W/cm(2), a peak output power of 2.7 mW is delivered to the implant with an active PV cell dimension of 1.5 × 1.5 × 0.06 mm(3). This corresponds to a volumetric power output density of ~20 mW/mm(3), significantly higher than power densities achievable using inductively coupled coil-based approaches used in other medical implant systems. This approach paves the way for further ministration of bionic implants.

Lipopolysaccharide-induced cell cycle arrest in macrophages occurs independently of nitric oxide synthase II induction.

Lipopolysaccharide (LPS, a Gram-negative bacterium cell wall component) is a potent macrophage activator that inhibits macrophage proliferation and stimulates production of nitric oxide (NO) via NO synthase II (NOSII). We investigated whether NO mediates the LPS-stimulated cell cycle arrest in mouse bone marrow-derived macrophages (BMM). The addition of the NO donor DETA NONOate (200 microM) inhibited BMM proliferation by approx. 80%. However, despite NO being an antimitogen, LPS was as potent at inhibiting proliferation in BMM derived from NOSII-/- mice as from wild-type mice. Consistent with these findings, LPS-induced cell cycle arrest in normal BMM was not reversed by the addition of the NOSII inhibitor S-methylisothiourea. Moreover, in both normal and NOSII-/- BMM, LPS inhibited the expression of cyclin D1, a protein that is essential for proliferation in many cell types. Despite inhibiting proliferation DETA NONOate had no effect on cyclin D1 expression. Our data indicate that while both LPS and NO inhibit BMM proliferation, LPS inhibition of BMM proliferation can occur independently of NOSII induction.

Inducible nitric oxide synthase (iNOS) activity promotes ischaemic skin flap survival.

We have examined the role of nitric oxide (NO) in a model of functional angiogenesis in which survival of a skin flap depends entirely on angiogenesis to provide an arterial blood supply to maintain tissue viability. The different effects of nitric oxide synthase (NOS) inhibitors on rat skin flap survival appeared to be explained on the basis of their NOS isoform selectivity. Skin flap survival was decreased by iNOS-selective (inducible NOS) inhibitors, S-methyl-isothiourea, aminoguanidine and aminoethylthiorea; unaffected by the non-selective inhibitor nitro-imino-L-ornithine; and enhanced by the cNOS (constitutive NOS, that is endothelial NOS (eNOS) and neuronal NOS (nNOS)) inhibitor, nitro-L-arginine methyl ester. Skin flap survival was reduced in mice with targeted disruption of the iNOS gene (iNOS knockout mice), and the administration of nitro-L-arginine methyl ester significantly increased flap survival in iNOS knockout mice (P<0.05). iNOS immunoreactivity was identified in mast cells in the angiogenic region. Immunoreactive vascular endothelial growth factor (VEGF) and basic fibroblast growth factor were also localized to mast cells. The combination of interferon-gamma and tumour necrosis factor-alpha induced NO production and increased VEGF levels in mast cells cultured from bone marrow of wild-type, but not iNOS KO mice. The increased tissue survival associated with the capacity for iNOS expression may be related to iNOS-dependent enhancement of VEGF levels and an ensuing angiogenic response. Our results provide both pharmacological and genetic evidence that iNOS activity promotes survival of ischaemic tissue.

Role of priming stresses and Hsp70 in protection from ischemia-reperfusion injury in cardiac and skeletal muscle.

Ischemia-reperfusion injury limits the survival of muscle involved in tissue trauma or transfers during microsurgical reconstruction. Priming stresses such as ischemic preconditioning or mild hyperthermia have frequently been associated with improved survival of ischemic-reperfused cardiac muscle, such protection coinciding with induction of the stress-related heat shock protein 70 (Hsp70). Little is known about the response of skeletal muscle to priming stresses. This review summarizes the current knowledge on the use of priming stresses as protective strategies against the consequences of ischemia-reperfusion in cardiac and skeletal muscle and the potential role of Hsp70.

Expression of leukemia inhibitory factor in human nerve following injury.

In animal models of peripheral nerve injury, leukemia inhibitory factor (LIF) is normally expressed at very low levels. Following nerve injury, its expression is rapidly increased in the nerve at the injury site and promotes both sensory and motor neuron survival. Once normal nerve function is restored, LIF expression returns to negligible levels. For this reason, LIF is considered to be a peripheral nerve trauma factor. We wished to determine whether LIF is also upregulated in human nerves following trauma and whether it is expressed in neuromas of varying age. Immunohistochemical staining for the presence of LIF was performed on injured and control human nerves from a number of subjects. Results demonstrate that LIF expression is increased in nerves within hours of injury and, in the case of neuroma formation, can persist for several years. LIF immunoreactivity was consistently found in Schwann cells, in peripheral nerve axons, and, at stages when an inflammatory response was present, also in neutrophils, mast cells, macrophages, and blood vessel walls. The level of staining within the connective tissue of injured nerves was elevated compared to control nerves, which may be due to the presence of LIF bound to the soluble secreted form of the LIF receptor. Whether the continued expression of LIF is unhealed injured nerves promotes the development of neuromas remains to be resolved.

Stimulating effect of an arteriovenous shunt on the in vivo growth of isografted fibroblasts: a preliminary report.

Isogenous fibroblasts derived from the skin of inbred Sprague-Dawley rats were cultured in vitro, labeled with bisbenzamide (BB) or carboxyfluorescein diacetate (CFDA), and seeded into polycarbonate growth chambers. After 24 h incubation in vitro, the chambers, either empty or containing an arteriovenous (AV) shunt, were implanted subcutaneously into the inguinal region of Sprague-Dawley rats and examined by fluorescence microscopy 2 or 7 days later. The AV shunt remained patent in all experiments. The density of labeled cells on the chamber surface in all chambers decreased in the first 2 days after insertion. At 7 days, the cell density in the empty chambers had not altered from the 2-day level, but the density in the AV shunt containing chambers had increased to almost three times the day 2 level (p = 0.013). It appears that an AV shunt can induce a significant proliferation of fibroblasts implanted adjacent to it. For at least 7 days after labeling, BB and CFDA provide a simple and effective method of quantitative detection of implanted fibroblasts. It is concluded that nutrients from the AV shunt implanted in a growth chamber result in a significant increase in the number of viable, matrix-synthesizing cells, compared with AV shunt-free controls.

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.

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.